Preliminary observations on phenotypic differences between complete and partial globozoospermia

Dear Editor, Globozoospermia syndrome (Mendelian Inheritance in Man [MIM]:102530) is a rare sperm malformation disorder with an incidence rate of <0.1% in males.1 The most prominent feature of globozoospermia is the round shape of the sperm head, accompanied by the absence of an acrosome or the presence of a residual, dysfunctional acrosome.1 Globozoospermia is primarily caused by gene missense variants, with the dpy-19-like protein II (DPY19L2), spermatogenesis-associated 16 (SPATA16), and serine peptidase inhibitor Kazal type 2 (SPINK2) being the common pathogenic genes.2,3 Globozoospermia can be classified into type I (100% round-headed sperm) and type II (<100% round-headed sperm), and the specific sperm characteristics and functions may differ between the two types.2 Due to the lack of an acrosome or an abnormal acrosome function, sperm cannot secrete acrosomal enzymes to penetrate the cumulus oophorus and enter the oocyte, making natural conception impossible for patients with globozoospermia. Reports have shown that intracytoplasmic sperm injection (ICSI) technology can enable successful pregnancies in such patients.4 However, a study indicates that a combination of ICSI and artificial oocyte activation (AOA) is necessary for successful conception.5 The discrepancies in these research findings may be attributed to the different types of globozoospermia. This study analyzed the differences in structure, function, and missense variant genes between the two types of globozoospermia. We used ICSI technology alone for assisted reproduction in patients with type II globozoospermia and analyzed their pregnancy outcomes. The findings will provide new insights into the diagnosis and treatment of globozoospermia. The two patients involved in this study were infertile males who visited Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics, Fujian Medical University (Fuzhou, China) between January 2024 and September 2025. They had no prior pregnancies achieved, and their urogenital systems, including erectile and ejaculatory functions, were normal. The patients had no other reproductive system diseases, and their endocrine levels were normal. They came to our hospital for assisted reproduction after semen analysis revealed decreased sperm number or sperm motility. Through sperm morphological analysis, we determined that one patient had complete globozoospermia (type I) and the other had partial globozoospermia (type II). Transmission electron microscopy (Koninklijke Philips Electronics N.V., Amsterdam, The Netherlands) was used to analyze ultrastructural changes in sperm. Computer-assisted sperm analysis (Beiang Pharmaceutical Technology Co., Ltd., Shanghai, China) was employed to detect sperm kinetic parameters. The functional parameters of sperm, such as sperm membrane integrity, nuclear maturity, and DNA integrity, were also assessed. Biochemical components in seminal plasma and levels of sex hormones in the blood were measured. Whole-exome sequencing (WES) was performed to identify exonic variants, which were compared with the reference genome GRCh37.P13 to determine mutated genes. Bidirectional Sanger sequencing was performed via the dideoxy chain termination method. Following polymerase chain reaction (PCR) amplification of target gene fragments, DNA chain extension was terminated with fluorescently labeled dideoxynucleotides (ddNTP), and capillary electrophoresis was conducted on an ABI 3500 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA) to separate fragments and read base sequences for verification of identified gene variants. Additionally, ICSI technology was used for assisted reproduction in the patient with type II globozoospermia. This study was approved by the Ethics Committee of Fujian Maternal and Child Health Hospital (Approval No. 2023KY093). Informed consent was obtained from the patients for the study, and the study obtained permission to publicly publish the data. All methods were carried out in accordance with the relevant guidelines and national regulations. The results showed that the baseline characteristics of the two patients were similar, with normal biochemical components in seminal plasma and normal sex hormone levels (Table 1). Analyses revealed that the sperm count, sperm concentration, and semen volume of the type I patient were normal, but sperm motility was below the reference value. The type II patient had lower sperm concentration and motility than the reference values, while the other parameters were normal (Table 1). Transmission electron microscopy results revealed that both types of round-headed sperm exhibited morphological abnormalities in the sperm head and showed significant differences in mitochondrial arrangement in the midpiece and in the disorganization of the “9+2” microtubule structure in the sperm flagellum. Specifically, type I sperm displayed no residual acrosome in the head, granulation in the nuclear region, swollen mitochondria in the neck, normal mitochondrial arrangement in the tail, and a normal “9+2” microtubule structure in the flagellum. By contrast, type II sperm exhibited partial residual or complete acrosome, vacuoles in the nuclear region, disordered or absent mitochondrial arrangement in the tail, and partial or complete disorganization of the “9+2” microtubule structure in the flagellum (Table 1 and Supplementary Figure 1). In type II sperm, 5.5% of the acrosomes were intact, and 11.5% of the mitochondria were normal, similar to the results of Gatimel et al.6 on acrosome sperm. The membrane integrity of type I sperm was normal, while that of type II sperm was below the reference value. The nuclear maturity of both types of sperm was lower than that of normal sperm, with no significant difference between the two. The DNA fragmentation index of both types was higher than the reference value, with type I sperm showing better integrity than type II (Table 1 and Supplementary Figure 2). In summary, the two types of globozoospermia exhibited significant differences not only in the proportion of round-headed sperm but also in acrosomal remnants, nuclear maturity, DNA integrity, mitochondrial arrangement, and flagellar microtubule structure.Table 1: Differences in basic information and sperm function parameters between the two patients with distinct types of globozoospermiaSupplementary Figure 1: Ultrastructural analysis of the two types of globozoospermia using transmission electron microscopy. (a) In cases of complete globozoospermia, the acrosome of the head disappears and the mitochondria in the neck swell. (b) In partial globular spermatozoa, vacuolization of the nuclear region and residual acrosomal components in the head can be observed (c) In partial acrosomal insufficiency, a complete acrosomal head can be observed. (d) In the sperm tails of patients with complete globozoospermia, normal mitochondrial arrangement can be observed. (e) Disordered mitochondrial arrangement can be observed in the tail of sperm with partial globozoospermia. (f) Mitochondrial structural defects can be observed in the tail of sperm with partial globozoospermia. (g) A normal “9+2” flagellum structure can be seen in complete globozoospermia. (h) A roughly normal “9+2” structure can be seen in partial globozoospermia. (i) A disordered “9+2” structure can be seen in partial globozoospermia.Supplementary Figure 2: Sperm morphology and function analysis of the two types of globozoospermia. (a) Sperm morphology analysis. Used Papanicolaou staining method. (b) Sperm maturity analysis. Used aniline blue staining method and blue indicates immature. (c) Sperm cell membrane integrity analysis. Used Eosin–Nigrosin staining method and black indicates incomplete membrane integrity. (d) Sperm DNA integrity analysis. Used sperm chromatin dispersion assay and no halo or a small halo indicates DNA fragmentation.Globozoospermia is inherited in an autosomal recessive manner.7 WES revealed that patient I had a deletion in the DPY19L2 gene (NM_173812.5), while patient II exhibited a missense variant in the cilia- and flagella-associated protein 58 (CFAP58) gene (NM_001008723.2). Specifically, the CFAP58 variant occurs at position c.1855C>T (p.Arg619Trp), and the DPY19L2 gene is completely absent. Both genes were predicted to be deleterious by gene prediction library. Homozygous loss of the DPY19L2 gene, which encodes a transmembrane domain protein, is a common cause of globozoospermia.4 This is consistent with the genetic test results of patient I. Interestingly, previous studies have shown that CFAP58 gene variants like those detected in patient II are pathogenic for multiple morphological abnormalities of the flagella (MMAF).8CFAP58 variants have not been reported in studies of globozoospermia. Missense variants in the DPY19L2 gene can lead to instability in the connection between the sperm nuclear dense layer and the nuclear membrane, which prevents the acrosome from attaching to the sperm nucleus. This results in failed nuclear formation, abnormal vesicle transport, and, ultimately, the inability to form the acrosome. Missense variants in the CFAP58 gene have been reported to be associated with sperm flagella formation, and these variants can cause disorganization of the “9+2” structure of the flagellum.8 The missense variants in both the DPY19L2 and CFAP58 genes leading to globozoospermia suggest that the formation of the sperm head and tail occurs simultaneously and that genes affecting sperm structural changes contribute to the development of globozoospermia.9 Existing studies indicate that conventional ICSI can facilitate successful pregnancies in globozoospermia patients, although the pregnancy rate remains low.5 Other research, however, has demonstrated that combining ICSI with AOA yields superior pregnancy outcomes compared to ICSI alone.5 This discrepancy arises because injecting acrosome-deficient sperm via ICSI alone fails to trigger the Ca2+ oscillations required for oocyte activation. AOA addresses this by injecting a small quantity of Ca2+ oscillation inducer along with round-headed sperm to activate the oocyte. Nevertheless, the safety of AOA remains controversial because it introduces exogenous substances into the oocyte. We retrieved five oocytes from the type II globozoospermia patient’s spouse following ovulation induction, three of which were mature. After ICSI, two high-quality embryos were cultured on the third day, and we transferred two embryos into the uterus, resulting in one successful pregnancy. This result indicates that type II globozoospermia patients can have successful pregnancies using only ICSI without the need for AOA. This approach avoids the risk of introducing exogenous substances into oocytes. Pregnancy outcome data for type I globozoospermia are not available in this study because patient I declined assisted reproductive technology. In summary, this study detected a novel candidate variant in type II globozoospermia and analyzed the differences in morphological, functional, and genetic associations among patients with different types of the condition. These findings provide important reference for clinical diagnosis (e.g., genetic screening indications) and the selection of assisted reproductive strategies (e.g., ICSI protocol selection). AUTHOR CONTRIBUTIONS GXC designed the research, performed the research, analyzed the data, and wrote the manuscript. HLX and LY collected the data and searched the literature. YS and BHZ finished language editing, designed the project, and guided the whole process. All authors read and approved the final manuscript. COMPETING INTERESTS All authors declare no competing interests.