FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility in mice.

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

[Intracytoplasmic sperm injection for patients with special types of teratozoospermia: Analysis of outcomes].

OBJECTIVE: To investigate the outcomes of intracytoplasmic sperm injection (ICSI) in the treatment of special types of teratozoospermia such as globozoospermia, acephalic spermatozoa syndrome (ASS) and multiple morphological abnormalities of sperm flagella (MMAF). METHODS: We retrospectively analyzed the clinical data on 7 cases of globozoospermia (group A), 6 cases of ASS (group B) and 21 cases of MMAF (group C) treated by ICSI from January 2011 to January 2021, all confirmed with pathogenic or likely pathogenic gene variations. We compared the age, body mass index (BMI), sperm parameters, number of mature oocytes, and rates of fertilization, high-quality embryos, clinical pregnancy, live birth and spontaneous abortion among the three groups of patients. RESULTS: There were no statistically significant differences in the age, BMI and number of metaphase Ⅱ (MⅡ) oocytes among the three groups (P > 0.05). Sperm concentration and motility were dramatically higher (P < 0.01) while the rates of fertilization, clinical pregnancy and live birth remarkably lower in group A than in B and C (P < 0.01). No statistically significant difference was observed in the spontaneous abortion rate among the three groups (P > 0.05). CONCLUSION: ICSI can achieve relatively satisfactory outcomes of clinical pregnancy in patients with ASS or MMAF, but only a low fertilization rate or no fertilization at all in those with globozoospermia even if treated by artificial oocyte activation.

A loss-of-function variant in SSFA2 causes male infertility with globozoospermia and failed oocyte activation.

Globozoospermia (OMIM: 102530) is a rare type of teratozoospermia (< 0.1%). The etiology of globozoospermia is complicated and has not been fully revealed. Here, we report an infertile patient with globozoospermia. Variational analysis revealed a homozygous missense variant in the SSFA2 gene (NM_001130445.3: c.3671G > A; p.R1224Q) in the patient. This variant significantly reduced the protein expression of SSFA2. Immunofluorescence staining showed positive SSFA2 expression in the acrosome of human sperm. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and Coimmunoprecipitation (Co-IP) analyses identified that GSTM3 and Actin interact with SSFA2. Further investigation revealed that for the patient, regular intracytoplasmic sperm injection (ICSI) treatment had a poor prognosis. However, Artificial oocyte activation (AOA) by a calcium ionophore (A23187) after ICSI successfully rescued the oocyte activation failure for the patient with the SSFA2 variant, and the couple achieved a live birth. This study revealed that SSFA2 plays an important role in acrosome formation, and the homozygous c.3671G > A loss-of-function variant in SSFA2 caused globozoospermia. SSFA2 may represent a new gene in the genetic diagnosis of globozoospermia, especially the successful outcome of AOA-ICSI treatment for couples, which has potential value for clinicians in their treatment regimen selections.

PRSS55 is a novel potential causative gene for human male infertility.

RESEARCH QUESTION: Testis-specific PRSS55 is a chymotrypsin-like serine protease that is highly conserved among mammalian species. The essential role of Prss55 in mouse male fertility has been established. What is the role of PRSS55 in human reproduction? DESIGN: Whole exome sequencing was used to identify the genetic cause in an infertile male with teratozoospermia. Papanicolaou staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to explore morphological defects in the patient's spermatozoa. Immunofluorescence staining and western blot analysis were conducted to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist the patient with fertilization. RESULTS: Sanger sequencing of the pedigree demonstrated that the infertile man carried a novel homozygous mutation in PRSS55 (c.575C>T [p.A192V]). Morphological defects in the sperm head, neck, midpiece and tail were demonstrated by Papanicolaou staining, SEM and TEM. Immunofluorescence staining and western blotting of the patient's spermatozoa showed that the point mutation changed the conformation of PRSS55 and caused a sharp decrease in the PRSS55 protein concentration. The expression and subcellular localization of PRSS55 in the testis and spermatozoa of mice and humans showed that PRSS55 was expressed in the head and flagella of spermatids and epididymal spermatozoa. Moreover, ICSI treatment for this kind of infertile patient was shown to be effective. CONCLUSIONS: These findings revealed a novel mutation in PRSS55 in an infertile patient, suggesting for the first time the crucial role of PRSS55 in human fertility. This study provides new insight into genetic counselling diagnoses and subsequent treatment for male infertility.

Genome-wide compound heterozygote analysis highlights DPY19L2 alleles in a non-consanguineous Spanish family with total globozoospermia.

RESEARCH QUESTION: Would the use of genome-wide genotyping be an advantageous strategy to identify the molecular aetiology of two brothers from a non-consanguineous family, clinically diagnosed with total globozoospermia? DESIGN: Two related Spanish globozoospermic patients were studied. Eight first- and second-degree family members were also included in the study. The clinical procedure included anamnesis, physical examination and semen analyses. Acrosome visualization was performed by fluorescein isothiocyanate-Pisum sativum agglutinin labelling and ultrastructural electron microscope sperm analysis. Sperm DNA fragmentation was determined by TUNEL and SCD. Molecular analysis included: the detection of deletion of the DPY19L2 gene by a BPa (break point "a") gap-polymerase chain reaction, and genotyping by using a high-throughput genome-wide genotyping platform and a genotype imputation strategy. RESULTS: The biological characteristics of the two globozoospermic siblings included round-headed spermatozoa without an acrosome; ultrastructural defects in spermatozoa; increased sperm fragmentation and aneuploidies, inability of spermatozoa to activate oocytes (correctable with artificial activation) and good developmental potential of embryos generated by IVF/intracytoplasmic sperm injection. This genetic study focused on a genome-wide compound heterozygote analysis that identified two deleterious rare coding variants in the DPY19L2 gene [rs771726551 (c.431T>A exon 3) and rs147579680 (c.869G>A exon 8)]. CONCLUSION: A genome-wide compound heterozygote analysis strategy should be considered for molecular screening in globozoospermia and other rare congenital diseases, particularly in cases from non-consanguineous families.

Awareness and reporting of globozoospermia among in vitro fertilization and andrology laboratories: A national survey.

Globozoospermia (GZ) is a rare disorder found in less than 0.1% of infertile men in which spermatozoa lack acrosomes necessary for penetration of an oocyte. While methods have been demonstrated to allow globozoospermic men to achieve a viable pregnancy with their partner, the Wold Health Organization considers identifying and reporting GZ on semen analysis to be 'important'. Our study aims to determine if and to what extent in vitro fertilization (IVF) laboratories nationwide recognize and report GZ on semen analysis reports. We constructed an IRB-approved survey sent nationwide to IVF and andrology clinic laboratory directors listed by the Society for Assisted Reproductive Technology and/or the American Society for Reproductive Medicine. Results from the survey were de-identified for analysis. A total of 490 surveys were sent with a response rate of 10% (n = 51). Most respondents (66%) practiced in a private, rather than academic, setting. A majority of respondents were confident in their technicians' knowledge of GZ (86%) and ability to identify it on a sample (94%). However, only half of respondents noted a space to report the concern for GZ to the ordering physician, and 25% of respondents did not feel their clinic was able to identify patients where there is a concern for GZ. Similarly, 84% of respondents did not report a percent of acrosome-deficient sperm. Less than half of respondents reported that their clinic has previously diagnosed GZ. Though the majority of respondents felt that their laboratory technicians would be able to identify GZ, a significant minority felt that their clinic did not have means to be able to report concern for GZ. This may be due to the absence of a proper channel to report a concern for GZ, a lack of knowledge about the condition, or failure to distinguish GZ from a broader reported percentage of morphologically abnormal sperm. Given evidence that the diagnosis of GZ may be under-reported in the United States, there should be a national standard for laboratory technicians to be trained to recognize GZ and be able to report their suspicion to the ordering clinician.

The molecular mechanisms underlying acrosome biogenesis elucidated by gene-manipulated mice†.

Sexual reproduction requires the fusion of two gametes in a multistep and multifactorial process termed fertilization. One of the main steps that ensures successful fertilization is acrosome reaction. The acrosome, a special kind of organelle with a cap-like structure that covers the anterior portion of sperm head, plays a key role in the process. Acrosome biogenesis begins with the initial stage of spermatid development, and it is typically divided into four successive phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. The run smoothly of above processes needs an active and specific coordination between the all kinds of organelles (endoplasmic reticulum, trans-Golgi network, and nucleus) and cytoplasmic structures (acroplaxome and manchette). During the past two decades, an increasing number of genes have been discovered to be involved in modulating acrosome formation. Most of these proteins interact with each other and show a complicated molecular regulatory mechanism to facilitate the occurrence of this event. This review focuses on the progresses of studying acrosome biogenesis using gene-manipulated mice and highlights an emerging molecular basis of mammalian acrosome formation.

Translational aspects of novel findings in genetics of male infertility-status quo 2021.

INTRODUCTION: Male factor infertility concerns 7-10% of men and among these 40-60% remain unexplained. SOURCES OF DATA: This review is based on recent published literature regarding the genetic causes of male infertility. AREAS OF AGREEMENT: Screening for karyotype abnormalities, biallelic pathogenic variants in the CFTR gene and Y-chromosomal microdeletions have been routine in andrology practice for >20 years, explaining ~10% of infertility cases. Rare specific conditions, such as congenital hypogonadotropic hypogonadism, disorders of sex development and defects of sperm morphology and motility, are caused by pathogenic variants in recurrently affected genes, which facilitate high diagnostic yield (40-60%) of targeted gene panel-based testing. AREAS OF CONTROVERSY: Progress in mapping monogenic causes of quantitative spermatogenic failure, the major form of male infertility, has been slower. No 'recurrently' mutated key gene has been identified and worldwide, a few hundred patients in total have been assigned a possible monogenic cause. GROWING POINTS: Given the high genetic heterogeneity, an optimal approach to screen for heterogenous genetic causes of spermatogenic failure is sequencing exomes or in perspective, genomes. Clinical guidelines developed by multidisciplinary experts are needed for smooth integration of expanded molecular diagnostics in the routine management of infertile men. AREAS TIMELY FOR DEVELOPING RESEARCH: Di-/oligogenic causes, structural and common variants implicated in multifactorial inheritance may explain the 'hidden' genetic factors. It is also critical to understand how the recently identified diverse genetic factors of infertility link to general male health concerns across lifespan and how the clinical assessment could benefit from this knowledge.

Loss of SPACA1 function causes autosomal recessive globozoospermia by damaging the acrosome-acroplaxome complex.

STUDY QUESTION: Is the sperm acrosome membrane-associated protein 1 (SPACA1) gene critical to human globozoospermia? SUMMARY ANSWER: The biallelic loss-of-function (variant of SPACA1) causes globozoospermia as a result of acrosome-acroplaxome complex damage. WHAT IS KNOWN ALREADY: SPACA1 expression decreases in patients with globozoospermia. Spaca1 gene-disrupted mice have abnormally shaped sperm heads that resemble those of human globozoospermia. STUDY DESIGN, SIZE, DURATION: We recruited a consanguineous family with two brothers affected by infertility as a consequence of globozoospermia. The semen analysis data and ART outcomes were collected. Exome sequencing (ES) was used to identify potential pathogenic variants. Protein-protein interaction (PPI) technologies and proteomic analysis were utilized to explore the pathogenic mechanism. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two globozoospermic brothers and their consanguineous parents were recruited to identify the potential pathogenic variant through ES. A homozygous nonsense variant in the SPACA1 gene in both brothers inherited from the heterozygous parents was identified. Twenty normal fertile males were recruited as controls. Sperm ultrastructure was observed with transmission electron microscopy. Western blotting was performed to measure SPACA1 expression level in the sperm from the patients. Mass spectrometry (MS) analyses were used to identify differentially expressed proteins and to investigate proteins that interact with SPACA1. Co-immunoprecipitation (co-IP), yeast two-hybrid (Y2H) and immunofluorescence colocalization assays were used to confirm the PPI. MAIN RESULTS AND THE ROLE OF CHANCE: A nonsense variant (NM_030960.2: c.53G>A; p. Trp18*) in the SPACA1 gene was identified as the pathogenic variant in a family with globozoospermia. Patient IV:1 and Patient IV:2 had a phenotype very similar to that of Spaca1 gene-disrupted mice. The nonsense variant in SPACA1 led to premature transcriptional termination in the signal peptide, which was confirmed by western blotting. MS-based proteomics analysis showed that eight interactors of SPACA1 were differentially expressed in the patients' sperm, including actin-like Protein 7A (ACTL7A), an important component of the acrosome-acroplaxome complex. The PPI of SPACA1 and ACTL7A was confirmed via co-IP and Y2H assays. Immunofluorescence showed that SPACA1 and ACTL7A colocalized in mature sperm, revealing that these proteins were coexpressed spatially. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of globozoospermia, only two patients from one family harbouring the SPACA1 variant were found. Future studies should evaluate SPACA1 variants in larger cohorts to corroborate this finding. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that the SPACA1 gene was critical for globozoospermia, which expanded the spectrum of causative genes for globozoospermia. This study also provided evidence for ICSI clinical outcomes for patients with SPACA1-deficient globozoospermia, which may guide clinical treatment strategies. Furthermore, this study explored the pathogenesis of globozoospermia caused by SPACA1 deficiency. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Precision Medical Research of National Key Research and Development Program (2018YFC1002400), National Natural Science Foundation of China (81873724), and Natural Science Foundation of Shanghai (20ZR1472700). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Vps13b is required for acrosome biogenesis through functions in Golgi dynamic and membrane trafficking.

The sperm acrosome is a lysosome-related organelle that develops using membrane trafficking from the Golgi apparatus as well as the endolysosomal compartment. How vesicular trafficking is regulated in spermatids to form the acrosome remains to be elucidated. VPS13B, a RAB6-interactor, was recently shown involved in endomembrane trafficking. Here, we report the generation of the first Vps13b-knockout mouse model and show that male mutant mice are infertile due to oligoasthenoteratozoospermia. This phenotype was explained by a failure of Vps13b deficient spermatids to form an acrosome. In wild-type spermatids, immunostaining of Vps13b and Rab6 revealed that they transiently locate to the acrosomal inner membrane. Spermatids lacking Vps13b did not present with the Golgi structure that characterizes wild-type spermatids and showed abnormal targeting of PNA- and Rab6-positive Golgi-derived vesicles to Eea1- and Lamp2-positive structures. Altogether, our results uncover a function of Vps13b in the regulation of the vesicular transport between Golgi apparatus, acrosome, and endolysosome.

Single-center thorough evaluation and targeted treatment of globozoospermic men.

PURPOSE: To characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment. METHODS: We assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA). RESULTS: Ultrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome. CONCLUSIONS: Sperm bioassays and genomic studies can be used to characterize this gamete's capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.

Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice.

Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1-/- mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted by a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and Atg7 deacetylation was disrupted in spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomal vesicles. Taken together, these findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis.

Exome sequencing reveals novel causes as well as new candidate genes for human globozoospermia.

STUDY QUESTION: Can exome sequencing identify new genetic causes of globozoospermia? SUMMARY ANSWER: Exome sequencing in 15 cases of unexplained globozoospermia revealed deleterious mutations in seven new genes, of which two have been validated as causing globozoospermia when knocked out in mouse models. WHAT IS KNOWN ALREADY: Globozoospermia is a rare form of male infertility characterised by round-headed sperm and malformation of the acrosome. Although pathogenic variants in DPY19L2 and SPATA16 are known causes of globozoospermia and explain up to 70% of all cases, genetic causality remains unexplained in the remaining patients. STUDY DESIGN, SIZE, DURATION: After pre-screening 16 men for mutations in known globozoospermia genes DPY19L2 and SPATA16, exome sequencing was performed in 15 males with globozoospermia or acrosomal hypoplasia of unknown aetiology. PARTICIPANTS/MATERIALS, SETTING, METHOD: Targeted next-generation sequencing and Sanger sequencing was performed for all 16 patients to screen for single-nucleotide variants and copy number variations in DPY19L2 and SPATA16. After exclusion of one patient with DPY19L2 mutations, we performed exome sequencing for the 15 remaining subjects. We prioritised recessive and X-linked protein-altering variants with an allele frequency of <0.5% in the population database GnomAD in genes with an enhanced expression in the testis. All identified candidate variants were confirmed in patients and, where possible, in family members using Sanger sequencing. Ultrastructural examination of semen from one of the patients allowed for a precise phenotypic characterisation of abnormal spermatozoa. MAIN RESULTS AND ROLE OF CHANCE: After prioritisation and validation, we identified possibly causative variants in eight of 15 patients investigated by exome sequencing. The analysis revealed homozygous nonsense mutations in ZPBP and CCDC62 in two unrelated patients, as well as rare missense mutations in C2CD6 (also known as ALS2CR11), CCIN, C7orf61 and DHNA17 and a frameshift mutation in GGN in six other patients. All variants identified through exome sequencing, except for the variants in DNAH17, were located in a region of homozygosity. Familial segregation of the nonsense variant in ZPBP revealed two fertile brothers and the patient's mother to be heterozygous carriers. Paternal DNA was unavailable. Immunohistochemistry confirmed that ZPBP localises to the acrosome in human spermatozoa. Ultrastructural analysis of spermatozoa in the patient with the C7orf61 mutation revealed a mixture of round heads with no acrosomes (globozoospermia) and ovoid or irregular heads with small acrosomes frequently detached from the sperm head (acrosomal hypoplasia). LIMITATIONS, REASONS FOR CAUTION: Stringent filtering criteria were used in the exome data analysis which could result in possible pathogenic variants remaining undetected. Additionally, functional follow-up is needed for several candidate genes to confirm the impact of these mutations on normal spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed an important role for mutations in ZPBP and CCDC62 in human globozoospermia as well as five new candidate genes. These findings provide a more comprehensive understanding of the genetics of male infertility and bring us closer to a complete molecular diagnosis for globozoospermia patients which would help to predict the success of reproductive treatments. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by The Netherlands Organisation for Scientific Research (918-15-667); National Health and Medical Research Council of Australia (APP1120356) and the National Council for Scientific Research (CONICET), Argentina, PIP grant 11220120100279CO. The authors have nothing to disclose.

Globozoospermia syndrome: An update.

Among the factors involved in male infertility, there is a rare morphology disorder called "globozoospermia" that is classified into total globozoospermia and partial globozoospermia (type I and type II, respectively). This syndrome is primarily characterised by the presence of round-headed spermatozoa with cytoskeleton defects around the nucleus and no acrosome. Current data support the negative correlation between globozoospermia and conventional intracytoplasmic sperm injection (ICSI) outcomes, revealing the need for the management of patients undergoing assisted reproduction technology (ART) through more effective treatment techniques. This review highlights the most important characteristics of globozoospermia such as sperm parameters, DNA/chromatin integrity and sperm DNA fragmentation (SDF), as well as genetic features based on the latest knowledge. Additionally, we looked into current progress on fertilisation potential and possible treatment strategies for patients presenting with globozoospermia.

Identification of a New QTL Region on Mouse Chromosome 1 Responsible for Male Hypofertility: Phenotype Characterization and Candidate Genes.

Male fertility disorders often have their origin in disturbed spermatogenesis, which can be induced by genetic factors. In this study, we used interspecific recombinant congenic mouse strains (IRCS) to identify genes responsible for male infertility. Using ultrasonography, in vivo and in vitro fertilization (IVF) and electron microscopy, the phenotyping of several IRCS carrying mouse chromosome 1 segments of Mus spretus origin revealed a decrease in the ability of sperm to fertilize. This teratozoospermia included the abnormal anchoring of the acrosome to the nucleus and a persistence of residual bodies at the level of epididymal sperm midpiece. We identified a quantitative trait locus (QTL) responsible for these phenotypes and we have proposed a short list of candidate genes specifically expressed in spermatids. The future functional validation of candidate genes should allow the identification of new genes and mechanisms involved in male infertility.

[Detection of DPY19L2 gene mutation in 2 cases of globozoospermia].

OBJECTIVE: To investigate the mutation of the DPY19L2 gene in patients with globozoospermia. METHODS: We collected the clinical data and peripheral blood from 2 patients with globozoospermia and screened for mutation of the DPY19L2 gene by PCR amplification and DNA sequencing technology. RESULTS: The sperm from the 2 globozoospermia patients were round morphologically under the light microscope, with deeply stained nuclei but no acrosome. Electron microscopy showed the sperm with a large round head but no acrosomal structure, the nuclei enveloped by a single layer of membrane and the cytoplasm dispersed. PCR amplification revealed homozygous deletion of Exon 5, Exon6 and Exon15 in the DPY19L2 gene in both the patients. CONCLUSIONS: This study proved that the homozygous mutation of DPY19L2 could lead to globozoospermia, which has an important significance for researches on the molecular mechanisms and gene diagnosis of the disease as well as for clinicians in genetic counseling and treatment.

[Outcomes of assisted reproductive technology for patients with different types of teratozoospermia].

OBJECTIVE: To discuss the outcomes of ICSI in infertile patients with globozoospermia (GS), acephalic spermatozoa syndrome (ASS) or teratozoospermia with miniacrosome and irregular-headed sperm defect (TMRHS). METHODS: This retrospective study included 3 cases of GS, 3 cases of ASS and 2 cases of TMRHS undergoing ICSI. We analyzed the rates of fertilization, cleavage, blastocyst formation, implantation, clinical pregnancy and live birth in the three groups of patients. RESULTS: The patients of the GS and ASS groups all achieved clinical pregnancies and healthy births, but those of the TMRHS group showed a lower fertilization rate than the other two groups and achieved no clinical pregnancy. CONCLUSIONS: ICSI could achieve successful clinical pregnancy in infertile patients with globozoospermia or acephalic spermatozoa syndrome, but no satisfactory clinical outcome in those with miniacrosome and irregular-headed sperm defect, though it has to be further proved by more studies with larger-sized samples.

A successful healthy childbirth and an ongoing evolutive pregnancy in a case of partial globozoospermia by hyaluronic acid sperm selection.

We here report a successful healthy childbirth and an ongoing evolutive pregnancy in a case of partial globozoospermia after selection of mature spermatozoa bound to hyaluronic acid (HA). The couple underwent two in vitro fertilisation (IVF) cycles. In the first attempt, 14 MII oocytes were retrieved. Randomly, seven oocytes were injected by conventional PVP-ICSI and seven by HA-ICSI. Fertilised oocytes were 2/7 and 4/7 after PVP-ICSI and HA-ICSI respectively. Transfer of two grade A embryos from HA-ICSI lead to birth of a healthy baby. The surplus embryo of the HA-ICSI group was vitrified at blastocyst stage. The two embryos from PVP-ICSI arrested their development. Two years after the childbirth, the vitrified blastocyst was transferred into the uterus, but implant failed. In the second cycle, 14 MII oocytes were retrieved and they were injected by HA-ICSI. Fertilised oocytes were 10 out of 14 injected oocytes. On day 5, two blastocysts were transferred into uterus and a single evolutive pregnancy is ongoing. On day 6, one blastocyst was vitrified. The remaining surplus embryos arrested their development. A healthy childbirth and an ongoing evolutive pregnancy in two consecutive ICSI attempts of the same couple suggest that HA sperm selection might assist in cases with partial globozoospermia.

Comparison of sperm morphology and nuclear sperm quality in SPATA16- and DPY19L2-mutated globozoospermic patients.

The aim of this study was to compare the sperm morphology and nuclear sperm quality (sperm aneuploidy and DNA fragmentation) in two groups of globozoospermic patients: DPY19L2-mutated patients (n = 6) and SPATA16-mutated patients (n = 2). Results for these two groups were also compared to a group of fertile men (n = 25). Fluorescence in situ hybridisation was performed for chromosomes X, Y and 18. Sperm DNA fragmentation was evaluated by TUNEL assay. Sanger sequencing was performed for mutations screening of DPY19L2 and SPATA16 genes. Sperm analysis revealed a classic phenotype of total globozoospermia in DPY19L2-mutated group and a particular phenotype characterised by a predominance of double/multiple round-headed (39.00 ± 4.2%) and multi-tailed spermatozoa (26.00 ± 16.97%) in SPATA16-mutated group. FISH analysis showed a significantly higher aneuploidy rate in globozoospermic patients compared to controls (p < 0.05), and a higher rate was observed in SPATA16-mutated group compared to DPY19L2-mutated group (p < 0.05). DNA fragmentation index was significantly higher in globozoospermic men compared to controls (p < 0.001), and there is no statistically significant difference between the two globozoospermic groups. We showed that SPATA16 defects could be associated with an abnormal meiosis leading to a particular morphological sperm defect of double/multiple round-headed and multi-flagella and a higher sperm aneuploidy rate than in case of DPY19L2-defects in classic globozoospermia.

Embryos derived from couples with consanguineous marriages with globozoospermia should be screened for gender or DPY19L2 deletion.

Globozoospermia or round-headed spermatozoa are a rare type of infertility which accounts for <0.1% of male infertility. Several genes are associated with this disease, including DPY19L2, SPATA16, PICK1 and CCIN that DPY19L2 accounts for 75% of globozoospermia. Isfahan Fertility and Infertility Center (IFIC) is a referral centre for globozoospermia, and individuals with globozoospermia are routinely screened for DPY19L2 deletion. In the present study, we have screened six couples with globozoospermia and consanguineous marriages. Genomic DNA both female and male partners were screened for DPY19L2 deletion for exons 1, 11 and 22 as exons most prone to non-homologous recombination. In addition, qPCR was carried out on genomic samples of their partners to determine whether they are heterozygous for DPY19L2 deletion. The results revealed that one female was heterozygous for DPY19L2 deletion. Therefore, this couple decided to undergo intracytoplasmic sperm injection and gender selection and two XX embryos were transferred for this couple and two healthy girls were born. In conclusion, we advise for the couples with DPY19L2-globozoospermia and consanguineous marriages to be screened for DPY19L2 deletion in the hope of reducing occurrence of globozoospermia in future progeny.