Human Fertilization Failure Research Articles

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.

Gene mutations associated with fertilization failure after in vitro fertilization/intracytoplasmic sperm injection.

Fertilization failure during assisted reproductive technologies (ART) is often unpredictable, as this failure is encountered only after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been performed. The etiology of fertilization failure remains elusive. More and more mutations of genes are found to be involved in human fertilization failure in infertile patients as high throughput sequencing techniques are becoming widely applied. In this review, the mutations of nine important genes expressed in sperm or oocytes, PLCZ1, ACTL7A, ACTL9, DNAH17, WEE2, TUBB8, NLRP5, ZP2, and TLE6, were summarized and discussed. These abnormalities mainly have shown Mendelian patterns of inheritance, including dominant and recessive inheritance, although de novo mutations were present in some cases. The review revealed the crucial roles of each reported gene in the fertilization process and summarized all known mutations and their corresponding phenotypes. The review suggested the mutations might become promising targets for precision treatments in reproductive medicine. Moreover, our work will provide some helpful clues for genetic counseling, risk prediction, and optimizing clinical treatments for human infertility by supplying the useful and timely information on the genetic causes leading to fertilization failure.

Mutation analysis of the TUBB8 gene in primary infertile women with oocyte maturation arrest

BackgroundOocyte maturation arrest at metaphase I leads to fertilization failure in humans. In early embryos, the tubulin beta 8 class VIII (TUBB8) encodes a β-tubulin isotype and aids in the assembling of the human oocyte spindle. Mutations in the TUBB8 potentially interfere with human oocyte maturation—a crucial prerequisite for fertilization and subsequent embryonic development. This study aims to investigate the novel mutations in TUBB8 and their prevalence.ResultsHundred fertile women (controls) and eleven infertile women with oocyte maturation arrest were chosen for the study. A total of five TUBB8 heterozygous/homozygous mutations were found in eleven infertile females (p.A313V, p.C239W, p.R251Q, p.P358L, and p.G96R). The Exome Aggregation Consortium (ExAC), SIFT, and PolyPhen-2 analyses revealed that p. A313V has unknown pathogenicity and p.C239W, p.R251Q, p.P358L, and p.G96R have possible pathogenicity. The wild-type (WT) and four mutant gene constructs were transfected to Hela cells. The Western blot analysis indicates that the TUBB8 expression of the p.C239W, p.R251Q, and p.G96R mutations was significantly decreased than that of WT. The immunofluorescence assay showed that the Hela cells transfected with either p.C239W, p.R251Q, or p.G96R mutations exhibited the disrupted microtubule structure, revealing a significant difference in the organization of the microtubule network compared to the WT.ConclusionsWe identified three novel variants and two reported variants out of 11 infertile women with oocyte metaphase I arrest. According to the present data, TUBB8 gene variants account for 31.96% of all participants (109/341) with oocyte maturation arrest.

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

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.

Novel homozygous variations in PLCZ1 lead to poor or failed fertilization characterized by abnormal localization patterns of PLCζ in sperm.

Total fertilization failure (TFF), which is the failure of fertilization in all oocytes, occurs in 1%-3% of intracytoplasmic sperm injection (ICSI) cycles. However, the sperm-related factors that cause fertilization failure in humans are still largely unknown. Here, we identified three novel homozygous variations in the PLCZ1 gene in a recessive inheritance pattern in three consanguineous families, which all located in a key catalytic domain, and predicted to modify its secondary structure and thus impair its hydrolytic activity. Moreover, immunofluorescent staining revealed that PLCζ in mutant sperm exhibited abnormal localization patterns. ICSI-AOA resulted in an increased rate of normal fertilization compared with previous ICSI cycles (75.0% vs 2.2%, P < .001). In summary, we identified three novel homozygous variations in PLCZ1 that led to poor or failed fertilization that could be overcame by ICSI-AOA.

Novel mutations in WEE2: Expanding the spectrum of mutations responsible for human fertilization failure.

Successful fertilization is fundamental for sexual reproduction. After undergoing a series of molecular and morphological changes, the haploid sperm fuses with the haploid oocyte to create a diploid zygote. Defects in this process might lead to human fertilization failure. We have previously found homozygous mutations in WEE2 that are responsible for human fertilization failure, but the genetic basis of human fertilization failure requires further investigation. In the present study, we screened for WEE2 mutations in a new cohort of patients with fertilization failure. Through Sanger sequencing of WEE2 exons, we identified seven novel mutations and two reported mutations in WEE2 from six affected individuals. Morphologically normal PB1 oocytes can be retrieved from all patients. However, most of the oocytes cannot be fertilized successfully. These findings confirmed our previous research and expanded the mutational spectrum of WEE2, making it a potential genetic diagnostic marker for those suffering from human fertilization failure.

Homozygous Mutations in WEE2 Cause Fertilization Failure and Female Infertility

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted invivo. In clinical practice, some infertility case subjects suffer from recurrent failure of invitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein invitro and in affected individuals' oocytes invivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 invitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts invitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases.

Is there an association between PAWP/WBP2NL sequence, expression, and distribution in sperm cells and fertilization failures in ICSI cycles?

Successful fertilization in mammals depends on the sperm's ability to initiate intracellular Ca2+ oscillations in the egg, a phenomenon that is elicited by Sperm-oocyte activating factors (SOAFs), whose quantitative and/or qualitative defect might result in fertilization failure. One such proposed factor is Post-acrosomal WW domain-binding protein (PAWP/WBP2NL), although its ability to activate human oocytes has been questioned and its implication in human fertilization failure remains unknown. Here, we sought to determine if PAWP/WBP2NL expression and distribution in sperm cells associate with low/complete fertilization failure in males participating in intracytoplasmic sperm injection (ICSI) cycles. This prospective study was conducted on eight couples referred for elective ICSI with either the woman's own (n = 4) or a donor eggs (n = 4). Eight sperm donor samples used in ICSI, which resulted in normal fertilization rates, were used as the control group. For each male patient and donor sperm, PAWP/WBP2NL sequence, protein expression, and cellular distribution were analyzed by PCR amplification-sequencing, Western blot, and immunofluorescence, respectively. PAWP/WBP2NL was present in all samples, and no significant differences were detected between patients with fertilization failure and donors in sequence variants or mean protein expression, or in the proportion of PAWP/WBP2NL-positive sperm. In conclusion, no clear association between PAWP/WBP2NL protein expression in sperm and fertilization outcome in ICSI were observed from this cohort.

Novel strategy of ICSI – selection of sperm with morphologically normal midpiece by using IMSI

OBJECTIVE: The sperm aster is a radial microtubule array that is organized from the sperm centrosome and that induces pronuclear apposition and syngamy in human fertilization. Cytological analyses of human eggs folllowing ICSI suggests that fertilization failure in humans can be caused by failure of sperm aster formation, even with successful oocyte activation. These reports suggest that a novel strategy is needed for treating sterility with poor sperm centrosomal function. It is reported that microinjection with motile sperm strictly selected by high power light microscopy (6,000 times) improves the pregnancy rate in couples with repeated ICSI failures.

The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.

P-489: Possible role of proteasomes during proximal centriole release at fertilization in mammals: Implications for fertilization failure in humans

Cytoskeletal organization during fertilization is similar in bovine and humans in that they both require the participation of a paternally derived centriole. After sperm penetration, the zygotic centrosome organizes a microtubule array that is responsible for bringing the parental genomes together prior to first mitosis. For this purpose, the sperm-borne proximal centriole must be released from the surrounding dense structures of the sperm tail-connecting piece. The proteasomal machinery has been shown to take part in multiple steps of fertilization in different species.

Defective sperm decondensation: a cause for fertilization failure.

The study aimed to evaluate the role of chromatin packaging (CMA3 staining), sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies in oocytes that failed in vitro fertilization (IVF). The percentage CMA3 staining categorized the data into three groups, < 44%, n = 10; > or = 44-59%, n = 10; and > or = 60%, n = 29. Morphology groups were < or = 4% (n = 11); > 4-14% (n = 19); and > 4% (n = 19). One hundred and seventy-two oocytes that failed IVF were evaluated for sperm-zona binding, ooplasma penetration and sperm decondensation. Odds ratio analyses indicated that being in the > or = 60% CMA3 staining group resulted in a 15.6 fold increase in the risk of decondensation failure, relative to CMA3, staining of < 44%. For morphology, there was a 2.17 fold decrease in the risk of fertilization failure in the morphology group with > 4-14% normal cells, while it increased 2.45 fold for the morphology group with < or = 4% normal cells. Using CMA3 fluorescence to discriminate, 51% of the oocytes in the group with elevated CMA3 fluorescence had no sperm in the ooplasma compared to 32% and 16% penetration failure in the CMA3 staining groups > or = 44-59% and < 44%, respectively. Sperm chromatin packaging quality and sperm morphology assessments are useful clinical indicators of human fertilization failure. Immunofluorescence techniques could be used to provide a clear diagnosis of failed fertilization.

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.

Cellular events during fertilization failure in humans

The overall fertilization rate indicates the overall effectiveness of IVF and ICSI, but it does not provide information on results for individual couples. As extensively described (1–3), fertilization failure in mammals may have different etiologies. Immunofluorescence is a useful tool for exploring the cellular aspects of fertilization failure (4).