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Study Question: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility?, Summary Answer: 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., What Is Known Already: 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., Study Design, Size, Duration: 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., Participants/materials, Setting, Methods: 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., Main Results and the Role of Chance: 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., Limitations, Reasons for Caution: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility., Wider Implications of the Findings: 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., Study Funding/competing Interest(s): 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., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility?, Summary Answer: 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., What Is Known Already: 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., Study Design, Size, Duration: 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., Participants/materials, Setting, Methods: 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., Main Results and the Role of Chance: 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., Limitations, Reasons for Caution: 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., Wider Implications of the Findings: : 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., Study Funding/competing Interest(s): This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Is a novel homozygous phospholipase C zeta (PLCζ), c.1658 G>C; p. R553P mutation in the C2 domain associated with the outcomes of recurrent fertilization failure after ICSI?, Summary Answer: PLCζ, c.1658 G>C led to defective human oocyte activation and fertilization failure, while this mutation in the C2 domain of PLCζ did not compromise concentration, motility and chromosome ploidy of sperm., What Is Known Already: Sperm-specific PLCζ is now widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations, which are essential for egg activation during mammalian fertilization. Thus far, few genetic studies have shown that different point mutations in the PLCζ gene are associated with male infertility., Study Design, Size, Duration: This was a basic medical research to assess pathogenicity for novel mutation in the C2 domain of PLCζ during human fertilization., Participants/materials, Setting, Methods: Single-cell omics were applied to analyze the DNA methylation state of the fertilization failure oocytes and the ploidy of the patient's sperm. Whole genome sequencing data for the patient were analyzed for mutations in PLCζ. Sanger sequencing confirmed the presence of a rare variant, and then the mutant and wild-type PLCζ mRNA were injected to observe oocyte activation., Main Results and the Role of Chance: The fertilization failure oocytes (n = 4) were triploid and lacking proper DNA demethylation. The whole genome sequencing analysis revealed a novel missense homozygous mutation in PLCζ, c.1658 G>C; p. R553P, which leads to the conversion of arginine 553 to proline. This point mutation does not affect the production of the corresponding protein in sperm. However, microinjection of the mRNA transcribed from the PLCζ R553P mutation gene failed to trigger oocyte activation and the subsequent embryo development., Limitations, Reasons for Caution: Only one patient with PLCζ mutations was available because of its rare incidence., Wider Implications of the Findings: Notably, we discovered a novel homozygous mutation in PLCζ, which results in an abnormal conformation at the C2 domain of the PLCζ protein. Our findings indicate an essential role of PLCζ in human fertilization and the requirement of a normal structure of C2 domain in PLCζ-mediated physiological function., Study Funding/competing Interest(s): This project is funded by the National Natural Science Foundation of China (31571544, 31871482, 31871447) and National Key Research and Development Program (2018YFC1004000, 2017YFA0103801). All authors declared no competing interests., Trial Registration Number: Not applicable., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Study Question: Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations?, Summary Answer: We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest., What Is Known Already: We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest., Study Design, Size, Duration: We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation., Participants/materials, Setting, Methods: Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling., Main Results and the Role of Chance: Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage., Limitations, Reasons for Caution: The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations., Wider Implications of the Findings: The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients' functional PB1 oocytes., Study Funding/competing Interests: National Key R&D Program of China (2016YFC1000600); Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success?, Summary Answer: Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF., What Is Known Already: In human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception., Study Design, Size, Duration: Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study., Participants/materials, Setting, Methods: Samples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis., Main Results and the Role of Chance: A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF., Limitations, Reasons for Caution: A key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the ∼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx., Wider Implications of the Findings: These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Background: Cumulus cell (CC) gene expression is suggested as a non-invasive analysis method to predict oocyte competence. There are, however, important between-patient differences in CC gene expression. These can be compensated when expression results are combined with patient and cycle characteristics using a multiple regression analysis model., Methods: From ICSI patients stimulated with GnRH antagonist and recombinant FSH (n= 25) or GnRH agonist and highly purified menotrophin (n= 20), CC were collected and oocytes were individually fertilized and cultured. CC were analyzed for the expression of Syndecan 4 (SDC4), Prostaglandin-endoperoxide synthase 2 (PTGS2), Versican (VCAN), Activated leukocyte cell adhesion molecule, Gremlin 1, transient receptor potential cation channel, subfamily M, member 7 (TRPM7), Calmodulin 2 and Inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) using quantitative PCR. Results were analyzed in relation to the stimulation protocol. Within-patient variation in gene expression was related to oocyte maturity and developmental potential. Models predictive for normal embryo or blastocyst development and pregnancy in single embryo transfer cycles were developed., Results: Mature oocytes have higher PTGS2 and lower VCAN expression in their cumulus. All genes except VCAN had a positive correlation with good embryo or blastocyst morphology and were used to develop predictive models for embryo or blastocyst development (P< 0.01). Specific models were obtained for the two stimulation protocols. In both groups, better cleavage-stage embryo prediction relied on TRPM7 and ITPKA expression and pregnancy prediction relied on SDC4 and VCAN expression. In the current data set, the use of CC expression for pregnancy prediction resulted in a sensitivity of >70% and a specificity of >90%., Conclusions: Multivariable models based on CC gene expression can be used to predict embryo development and pregnancy.

With rare exceptions, mammalian mitochondria are inherited through the female. This probably serves to minimize lethal cytoplasmic gene competition and to prevent the inheritance of sperm mitochondrial DNA that has been subject to degradation by free radicals. In general, organisms are intolerant of mitochondrial heteroplasmy and, when this occurs in humans, it frequently presents as progressive and lethal bioenergetic or neurological disease. The mitochondria of spermatozoa are specifically destroyed by proteolysis in early embryonic development, in mice at the 4- to 8-cell transition. While there are concerns in human assisted reproduction that microinjection of abnormal or immature sperm cells could lead to lasting harm in the offspring through transmission of abnormal mitochondria, there is no clinical evidence to support this. There is more potential for harm through attempts to 'rescue' poor quality oocytes by cytoplasmic or nuclear transfer, as it is not currently possible to control the final fate of the donated mitochondria in relation to nuclear-mitochondrial interactions or the embryonic axes. Moreover, the balance between nuclear and mitochondrial genes and the role of cytoplasmic factors in epigenesis are still poorly understood. The future challenge for biologists is to comprehend the nature of the selective destruction of paternal mitochondria, as it appears to be a species-specific recognition phenomenon.

An evolutionary hypothesis based on an 'antagonist pleiotropy' or 'disposable soma' mechanism is put forward to explain differences in longevity between species, strains, and sexes. Data from several congenic mouse strains and mammalian species suggest that there may be an association between cleavage rate of concepti and longevity, in such a way that concepti from species, strains or the sex (male) with the fastest cleavage rates have shorter life spans. The major histocompatibility complex (MHC) and, in particular, the conceptus development gene (Ped) together with several Y-linked genes that are expressed during the preimplantation stages of development may play an important role in determining or modulating longevity in mammals. Notwithstanding, effects of other loci as well as environmental factors on conceptus development and longevity cannot be ignored.

We have studied the effect of moderately impaired maternal insulin secretion on oocyte chromosomal constitution, fertilization and zygote DNA synthesis. Female mice were injected with a single dose of streptozotocin (65 mg/kg) 14 days before fertilization/ovulation. Zygotes/oocytes were recovered from control and subdiabetic mice on day 1 of pregnancy. Compared with control animals, subdiabetic females showed a significant difference in the proportion of zygotes/oocytes. The subdiabetic mothers had a lower percentage of zygotes and a higher percentage of unfertilized and degenerated oocytes in comparison with control animals. The investigation of [3H]thymidine incorporation did not show any influence of the maternal subdiabetes on the initial zygote DNA synthesis. An analysis of the ovulated oocytes at the metaphase II stage isolated from subdiabetic mice did not reveal increased chromosomal anomalies in comparison with the controls. Control and subdiabetic mothers had a similar percentage of oocytes with a normal haploid set of chromosomes, and the incidence of aneuploidy/diploidy did not differ significantly. These observations suggest that insulin changes in subdiabetic mothers had a deleterious influence on oocyte fertilization in mice, but apparently they did not have any effect on the nuclear events.

The purpose of this study was to assess the genetic status of abnormal zygotes following assisted fertilization. Dispermic, monopronucleated and digynic zygotes were allowed to cleave intact or after enucleation, and on the biopsied blastomeres, multiplex polymerase chain reaction and fluorescent in-situ hybridization were performed. It was found that the distal pronucleus was usually male in origin in dispermic embryos, and that the sex ratio was restored when they were enucleated; however, they became mosaic at metaphase and their genetic heterogeneity was not restored after enucleation. Monopronucleated zygotes derived from standard in-vitro insemination can be transferred to the patient, since they usually showed normal diploid complement in their cells. On the contrary, single-pronucleated zygotes derived from intracytoplasmic sperm injection were usually activated parthenogenetically, but not fertilized. Digynic embryos, unlike dispermic ones, had a very low incidence of mosaicism, and when present, such mosaicism originated at a later embryo division. Most of the digynic embryos were triploid, indicating that the first division was normal and bipolar; moreover, when the female pronucleus was removed, they became diploid and their genetic status was considered normal. The recognition and understanding of fertilization abnormalities allow the identification of methods leading to their avoidance or correction.

In this work we report the possibility that oocyte immaturity is associated with premature chromosome condensation (PCC) after in-vitro fertilization (IVF). Using a murine model, we have related PCC and endoreduplicated-like oocytes to oocyte immaturity as a basis for a prognosis in oocyte immaturity problems. The cytogenetic analysis was performed in 511 embryos obtained from immature oocytes that were directly fertilized in vitro and in 1363 embryos obtained from immature oocytes that were matured in vitro with different concentrations of human chorionic gonadotrophin (HCG) added to the culture medium. As a control we used 507 embryos obtained from freshly ovulated oocytes. PCC at the G1-phase-(G1-PCC) was observed only when immature oocytes were immediately fertilized in vitro (45.4%) and PCC at the S-phase (S-PCC) only when using in-vitro matured oocytes with the highest HCG concentration (3.3%). Neither G1-PCC nor S-PCC were found in the control group. Endoreduplicated-like oocytes appeared in a significant percentage (27.3%) only in the immature group. Immature oocytes yielded a low fertilization rate (16.6%) while in-vitro maturation seemed to confer a higher fertilization capacity compared to the control group (90.1% versus 78.2%). The possible correlation between PCC and oocyte immaturity provides new prospects in the determination of human IVF failures of unknown origin. Thus, when a problem of oocyte immaturity is diagnosed through the presence of PCC, a special programme of in vitro oocyte maturation, such as a longer preincubation time or addition of hormones to the media, would be recommended.

Cytogenetic studies were made on 328 spermatozoa from three individuals using either fresh semen samples capacitated in Biggers, Whitten and Whittingham (BWW) medium plus human serum albumin (BWW + HSA) or semen samples preserved at 4 degrees C in TEST-Yolk buffer. A total of 261 sperm karyotypes were obtained in a series of experiments in which half of each sample was capacitated in BWW + HSA and the other half in TEST-Yolk buffer at 4 degrees C for 2 days; 123 and 138 sperm karyotypes were obtained from the two capacitation methods respectively. Neither the frequency of sperm chromosomal abnormalities nor the sex ratio was significantly different after each capacitation methods. In one individual, however, the sex ratio (19X:32Y in the fresh sample and 49X:28Y in the preserved sample) did show a significant difference. In three experiments with semen samples from a single individual capacitated at 4 degrees C for 1, 2 or 3 days in TEST-Yolk buffer we obtained 33, 30 and 34 sperm karyotypes respectively. No significant differences in the sex ratio was exhibited between these experiments; the number of chromosome anomalies was too low to allow statistical analysis. Our results suggest that TEST-Yolk capacitation for 1, 2 or 3 days does not induce significant variations in the frequency and type of chromosomal abnormalities in human spermatozoa.