Novel Biallelic TLE6 Variants Induce Preimplantation Embryonic Lethality That Cannot be Rescued by IVF or ICSI

Abstract Background: Preimplantation embryonic lethality is a rare cause of primary female infertility. Transducin-like enhancer of split 6 (TLE6) is a maternal effect gene which encodes a component of the subcortical maternal complex located in the oocytes and early embryos. It has been reported that biallelic variants in TLE6 can lead to preimplantation embryonic lethality. However, the incidence of TLE6 variants in patients with preimplantation embryonic lethality is not fully understood.Methods: Whole-exome sequencing and bioinformatics analyses were used to analyze a cohort of 28 patients with preimplantation embryonic lethality. We retrospectively analyzed the process and outcome of their attempts at in vitro fertilization and intracytoplasmic sperm injection. Results: In this study, four patients (14.29 %, 4/28) from three unrelated families in a cohort of 28 individuals with preimplantation embryonic lethality were identified as carrying biallelic TLE6 variants, including two homozygous variants and one compound heterozygous variant. These novel frameshift variants in TLE6 caused truncation of the TLE6 protein likely impairing its function. Immunofluorescence staining of oocytes for TLE6 indicated that it is localized in the oocyte cytoplasm, and that TLE6 was almost absent in the oocytes of the patients carrying biallelic TLE6 variants compared with normal control oocytes. A retrospective analysis showed that the four affected individuals underwent a total of nine in vitro fertilization and intracytoplasmic sperm injection attempts, in which a total of 119 metaphase II oocytes were retrieved, but none of them obtained high-quality blastocysts or obtained a successful pregnancy. However, one of these patients became pregnant on the first attempt using donated oocytes.Conclusions: Biallelic TLE6 variants cause preimplantation embryonic lethality that cannot be rescued by in vitro fertilization or intracytoplasmic sperm injection. Thus, oocyte donation may be the preferred treatment for patients harboring biallelic TLE6 variants.

Download Full-text

Identification of Novel Biallelic TLE6 Variants in Female Infertility With Preimplantation Embryonic Lethality

Frontiers in Genetics ◽

10.3389/fgene.2021.666136 ◽

2021 ◽

Vol 12 ◽

Author(s):

Manyu Zhang ◽

Chunyu Liu ◽

Beili Chen ◽

Mingrong Lv ◽

Huijuan Zou ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Essential Role ◽

Bioinformatics Analysis ◽

Oocyte Donation ◽

Embryonic Lethality ◽

Female Infertility ◽

Whole Exome ◽

Enhancer Of Split ◽

Lower Expression

Preimplantation embryonic lethality is a rare cause of primary female infertility. It has been reported that variants in the transducin-like enhancer of split 6 (TLE6) gene can lead to preimplantation embryonic lethality. However, the incidence of TLE6 variants in patients with preimplantation embryonic lethality is not fully understood. In this study, we identified four patients carrying novel biallelic TLE6 variants in a cohort of 28 patients with preimplantation embryonic lethality by whole-exome sequencing and bioinformatics analysis, accounting for 14.29% (4/28) of the cohort. Immunofluorescence showed that the TLE6 levels in oocytes from patients were much lower than in normal control oocytes, suggesting that the variants result in the lower expression of the TLE6 protein in oocytes. In addition, a retrospective analysis showed that the four patients underwent a total of nine failures of in vitro fertilization and intracytoplasmic sperm injection attempts, and one of them became pregnant on the first attempt using donated oocytes. Our study extends the genetic spectrum of female infertility caused by variants in TLE6 and further confirms previously reported findings that TLE6 plays an essential role in early embryonic development. In such case, oocyte donation may be the preferred treatment.

Download Full-text

Mutations in ZP4 are associated with abnormal zona pellucida and female infertility

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-207170 ◽

2021 ◽

pp. jclinpath-2020-207170

Author(s):

Xiaoli Wei ◽

Youzhu Li ◽

Qicai Liu ◽

Wensheng Liu ◽

Xiaohong Yan ◽

...

Keyword(s):

Embryo Development ◽

Zona Pellucida ◽

Intracytoplasmic Sperm Injection ◽

Treatment Strategies ◽

Suction Pressure ◽

Human Female ◽

Female Reproduction ◽

Efficient Treatment ◽

Whole Exome

BackgroundThe zona pellucida (ZP) of human oocytes plays essential protective roles in sperm–egg interactions during fertilisation and embryo development. ZP4-null female rabbits exhibit a thin and irregular ZP, which severely impairs embryo development and fertility. However, the effects of ZP4 defect on human female reproduction remain unknown.Methods and resultsWe performed whole-exome sequencing in 26 female patients with abnormal (thin and irregular) ZP and identified heterozygous variants in ZP4 (OMIM: 613514) from 3 patients (approximately 11%). No ZP4 variant was found in the 30 control women with proven fertility. We constructed ZP4-mutated plasmids and found that the variants reduced the secretion of ZP4 in vitro. Lower suction pressure facilitated egg retrieval, and intracytoplasmic sperm injection (ICSI) was a desirable treatment for ZP4-mutated patients with abnormal ZP.ConclusionsWe identified ZP4 as a novel gene for human abnormal ZP and found that lower suction pressure and ICSI are efficient treatment strategies.

Download Full-text

Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro

Reproduction Fertility and Development ◽

10.1071/rd19217 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1778

Author(s):

Raul A. Gonzalez-Castro ◽

Fabio Amoroso-Sanches ◽

JoAnne E. Stokes ◽

James K. Graham ◽

Elaine M. Carnevale

Keyword(s):

Flow Cytometry ◽

Dna Fragmentation ◽

Fluorescence Intensity ◽

Intracytoplasmic Sperm Injection ◽

Binding Protein ◽

Sperm Parameters ◽

Oocyte Activation ◽

Oocyte Cytoplasm ◽

Principal Piece

Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.

Download Full-text

Distinct clinical and biological implications of CUX1 in myeloid neoplasms

Blood Advances ◽

10.1182/bloodadvances.2018028423 ◽

2019 ◽

Vol 3 (14) ◽

pp. 2164-2178 ◽

Cited By ~ 3

Author(s):

Mai Aly ◽

Zubaidah M. Ramdzan ◽

Yasunobu Nagata ◽

Suresh K. Balasubramanian ◽

Naoko Hosono ◽

...

Keyword(s):

Myeloid Leukemia ◽

Leukemic Cell ◽

Compound Heterozygous ◽

Experimental Conditions ◽

Leukemic Cell Lines ◽

Myeloid Neoplasms ◽

Whole Exome ◽

Functional Consequences ◽

Founder Events

Abstract Somatic mutations of the CUT-like homeobox 1 (CUX1) gene (CUX1MT) can be found in myeloid neoplasms (MNs), in particular, in myelodysplastic syndromes (MDSs). The CUX1 locus is also deleted in 3 of 4 MN cases with −7/del(7q). A cohort of 1480 MN patients was used to characterize clinical features and clonal hierarchy associated with CUX1MT and CUX1 deletions (CUX1DEL) and to analyze their functional consequences in vitro. CUX1MT were present in 4% of chronic MNs. CUX1DEL were preferentially found in advanced cases (6%). Most MDS and acute myeloid leukemia (AML) patients with −7/del(7q) and up to 15% of MDS patients and 5% of AML patients diploid for the CUX1 locus exhibited downmodulated CUX1 expression. In 75% of mutant cases, CUX1MT were heterozygous, whereas microdeletions and homozygous and compound-heterozygous mutations were less common. CUXMT/DEL were associated with worse survival compared with CUX1WT. Within the clonal hierarchy, 1 of 3 CUX1MT served as founder events often followed by secondary BCOR and ASXL1 subclonal hits, whereas TET2 was the most common ancestral lesion, followed by subclonal CUX1MT. Comet assay of patients’ bone marrow progenitor cells and leukemic cell lines performed in various experimental conditions revealed that frameshift mutations, hemizygous deletions, or experimental CUX1 knockdown decrease the repair of oxidized bases. These functional findings may explain why samples with either CUX1MT or low CUX1 expression coincided with significantly higher numbers of somatic hits by whole-exome sequencing. Our findings implicate the DNA repair dysfunction resulting from CUX1 lesions in the pathogenesis of MNs, in which they lead to a mutator phenotype.

Download Full-text

Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic sperm injection (ICSI)

Zygote ◽

10.1017/s0967199401001307 ◽

2001 ◽

Vol 9 (4) ◽

pp. 277-282 ◽

Cited By ~ 23

Author(s):

Yukiko Yamazaki ◽

Teruhiko Wakayama ◽

Ryuzo Yanagimachi

Keyword(s):

Oocyte Maturation ◽

Intracytoplasmic Sperm Injection ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Serum Free ◽

Nuclear Maturation ◽

Normal Term ◽

Developmental Capacity ◽

Oocyte Cytoplasm

The fertilisability and developmental capacity of mouse oocytes matured in vitro were examined by in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). While more than 50% of cumulus-enclosed oocytes were fertilised by IVF after maturation in serum-supplemented medium, none were fertilised when the oocytes matured without serum. By ICSI, the majority (78-94%) of the oocytes were fertilised regardless of the presence or absence of serum in oocyte maturation media. Although the majority (88-92%) of cumulus-free germinal vesicle oocytes underwent nuclear maturation in both serum-free and serum-containing media, those matured in the presence of serum were more readily fertilised by ICSI (43%) than those matured without it (3-5%). The cumulus-free oocytes co-cultured with cumulus cells but without serum were fertilised at 36%, suggesting some secreted factor promotes the oocyte's cytoplasmic maturation. The oocytes fertilised by ICSI developed into normal-term fetuses regardless of the presence or absence of serum or cumulus cells in oocyte maturation medium. These results lead us to conclude that (a) the cytoplasm of the oocytes can mature in serum-free medium and (b) the presence of both the serum and the cumulus cells in the medium surrounding maturing oocytes is beneficial for the development of the fertilisation- and development-competence of oocyte cytoplasm.

Download Full-text

Functionally pathogenic EARS2 variants in vitro may not manifest a phenotype in vivo

Neurology Genetics ◽

10.1212/nxg.0000000000000162 ◽

2017 ◽

Vol 3 (4) ◽

pp. e162 ◽

Cited By ~ 7

Author(s):

Nathan McNeill ◽

Alessia Nasca ◽

Aurelio Reyes ◽

Benjamin Lemoine ◽

Brandi Cantarel ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Protein Function ◽

In Silico ◽

In Silico Analysis ◽

Compound Heterozygous ◽

Functional Testing ◽

Whole Exome

Objective:To investigate the genetic etiology of a patient diagnosed with leukoencephalopathy, brain calcifications, and cysts (LCC).Methods:Whole-exome sequencing was performed on a patient with LCC and his unaffected family members. The variants were subject to in silico and in vitro functional testing to determine pathogenicity.Results:Whole-exome sequencing uncovered compound heterozygous mutations in EARS2, c.328G>A (p.G110S), and c.1045G>A (p.E349K). This gene has previously been implicated in the autosomal recessive leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The p.G110S mutation has been found in multiple patients with LTBL. In silico analysis supported pathogenicity in the second variant. In vitro functional testing showed a significant mitochondrial dysfunction demonstrated by an ∼11% decrease in the oxygen consumption rate and ∼43% decrease in the maximum respiratory rate in the patient's skin fibroblasts compared with the control. EARS2 protein levels were reduced to 30% of normal controls in the patient's fibroblasts. These deficiencies were corrected by the expression of the wild-type EARS2 protein. However, a further unrelated genetic investigation of our patient revealed the presence of biallelic variants in a small nucleolar RNA (SNORD118) responsible for LCC.Conclusions:Here, we report seemingly pathogenic EARS2 mutations in a single patient with LCC with no biochemical or neuroimaging presentations of LTBL. This patient illustrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant.ClinicalTrials.gov identifier:NCT00001671.

Download Full-text

Novel LAT Pathogenic Variants in a POI Family and Its Role in the Ovary

Frontiers in Genetics ◽

10.3389/fgene.2021.764160 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kun Chu ◽

Yi He ◽

Ziyuan Li ◽

Zhongxin Jiang ◽

Liang Wang ◽

...

Keyword(s):

Compound Heterozygous ◽

Ovarian Insufficiency ◽

Mutant Proteins ◽

Adverse Health Outcomes ◽

Pathogenic Variants ◽

Whole Exome ◽

Variant Analysis ◽

First Time

Premature ovarian insufficiency (POI) affects about 1% of women under 40 years and leads most often to definitive infertility with adverse health outcomes. Genetic factor has been reported to play an important role in POI. However, the genetic etiology remains unknown in the majority of the POI patients. Whole-exome sequencing and variant analysis were carried out in a POI pedigree. In vitro studies of the wild-type and mutant proteins were conducted in primary granulosa cells (GCs) and granulosa cell line. The result showed that the patients carried compound heterozygous nonsynonymous mutations (c.245C > T and c.181C > G) in LAT gene, which were identified to be transmitted from their parents. The two variants were assessed to affect residues that were conserved across different species examined, and were predicted to be deleterious by software predictions. Protein structure predicting result indicated that the two variants could alter their interactions with surrounding residues, which may change the internal structure of the LAT protein. Moreover, LAT protein expression in GCs was demonstrated for the first time, and further functional assays suggested that this mutation could reduce LAT expression and influence GC survival, which may contribute to the etiology of POI. In summary, we detect novel LAT pathogenic variants in a POI pedigree and report for the first time that LAT is present and functional in the GCs of the ovary. Our findings not only shed new light on the role of LAT in GCs, but also broaden the spectrum of genetic causes of POI.

Download Full-text

Compound heterozygous ZP1 mutations cause empty follicle syndrome in infertile sisters

10.1101/363911 ◽

2018 ◽

Cited By ~ 2

Author(s):

Ling Sun ◽

Xiang Fang ◽

Zhi-Heng Chen ◽

Han-Wang Zhang ◽

Xiao-Fang Peng ◽

...

Keyword(s):

Medical Treatment ◽

Zona Pellucida ◽

Ovarian Stimulation ◽

Bioinformatics Analysis ◽

Compound Heterozygous ◽

The Core ◽

Empty Follicle Syndrome ◽

Whole Exome ◽

Compound Heterozygous Variants

AbstractPurposeEmpty follicle syndrome (EFS) is a condition in which no oocyte is retrieved from mature follicles after proper ovarian stimulation in an in vitro fertilization (IVF) procedure. Genetic evidence accumulates for the etiology of recurrent EFS even with improved medical treatment which had avoided the pharmacological or iatrogenic problems. Here, this study investigated the genetic cause of recurrent EFS in a family with two infertile sisters.MethodsIn this work, we present two infertile sisters in a family with recurrent EFS after three cycles of standard ovarian stimulation with hCG and/or GnRHa therapy. We performed whole-exome sequencing and targeted sequencing in the core members of this family, and further bioinformatics analysis to identify pathogenesis of gene.ResultsWe identified compound heterozygous variants, c.161_165del (p.54fs) and c.1166_1173del (p.389fs), on zona pellucida glycoprotein 1 (ZP1) gene, which were shared with two infertile sisters. Cosegregation tests on the affected and unaffected members of this family confirmed that the allelic mutants were transmitted from either parent.ConclusionsThis EFS phenotype was distinct from the previously reported disruption of zona pellucida due to homozygous ZP1 defects. We thus propose that the specific mutations in ZP1 gene may render a causality for the recurrent EFS.

Download Full-text