New biallelic mutations in WEE2: expanding the spectrum of mutations that cause fertilization failure or poor fertilization

Abstract Purpose To study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization. Methods Thirty-one Chinese patients who underwent treatment with assisted reproductive technology and suffered from repeated (at least two times) total fertilization failure (TFF) or a low fertilization rate were enrolled. Genomic DNA was extracted from patients for whole-exome sequencing. Suspicious mutations were validated by Sanger sequencing. WEE2 protein levels in oocytes from affected patients were examined by immunofluorescence. Disruptive effects of mutations on WEE2 protein stability, subcellular localization, and kinase function were analyzed through western blotting, immunofluorescence, and flow cytometry in HeLa cells. Results Three of thirty-one (9.6%) enrolled patients had six compound heterozygous mutations of the WEE2 gene, and three of them were reported here for the first time (c.115_116insT, c.756_758delTGA, and c.C1459T). Oocytes from affected patients showed decreased WEE2 immunofluorescence signals. In vitro experiments showed that the mutant WEE2 gene caused reduced WEE2 protein levels or cellular compartment translocation in HeLa cells, leading to decreased levels of the phosphorylated Cdc2 protein. Compared with the wild-type WEE2 protein, the mutant WEE2 proteins were also found to have different effects on the cell cycle. Conclusion Three novel compound heterozygous WEE2 variants were found in patients with pronucleus formation failure. This study provides new evidence that WEE2 mutations result in loss of function, which could result in fertilization failure.

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The identification of novel mutations in PLCZ1 responsible for human fertilization failure and a therapeutic intervention by artificial oocyte activation

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa003 ◽

2020 ◽

Vol 26 (2) ◽

pp. 80-87 ◽

Cited By ~ 3

Author(s):

Jian Mu ◽

Zhihua Zhang ◽

Ling Wu ◽

Jing Fu ◽

Biaobang Chen ◽

...

Keyword(s):

Human Reproduction ◽

Missense Mutations ◽

Oocyte Activation ◽

Fertilization Failure ◽

Gene Encoding ◽

Human Fertilization ◽

Molecular Events ◽

Whole Exome ◽

Artificial Oocyte Activation ◽

Biallelic Mutations

Abstract Fertilization involves a series of molecular events immediately following egg–sperm fusion; Ca2+ oscillations are the earliest signaling event, and they initiate the downstream reactions including pronucleus formation. Successful human reproduction requires normal fertilization. In clinical IVF or ICSI attempts, some infertile couples suffer from recurrent fertilization failure. However, the genetic reasons for fertilization failure are largely unknown. Here, we recruited several couples diagnosed with fertilization failure even though their gametes are morphologically normal. Through whole-exome sequencing and Sanger sequencing, we identified biallelic mutations in gene-encoding phospholipase C zeta 1 (PLCZ1) in four independent males in couples diagnosed with fertilization failure. Western blotting showed that missense mutations decreased the level of PLCZ1 and that nonsense or frameshift mutations resulted in undetectable or truncated proteins. Expression of these mutations in mice significantly reduced the levels of oocyte activation. Artificial oocyte activation in patient oocytes could rescue the phenotype of fertilization failure and help establish pregnancy and lead to live birth. Our findings expand the spectrum of PLCZ1 mutations that are responsible for human fertilization failure and provide a potentially feasible therapeutic treatment for these patients.

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P–568 Homozygous Pathogenic Variants in ACTL9 Cause Fertilization Failure and Male Infertility in Human and Mouse

Human Reproduction ◽

10.1093/humrep/deab130.567 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Dai ◽

T Zhang ◽

J Guo ◽

Q Zhou ◽

Y Gu ◽

...

Keyword(s):

Mouse Model ◽

Male Infertility ◽

Therapeutic Option ◽

Calcium Ionophore ◽

Calcium Oscillations ◽

Oocyte Activation ◽

Fertilization Failure ◽

Pathogenic Variants ◽

Male Factors ◽

Poor Fertilization

Abstract Study question What are the other male factors that cause total fertilization failure (TFF) excepting for variants in PLCZ1? Summary answer Homozygous variants in ACTL9 (actin like 9) cause abnormal localization of PLCζ in a loosened perinuclear theca (PT) structure and leads to TFF. What is known already In previous studies, investigators have reported that the female factors in TFF after intracytoplasmic sperm injection (ICSI) include pathogenic variants in WEE2, TLE6, and TUBB8, whereas for male factors, pathogenic variants in PLCZ1 were reported to be the primary cause of TFF, which account for approximately 30% of couples with male factors in TFF excluding globozoospermia. Most recently, it was reported that pathogenic variants in ACTL7A led to reduced expression and abnormal localization of PLCζ, thereby identifying this genetic variant as a potential cause of TFF. Study design, size, duration Fifty-four infertile couples with TFF or poor fertilization (fertilization rate of < 20%) at the Reproductive and Genetic Hospital of CITIC-Xiangya during January 2014 to June 2020 were recruited into this study. Participants/materials, setting, methods Male factors were identified in (MOAT). WES analysis was used to analyze the genetic factors of individuals with male factors. Sperm morphological study was conducted by H&E staining and TEM. Immunostaining of PLCζ was used to analyze the status of sperm-borne activation factor. A knock-in mouse model was generated by CRISPER-Cas9 technology. Sperm from homozygous Actl9 variant mice were analyzed by TEM and ICSI. ICSI with AOA was performed in couples with ACTL9 variants. Main results and the role of chance A total of 54 couples with TFF or poor fertilization were screened, with 21 couples determined to have a male infertility factor by MOAT. Whole-exome sequencing of these 21 male individuals identified three homozygous pathogenic variants in ACTL9 in three individuals. ACTL9 variations led to abnormal ultrastructure of the PT, with PLCζ absent in the head and present in the neck of the mutant sperm, which contributed to failed normal calcium oscillations in oocytes and subsequent TFF. The key roles of ACTL9 in the PT structure and TFF after ICSI were further confirmed in Actl9-mutated mouse model. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF and achieved live births in a couple with an ACTL9 variant. Limitations, reasons for caution The mechanism of how ACTL9 regulate PLCζ remains unknown. Wider implications of the findings: It provided a genetic marker and a therapeutic option for individuals who have undergone ICSI without successful fertilization. Trial registration number not applioable

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Melatonin rescues impaired penetration ability of human spermatozoa induced by mitochondrial dysfunction

Reproduction ◽

10.1530/rep-19-0231 ◽

2019 ◽

Vol 158 (5) ◽

pp. 465-475 ◽

Cited By ~ 3

Author(s):

Xue-Ying Zhang ◽

Yi-Meng Xiong ◽

Ya-Jing Tan ◽

Li Wang ◽

Rong Li ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dysfunction ◽

Male Fertility ◽

Sperm Quality ◽

Fertilization Failure ◽

Respiratory Capacity ◽

Fertilization Rates ◽

Penetration Ability ◽

Poor Fertilization ◽

Mitochondrial Respiratory

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.

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Novel mutations in PLCZ1 cause male infertility due to fertilization failure or poor fertilization

Human Reproduction ◽

10.1093/humrep/dez282 ◽

2020 ◽

Vol 35 (2) ◽

pp. 472-481 ◽

Cited By ~ 3

Author(s):

Zheng Yan ◽

Yong Fan ◽

Fei Wang ◽

Zhiguang Yan ◽

Menghui Li ◽

...

Keyword(s):

Male Infertility ◽

Conflicts Of Interest ◽

Missense Mutations ◽

Oocyte Activation ◽

Fertilization Failure ◽

Novel Mutations ◽

Primary Infertility ◽

Human Fertilization ◽

Poor Fertilization

Abstract STUDY QUESTION Do sperm-specific phospholipase C zeta (PLCZ1) mutations account for male infertility due to fertilization failure? SUMMARY ANSWER Six novel mutations and one reported mutation in PLCZ1 were identified in five of 14 independent families characterized by fertilization failure or poor fertilization, suggesting that these mutations may be responsible for fertilization failure in men exhibiting primary infertility. WHAT IS KNOWN ALREADY PLCZ1 is essential for the induction of intracellular calcium (Ca2+) oscillations and the initiation of oocyte activation during mammalian fertilization. However, genetic evidence linking PLCZ1 mutations with male infertility remains limited. STUDY DESIGN, SIZE, DURATION Fourteen unrelated primary infertility patients were recruited into this study from January 2016 to December 2018; the patients exhibited total fertilization failure or poor fertilization, as evidenced by ICSI and sperm-related oocyte activation deficiencies identified in mouse oocyte activation assays. PARTICIPANTS/MATERIALS, SETTING, METHODS Genomic DNA samples were extracted from the peripheral blood of patients. The whole exons of PLCZ1 were sequenced by Sanger sequencing. The PLCZ1 sequences were aligned by CodonCode software to identify rare variants. The ExAC database was used to search for the frequency of corresponding mutations. The pathogenicity of identified variants and their possible effects on the protein were assessed in silico. PLCZ1 protein levels in semen samples were evaluated by western blotting. Oocyte activation ability was assessed by the injection of wild-type and mutant PLCZ1 cRNAs into human mature metaphase II (MII) oocytes in vitro. MAIN RESULTS AND THE ROLE OF CHANCE We identified six novel mutations and one reported mutation in PLCZ1 among five affected individuals. In addition to four novel missense mutations, two new types of genetic variants were identified, including one in-frame deletion and one splicing mutation. Western blot analysis revealed that PLCZ1 protein expression was not observed in the semen samples from the five affected patients. Microinjection with the PLCZ1 cRNA variants was performed, and a significant decrease in the percentage of pronuclei was observed for four novel missense mutations and one novel in-frame deletion mutation, suggesting that these mutations have a deleterious influence on protein function. By artificial oocyte activation treatment, the fertilization failure phenotypes of four affected patients were successfully rescued and three healthy babies were delivered. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION We screened only the whole exons of PLCZ1. Additional possible mutations in the non-coding region of PLCZ1 should be further studied. WIDER IMPLICATIONS OF THE FINDINGS Our study not only further confirms the important role of PLCZ1 in human fertilization but also expands the mutational spectrum of PLCZ1 associated with male infertility, which provides a basis for assessing genetic variation in PLCZ1 as a potential diagnostic marker for infertile men suffering from fertilization failure. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Natural Foundation of China (81 571 486 and 81 771 649). All authors have no conflicts of interest to declare.

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Identification of Novel Mutations in CDC20: Expanding the Mutational Spectrum for Female Infertility

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.647130 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lin Zhao ◽

Yichun Guan ◽

Qingxia Meng ◽

Weijie Wang ◽

Ling Wu ◽

...

Keyword(s):

Embryonic Development ◽

Oocyte Maturation ◽

Female Infertility ◽

Early Embryonic Development ◽

Human Reproduction ◽

Human Oocyte ◽

Fertilization Failure ◽

Novel Mutations ◽

Maturation Arrest ◽

Biallelic Mutations

Oocyte maturation and fertilization are fundamental processes for successful human reproduction, and abnormalities in these processes will cause infertility. Recently, we identified biallelic mutations in CDC20 that are responsible for human oocyte maturation arrest, fertilization failure, and early embryonic development arrest. In this study, we screened for further CDC20 mutations in a new cohort of patients with abnormalities in oocyte maturation, fertilization, and early embryonic development. Through whole-exome sequencing, we identified the four novel mutations c.887G > A (p. Arg296Gln), c.964C > T (p.Arg322∗), c.1155G > C (p.Trp385Cys), and c.330 + 1G > A (p. Glu111Ilefs∗36) and one previously reported mutation c.965G > A (p.Arg322Gln) in CDC20 in four infertile individuals from three independent families. The patients had different phenotypes of oocyte maturation arrest and fertilization failure resulting from the different mutations. This study confirms our previous research and expands the spectrum of known mutations in CDC20, providing new evidence supporting the function of CDC20 in the genetic etiology of female infertility characterized by oocyte maturation arrest and fertilization failure.

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