Novel bi-allelic variants in ACTL7A are associated with male infertility and total fertilization failure.

What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. N/A.

Wang J ，Zhang J ，Sun X ，Lin Y ，Cai L ，Cui Y ，Liu J ，Liu M ，Yang X ... -  《-》

High rate of detected variants in male PLCZ1 and ACTL7A genes causing failed fertilization after ICSI.

What is the frequency of PLCZ1, ACTL7A, and ACTL9 variants in male patients showing fertilization failure after ICSI, and how effective is assisted oocyte activation (AOA) for them? Male patients with fertilization failure after ICSI manifest variants in PLCZ1 (29.09%), ACTL7A (14.81%), and ACTL9 (3.70%), which can be efficiently overcome by AOA treatment with ionomycin. Genetic variants in PLCZ1, and more recently, in ACTL7A, and ACTL9 male genes, have been associated with total fertilization failure or low fertilization after ICSI. A larger patient cohort is required to understand the frequency at which these variants occur, and to assess their effect on the calcium ion (Ca2+) release during oocyte activation. AOA, using ionomycin, can restore fertilization and pregnancy rates in patients with PLCZ1 variants, but it remains unknown how efficient this is for patients with ACTL7A and ACTL9 variants. This prospective study involved two patient cohorts. In the first setting, group 1 (N = 28, 2006-2020) underwent only PLCZ1 genetic screening, while group 2 (N = 27, 2020-2023) underwent PLCZ1, ACTL7A, and ACTL9 genetic screening. Patients were only recruited when they had a mean fertilization rate of ≤33.33% in at least one ICSI cycle with at least four MII oocytes. Patients underwent a mouse oocyte activation test (MOAT) and at least one ICSI-AOA cycle using calcium chloride (CaCl2) injection and double ionomycin exposure at our centre. All patients donated a saliva sample for genetic screening and a sperm sample for further diagnostic tests, including Ca2+ imaging. Genetic screening was performed via targeted next-generation sequencing. Identified variants were classified by applying the revised ACMG guidelines into a Bayesian framework and were confirmed by bidirectional Sanger sequencing. If variants of uncertain significance or likely pathogenic or pathogenic variants were found, patients underwent additional determination of the sperm Ca2+-releasing pattern in mouse (MOCA) and in IVM human (HOCA) oocytes. Additionally, ACTL7A immunofluorescence and acrosome ultrastructure analyses by transmission electron microscopy (TEM) were performed for patients with ACTL7A and/or ACTL9 variants. Overall, the frequency rate of PLCZ1 variants was 29.09%. Moreover, 14.81% of patients carried ACTL7A variants and 3.70% carried ACTL9 variants. Seven different PLCZ1 variants were identified (p.Ile74Thr, p.Gln94*, p.Arg141His, p.His233Leu, p.Lys322*, p.Ile379Thr, and p.Ser500Leu), five of which are novel. Interestingly, PLCZ1 variants p.Ser500Leu and p.His233Leu occurred in 14.55% and 9.09% of cases. Five different variants were found in ACTL7A (p.Tyr183His, p.Gly214Ser, p.Val340Met, p.Ser364Glnfs*9, p.Arg373Cys), four of them being identified for the first time. A novel variant in ACTL9 (p.Arg271Pro) was also described. Notably, both heterozygous and homozygous variants were identified.The MOCA and HOCA tests revealed abnormal or absent Ca2+ release during fertilization in all except one patient, including patients with PLCZ1 heterozygous variants. TEM analysis revealed abnormal acrosome ultrastructure in three patients with ACTL7A variants, but only patients with homozygous ACTL7A variants showed reduced fluorescence intensity in comparison to the control.AOA treatment significantly increased the fertilization rate in the 19 patients with detected variants (from 11.24% after conventional ICSI to 61.80% after ICSI-AOA), as well as positive hCG rate (from 10.64% to 60.00%) and live birth rate (from 6.38% to 37.14%), resulting in 13 healthy newborns. In particular, four live births and two ongoing pregnancies were produced using sperm from patients with ACTL7A variants. Genetic screening included exonic and outflanking intronic regions, which implies that deep intronic variants were missed. In addition, other male genes or possible female-related factors affecting the fertilization process remain to be investigated. Genetic screening of PLCZ1, ACTL7A, and ACTL9 offers a fast, cost-efficient, and easily implementable diagnostic test for total fertilization failure or low fertilization after ICSI, eliminating the need for complex diagnostic tests like MOAT or Ca2+ analysis. Nonetheless, HOCA remains the most sensitive functional test to reveal causality of uncertain significance variants. Interestingly, heterozygous PLCZ1 variants are sufficient to cause inadequate Ca2+ release during ICSI. Most importantly, AOA treatment using CaCl2 injection followed by double ionomycin exposure is highly effective for this patient group, including those with ACTL7A variants, who also display a Ca2+-release deficiency. This study was supported by the Flemish Fund for Scientific Research (FWO) (TBM-project grant T002223N awarded to B.H.) and by the Special Research Fund (BOF) (starting grant BOF.STG.2021.0042.01 awarded to B.H.). A.C.B., R.R.G., C.C., E.V.D.V., A.R., D.S., L.L., P.C., S.S., A.B., and F.V.M. have nothing to disclose. B.H. reports a research grant from FWO and BOF, and reports being a board member of the Belgian Ethical Committee on embryo research. N/A.

Cardona Barberán A ，Reddy Guggilla R ，Colenbier C ，Van der Velden E ，Rybouchkin A ，Stoop D ，Leybaert L ，Coucke P ，Symoens S ，Boel A ，Vanden Meerschaut F ，Heindryckx B ... -  《-》

Novel variants in ACTL7A and PLCZ1 are associated with male infertility and total fertilization failure.

Total fertilization failure (TFF), which refers to fertilization failure in all mature oocytes, accounting for 5%-10% of in vitro fertilization (IVF) cycles and 1%-3% of intracytoplasmic sperm injection (ICSI) cycles in human. In this study, we recruited three unrelated primary infertile men with repeated cycles of TFF and performed whole-exome sequencing to identify the potential pathogenic variants. We identified homozygous or compound-heterozygous variants of paternal-effect genes ACTL7A and PLCZ1 that followed a Mendelian recessive inheritance pattern. Novel homozygous nonsense variant in ACTL7A [c.C146G: p.S49*] was identified in case 1, who came from a consanguineous family. Ultrastructural observation of ACTL7A-mutated spermatozoa by transmission electron microscopy (TEM) indicated that apparent increased thickness of perinuclear matrix and the acrosome was detached from the nuclear envelop. Besides, two novel compound-heterozygous variants in PLCZ1 were identified in case 2 [c.1174+3A>C:p.?; c.A1274G:p.N425S] and case 3 [c.136-1G>C:p.?; c.G1358A:p.G453D]. Mutated spermatozoa from case 2 with reduced expression of PLCZ1 showed apparent acrosome detachment by TEM analysis. And ICSI with assisted oocyte activation (ICSI-AOA) treatment can partly rescue the TFF. Taken together, our findings revealed that novel biallelic variants in the paternal-effect genes ACTL7A and PLCZ1 were associated with human TFF, which expanding the spectrum of genetic causes and facilitating the genetic diagnosis of male infertility with TFF.

Zhao S ，Cui Y ，Guo S ，Liu B ，Bian Y ，Zhao S ，Chen Z ，Zhao 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 ... -  《-》