CALR3 defects disrupt sperm-zona pellucida binding in humans: new insights into male factor fertilization failure and relevant clinical therapeutic approaches.

Do biallelic deleterious variants of Calreticulin 3 (CALR3) cause fertilization failure (FF), resulting in male infertility in humans? Biallelic mutations in CALR3 were identified in two infertile men from unrelated families and were shown to cause FF associated with failed sperm-zona pellucida (ZP) binding. In male mice, the Calr3-knockout has been reported to cause male infertility and FF. However, the mechanism behind this remains unclear in humans. Sequencing studies were conducted in a research hospital on samples from Han Chinese families with primary infertility and sperm head deformations to identify the underlying genetic causes. Data from two infertile probands characterized by sperm head deformation were collected through in silico analysis. Sperm cells from the probands were characterized using light and electron microscopy and used to verify the pathogenicity of genetic factors through functional assays. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of FF. ICSI were administered to overcome CALR3-affected male infertility. Novel biallelic deleterious mutations in CALR3 were identified in two infertile men from unrelated families. We found one homozygous frameshift CALR3 mutation (M1: c.17_27del, p.V6Gfs*34) and one compound heterozygous CALR3 mutation (M2: c.943A>G, p.N315D; M3: c.544T>C, p.Y182H). These mutations are rare in the general population and cause acrosomal ultrastructural defects in affected sperm. Furthermore, spermatozoa from patients harbouring the CALR3 mutations were unable to bind to the sperm-ZP or they disrupted gamete fusion or prevented oocyte activation. Molecular assays have revealed that CALR3 is crucial for the maturation of the ZP binding protein in humans. Notably, the successful fertilization via SUZI and ICSI attempts for two patients, as well as the normal expression of PLCζ in the mutant sperm, suggests that ICSI is an optimal treatment for CALR3-deficient FF. The results are based on sperm-related findings from two patients. Further studies are required to gain insight into the developmental stage and function of CALR3 in human testis. Our findings highlight the underlying risk of FF associated with sperm defects and provide a valuable reference for personalized genetic counselling and clinical treatment of these patients. This study was supported by the National Key R&D Program of China (2021YFC2700901), Hefei Comprehensive National Science Center Medical-Industrial Integration Medical Equipment Innovation Research Platform Project (4801001202), the National Natural Science Foundation of China (82201803, 82371621, 82271639), Foundation of the Education Department of Anhui Province (gxgwfx2022007), Key Project of Natural Science Research of Anhui Educational Committee (2023AH053287), and the Clinical Medical Research Transformation Project of Anhui Province (202204295107020037). The authors declare no competing interests. N/A.

Gao Y ，Xue R ，Guo R ，Yang F ，Sha X ，Li Y ，Hua R ，Li G ，Shen Q ，Li K ，Liu W ，Xu Y ，Zhou P ，Wei Z ，Zhang Z ，Cao Y ，He X ，Wu H ... -  《-》

ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida.

Does a homozygous nonsense mutation in ACR lead to total fertilization failure (TFF) resulting in male infertility in humans? A novel homozygous nonsense mutation of ACR (c.167G>A, p.Trp56X) was identified in two infertile brothers and shown to cause human TFF. ACROSIN, encoded by ACR, is a major acrosomal enzyme expressed only in the acrosome of the sperm head. Inhibition of acrosin prevents sperm penetration of the zona pellucida (ZP) in several species, including humans. Acr-knockout in hamsters causes male infertility with completely blocked fertilization. Of note, there are no reports of ACR mutations associated with TFF in humans. Whole-exome sequencing (WES) was used for the identification of pathogenic genes for male factor TFF in eight involved couples. Data from eight infertile couples who had experienced TFF during their IVF or ICSI attempts were collected. Functional assays were used to verify the pathogenicity of the potential genetic factors identified by WES. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of TFF caused by deficiencies in ACROSIN. A novel homozygous nonsense mutation in ACR, c.167G>A, p.Trp56X, was identified in two additional primary infertile brothers whose parents were first cousins. This rare mutation caused ACROSIN deficiency and acrosomal ultrastructural defects in the affected sperm. Spermatozoa lacking ACROSIN were unable to penetrate the ZP, rather than hampering sperm binding, disrupting gamete fusion, or preventing oocyte activation. These findings were supported by the fertilization success of SUZI and ICSI attempts, as well as the normal expression of ACTL7A and PLCζ in the mutant sperm, suggesting that ICSI without remedial assisted oocyte activation is an optimal treatment for ARCOSIN-deficient TFF. The absence of another independent pedigree to support our argument is a limitation of this study. The findings expand our understanding of the genes involved in human TFF, providing information for appropriate genetic counseling and fertility guidance for these patients. This study was supported by the National Natural Science Foundation of China (grant no. 82201803, 81901541, 82271639, and 32000584), University Synergy Innovation Program of Anhui Province (GXXT-2019-044), and the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (grant no. 2019PT310002). The authors declare no conflicts of interest. N/A.

Hua R ，Xue R ，Liu Y ，Li Y ，Sha X ，Li K ，Gao Y ，Shen Q ，Lv M ，Xu Y ，Zhang Z ，He X ，Cao Y ，Wu H ... -  《-》

Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.

Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. N/A.

Zhang Q ，Jin H ，Long S ，Tang X ，Li J ，Liu W ，Han W ，Liao H ，Fu T ，Huang G ，Chen S ，Lin T ... -  《-》

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? New mutations in the PLCZ1 sequence are associated with FFs after ICSI. FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

Torra-Massana M ，Cornet-Bartolomé D ，Barragán M ，Durban M ，Ferrer-Vaquer A ，Zambelli F ，Rodriguez A ，Oliva R ，Vassena R ... -  《-》

Fertilization defects in sperm from Cysteine-rich secretory protein 2 (Crisp2) knockout mice: implications for fertility disorders.

We hypothesize that fertility disorders in patients with aberrant expression of Cysteine-RIch Secretory Protein 2 (CRISP2) could be linked to the proposed functional role of this protein in fertilization. Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation. Testicular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Interestingly, evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility. A mouse line carrying a deletion in the sixth exon of the Crisp2 gene was generated. The analyses of the reproductive phenotype of Crisp2(-/-) adult males included the evaluation of their fertility before and after being subjected to unilateral vasectomy, in vivo fertilization rates obtained after mating with either estrus or superovulated females, in vitro sperm fertilizing ability and different sperm functional parameters associated with capacitation such as tyrosine phosphorylation (by western blot), acrosome reaction (by Coomassie Blue staining), hyperactivation (by computer-assisted sperm analysis) and intracellular Ca(2+) levels (by flow cytometry). Crisp2(-/-) males presented normal fertility and in vivo fertilization rates when mated with estrus females. However, the mutant mice showed clear defects in those reproductive parameters compared with controls under more demanding conditions, i.e. when subjected to unilateral vasectomy to reduce the number of ejaculated sperm (n = 5; P< 0.05), or when mated with hormone-treated females containing a high number of eggs in the ampulla (n ≥ 5; P< 0.01). In vitro fertilization studies revealed that Crisp2(-/-) sperm exhibited deficiencies to penetrate the egg vestments (i.e. cumulus oophorus and zona pellucida) and to fuse with the egg (n ≥ 6; P< 0.01). Consistent with this, Crisp2-null sperm showed lower levels of hyperactivation (n = 7; P< 0.05), a vigorous motility required for penetration of the egg coats, as well as a dysregulation in intracellular Ca(2+) levels associated with capacitation (n = 5; P< 0.001). The analysis of the possible mechanisms involved in fertility disorders in men with abnormal expression of CRISP2 was carried out in Crisp2 knockout mice due to the ethical and technical problems inherent to the use of human gametes for fertilization studies. Our findings in mice showing that Crisp2(-/-) males exhibit fertility and fertilization defects under demanding conditions support fertilization defects in sperm as a mechanism underlying infertility in men with aberrant expression of CRISP2. Moreover, our observations in mice resemble the situation in humans where fertility disorders can or cannot be detected depending on the accumulation of own individual defects or the fertility status of the partner. Finally, the fact that reproductive defects in mice are masked by conventional mating highlights the need of using different experimental approaches to analyze male fertility. This study was supported by the World Health Organization (H9/TSA/037), the National Research Council of Argentina (PIP 2009-290), the National Agency for Scientific and Technological Promotion of Argentina (PICT 2011, 2023) and the Rene Baron Foundation to P.S.C. and by the MEXT of Japan to M.I. The authors declare that there are no conflicts of interest.

Brukman NG ，Miyata H ，Torres P ，Lombardo D ，Caramelo JJ ，Ikawa M ，Da Ros VG ，Cuasnicú PS ... -  《-》