A novel compound heterozygous mutation in TUBB8 causing early embryonic developmental arrest

Abstract

Purpose

Mutations in the β-tubulin isotype, TUBB8, can cause female infertility. Although several mutations of TUBB8 have been reported, the full spectrum for guiding genetics counseling still needs to be further explored. Here, we sought to identify novel variants in TUBB8 and their phenotypic effects on microtubule network structure in vitro.

Methods

Whole-exome sequence analysis was performed in two families with infertility to detect pathogenic variants, with validation by Sanger sequencing. All gene variants and protein structures were predicted in silico. Cells were transfected with wild-type and mutants, and immunofluorescence analysis was performed to visualize microtubule network changes.

Results

We detected a novel compound heterozygous mutation, c.915_916delCC (p.Arg306Serfs*21) and c.82C > T (p.His28Tyr), and a benign heterozygous variant c.1286C > T (p.Thr429Met) in TUBB8 in the two families. Female patients with p.Arg306Serfs*21 and p.His28Tyr were infertile with early embryonic developmental arrest. The female patient with p.Thr429Met gave birth to a healthy baby in the second in vitro fertilization frozen embryo transfer cycle. The p.Arg306Serfs*21 mutation was predicted to cause large structural alteration in the TUBB8 protein and was confirmed to produce a truncated and trace protein by western blot analysis. Immunofluorescence analysis of transfected HeLa cells showed that p.Arg306Serfs*21 significantly disrupted microtubule structure.

Conclusions

Our findings expand the known mutational spectrum of TUBB8 associated with early embryonic developmental arrest and female infertility.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-023-02734-x.

Introduction

Normal gamete maturation, sperm–oocyte fertilization, and embryonic development are essential to successful human reproduction [1, 2], and failure at any stage can lead to infertility. Two meiotic divisions are needed for oocytes to mature, and after fertilization, zygotes initiate mitosis to produce embryos for implantation. Although the oocyte meiotic spindle differs from the mitotic spindle in several respects [3, 4], microtubules are the main structural component in both [5]. Tubulins are basic microtubule proteins [6] that play an important role in spindle assembly and chromosome separation. Microtubules are dynamic polymers assembled from heterodimers of one α-tubulin and one β-tubulin polypeptide (α/β tubulin). The human genome contains seven α-tubulin and eight β-tubulin members, of which TUBB8 encodes a special β-tubulin isotype, a major component of the oocyte and early embryo spindle, exists only in primates [7–9]. Mutations in TUBB8 cause oocyte maturation defect 2 (OOMD2) and several consequent phenotypes including (i) metaphase I (MI) arrest [7, 10, 11], (ii) metaphase II (MII) arrest [12, 13], (iii) cleavage arrest [14, 15], (iv) early embryonic developmental arrest [16–18], (v) oocytes with large polar bodies [19, 20], and (vi) zygotes containing multiple pro-nuclei that subsequently arrest at an early stage [21, 22]. To date, a total of 139 TUBB8 variants have been reported, including 113 heterozygous mutations, 15 homozygous mutations, and 11 compound heterozygous variants (Supplemental Table 1). These three types of mutations in TUBB8 are all considered to primarily cause MI arrest, but some arrest during fertilization, cleavage, or early embryonic development also occurs.

Here, we report a novel compound heterozygous mutation that causes early embryonic developmental arrest as well as a benign heterozygous variant that leads to successful pregnancy. The new findings expand the known mutational spectrum in TUBB8 to inform genetic counseling.

Materials and methods

Human subjects

Two patients diagnosed with female infertility were recruited from Chenzhou No.1 People’s Hospital and the Second Xiangya Hospital, respectively. Peripheral blood samples of the probands (n = 2) and their family members (n = 10) were taken for DNA extraction. The studies were approved by the Ethics Committee of the Second Xiangya Hospital. All participants provided informed consent to participate in the research.

Exome and variant screening

Five-ml peripheral blood samples were obtained from two patients and their relatives. Genomic DNA was extracted from blood samples using DNA extraction kits (Tiangen Biotech, Beijing, China). Exome capture and sequencing were performed using Agilent SureSelect Whole-Exome capture (Agilent, Santa Clara, CA) and Illumina sequencing technology (Illumina, San Diego, CA). The Illumina bioinformatics analysis pipeline was used for data analysis. Basic bioinformatics analysis included mapping the raw FASTQ files to the human reference sequence (UCSC hg19). Single-nucleotide variants and short insertions and deletions were filtered according to the functional annotation using an in-house next-generation sequencing analysis platform and public databases. All suspected pathogenic gene variants were queried in the Human Gene Mutation Database (HGMD)(http://www.hgmd.cf.ac.uk) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar). SIFT, Polyphen2, Mutation Taster, FATHMM-MKL, PROVEAN, and ExAC (http://exac.broadinstitute.org/) were also applied to predict the functional effects of the variants. A TUBB8 variant was considered a candidate mutation if it had a frequency below 0.1% in three public databases: the 1000 Genomes variant database, the NHLBI exome sequencing project, and ExAC. Changes in protein structure and the conservation and tolerance of amino acid variants were predicted using Alphafold2, PyMOL 1.7.4 software, and MetaDome online.

Sanger sequencing

Candidate mutations in TUBB8 were examined in patients, immediate family members, and 150 normal controls by Sanger sequencing. TUBB8 exon’s amplification primers and sequencing primers were quoted from the References 18 (Supplemental Table 2). PCR amplification was performed using the Q5 High-Fidelity Polymerase (NEB, MA, USA). PCR products were sequenced for the initial screening by Tsingke Biotechnology Co., Ltd (Beijing, China). All PCR products were sequenced using the ABI 3730XL automated sequencer according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA), and the sequencing results were analyzed using Chromas software (v2.6.5, Technelysium Pty Ltd, South Brisbane, Australia).

Expression of wild-type and mutant TUBB8 in cultured cells

A full-length TUBB8 cDNA cloned in a pCDNA3.1 ( +) vector with a CMV promoter and an in-frame C-terminal FLAG tag was purchased from Chubo BioTech, Inc. Point mutations were generated by quick-change polymerase chain reaction for the expression of the wild-type (WT). For transient expression of wild-type or mutant TUBB8, approximately 20,000 HeLa cells were seeded into 24-well plate 16 h before transfection. 0.1 µg (low density) or 0.5 µg (high density) plasmids were transfected into HeLa cells using 0.5ul or 2.5 µl Lipofectamine 2000 per well according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Forty-eight hours after transfection, the cells were fixed, permeabilized, and labeled with antibodies targeting to the FLAG epitope (1:200, #14,793) (Cell Signaling Technology, Beverly, MA, USA) and α-tubulin (1:200, #3873) (Cell Signaling Technology, Beverly, MA, USA). To quantify microtubule phenotypes, over 200 cells expressing either wild-type or mutant TUBB8 were examined in each of the three independent experiments. All FLAG-positive cells were classified according to the level of expression of the transgene as judged by the fluorescence intensity and microtubule structure, where abnormal microtubule structures were visualized as diffuse, mottled FLAG labeling as previously described.

In order to detect whether the expression level of wild-type and mutant proteins is different, approximately 350,000 HeLa cells were seeded into 6-well plate 16 h before transfection. 2.5 µg plasmids (1.25 µg c.82C > T plasmids and 1.25 µg c.915_916delCC plasmids in p.H28Y & p.R306S*21 group) were transfected into HeLa cells using 15 µl Lipofectamine 2000 per well according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Forty-eight hours after transfection, cultured Hela cells were dissolved in RIPA lysis buffer (Beyotime, China) and proteinase inhibitor. The protein expression was detected by Western blot.

Results

Clinical characteristics and phenotypes of oocytes/embryos harboring TUBB8 mutations

Two patients with female infertility were identified. In family 1, the patient was diagnosed with secondary infertility at 29 years of age after spontaneous abortion during the first trimester 2 years previously. Ten oocytes were retrieved from three IVF cycles; of nine matured oocytes, seven fertilized normally, and the other two were three pro-nuclei. Five embryos were usable and vitrified (Table 1), three of which were high-quality. At the first frozen embryo transfer (FET), two blastocysts (3BB and 3BC) were thawed and transferred but failed. At the second FET, one blastocyst (6AA, Fig. 1a) was transferred, and the patient conceived and gave birth to a healthy baby.

Table 1.

Clinical characteristics of patients and their retrieved oocytes and embryos

Age (years)	Duration of infertility (years)	Previous IVF/ICSI cycles	Total no. of oocyte retrieved	PB1 oocyte	Oocyte with abnormal morphology	Fertilized oocyte	3PN	No. of cleavage embryos	Embryos arrested at early stage	Usable embryos	Outcome of ET
38	12	1	7	7	0	6	1	5	4	1 (4cIII)	Failure
29	2	3	10	9	1	9	2	9	1(3PN)	5 (3 are high-quality)	Delivered at the 2nd ET

Abbreviation: ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; and ET, embryo transfer

Fig. 1.

Genetic analysis of TUBB8 mutants. a Morphology of the blastocyst transferred in the second FET cycle from the patient in family 1. b Pedigree of the family 1 and Sanger sequencing of the family members. c Pedigree of the family 2 and Sanger sequencing of the family members. d Conservation analysis of altered amino acids in five primate species. e The graph represents the tolerance of TUBB8 to amino acid mutations. Each amino acid of TUBB8 is classified according to its tolerance score

In family 2, the proband had been diagnosed with primary infertility 12 years previously at 38 years of age and underwent one intracytoplasmic sperm injection (ICSI) cycle, from which seven oocytes were obtained. All seven oocytes matured and six were fertilized, five of which arrested at an early stage. One four-cell embryo was transferred on D3 but failed to produce a pregnancy (Table 1). The patient’s two sisters also suffered from primary infertility: the elder sister underwent three IVF/ICSI cycles but also failed to become pregnant due to early embryonic developmental arrest. Eventually, the patient conceived through oocyte donation.

Novel TUBB8 variants

Whole-exome sequencing analysis of the two probands with validation by Sanger sequencing implicated a heterozygous missense mutation c.1286C > T (p.Thr429Met) in family 1 and a novel compound heterozygous mutations c.915_916delCC (p.Arg306Serfs*21) and c.82C > T (p.His28Tyr) in family 2. In family 1, the TUBB8 p.Thr429Met mutation was detected in four family members (I-1,II-1,II-3,and III-1, family 1), two of whom were female with offspring (I-1, II-3, family 1, Fig. 1b). All three sisters in family 2 (II-2, II-3, and II-4, family 2) harbored the same compound heterozygous mutations and suffered primary infertility (Fig. 1c).

In silico analysis of TUBB8 variants

According to SIFT, Polyphen2, MutationTaster, PROVEAN, and FATHMM-MKL, the p.His28Tyr mutation was predicted to be damaging, while the p.Thr429Met mutation was predicted to be damaging or possibly damaging (Table 2). No prediction tool derived scores for the p.Arg306Serfs*21 mutation (Table 2), which is a frameshift mutation resulting in a premature stop codon that predicts a truncated protein from 444 to 326 amino acids and a terminal 21 amino acid sequence bearing no homology to the corresponding amino acid sequence of TUBB8. The residues altered by p.Arg306Serfs*21 in TUBB8 are highly evolutionarily conserved in primates, but the residues altered by p.His28Tyr and p.Thr429Met are not (Fig. 1d). p.28 (c.82–84) corresponds to a histidine residue with a tolerance score of 0.32, indicating intolerance to mutations at this site. Similarly, p.429 (c.1285–1287) corresponds to a threonine with a tolerance score of 0.53, indicating slight intolerance to the mutation at this locus (Fig. 1e).

Table 2.

Effects of TUBB8 mutations predicted with in silico tools

Mutations	Amino acid change	Genotype	SIFTa	Polyphen2b	Mutation tasterc	PROVEANd	Fathmm _MKLe	1000 genomesf	gnomad_ exon_totalg	gnomad_exon _east Asianh
c.915_916del	p.Arg306Serfs*21	com-het	NA	NA	NA	NA	NA	NA	NA	NA
c.82C > T	p.His28Tyr	com-het	Damaging (0.0)	Damaging (0.999)	Disease causing (1)	Damaging (− 2.63)	Damaging (0.900)	NA	2.596e-05	0.0004
c.1286C > T	p.Thr429Met	het	Damaging (0.0)	Probably damaging (0.989)	Disease causing (1)	Tolerable (− 1.72)	Damaging (0.805)	0.000199681	2.97e-05	0.0002

^aSIFT, sorting Intolerant from tolerant (http://sift.jcvi.org/)

^bPolyphen-2 (http://genetics.bwh.harvard.edu/pph2/)

^cMutation Taster (http://www.mutationtaster.org/)

^dPROVEAN (http://provean.jcvi.org/protein_batch_submit.php?species=human)

^eFATHMM-MKL (http://fathmm.biocompute.org.uk/fathmmMKL.htm)

^fFrequency of variation in the 1000 Genomes database

^gFrequency of variation in the total of exome gnomAD (genome Aggregation Database) (version 2.1)

^hFrequency of variation in the East Asian population of exome gnomAD (version 2.1)

Alterations in TUBB8 protein structure

The TUBB8 protein contains a tubulin domain (aa 47–244), a tubulin C-terminal domain (aa 246–383), a coiled-coil domain (aa 405–444), and other domains. The p.His28Tyr variant is located in the tubulin domain, p.Arg306Serfs*21 in the tubulin C-terminal domain, and p.Thr429Met in the coiled-coil domain (Fig. 2a). We predicted the three-dimensional structure of wild-type TUBB8 using Alphafold2 based on the Q76FS2 PDB template (Fig. 2b). The entire protein model of the p.Arg306Serfs*21 mutation was also predicted, especially the 21 aa C-terminal region (Fig. 2c). In p.Arg306Serfs*21, the original α-helix and β-fold regions near the mutation site were lost and resulted in the loose structure of the entire protein. For the two missense variants, p.His28Tyr and p.Thr429Met, the protein 3D structure and atomic model were rendered by PyMOL 1.7.4 software. It showed that there were no obvious effects on the protein structure and did not display changes in hydrogen bonding. Both the original His28 residue and replacement Tyr28 residue formed hydrogen bonds with Ile24 and Ser25, while the original Thr429 residue and the replacement Met429 residue formed hydrogen bonds with Glu432 (Fig. 2d).

Fig. 2.

Protein conformation predictions caused by variants in TUBB8 using Alphafold2 and PyMol. a The positions of altered alleles are shown on the TUBB8 gene, and the corresponding amino acids are indicated on the TUBB8 protein. b The 3D protein structure of wild-type TUBB8 predicted by Alphafold2. c The predicted protein conformation of p.Arg306Serfs*21 by Alphafold2 shows a large structural alteration. d TUBB8 variants were mapped to the atomic model using PyMol. Red dashed lines represent hydrogen bonds

TUBB8 variants disrupt microtubule structure in HeLa cells

To determine the influence of the TUBB8 mutations on microtubule behavior in vivo, we transfected FLAG-tagged constructs into HeLa cells (Fig. 3a). When exogenous TUBB8 proteins were expressed at a relatively low level, the wild-type and mutant proteins had little effect on the organization of the cytoplasmic microtubule network. High expression of p.His28Tyr or p.Thr429Met mutant TUBB8 was also typically associated with normal microtubule phenotypes, but p.Arg306Serfs*21 or p.Arg306Serfs*21 and p.His28Tyr mutations were significantly more frequently abnormal than wild-type (Fig. 3b) (P < 0.01). Moreover, the p.Arg306Serfs*21 (85.0%) mutation was significantly more frequently abnormal (Fig. 3b) (P < 0.05) than p.Arg306Serfs*21 and p.His28Tyr co-transfection (58.7%). It is worth noting that p.Arg306Serfs*21 shows a diffuse mottled stain throughout the cytoplasm, with no evidence of co-assembly into microtubules. Western blot analysis confirmed that p.Arg306Serfs*21 mutation resulted in a truncated protein (326 N-terminal amino acids rather than the full-length 444 amino acid polypeptide) (Fig. 3c) and a dramatic lowered expression (2.8%, P < 0.001) than that of wild-type (Fig. 3d), resulting in a severely impaired protein function following a loss-of-function mechanism. The TUBB8 expression of p.His28Tyr mutation also showed significantly decreased, while p.Thr429Met mutation showed no significant influence. The expression of mutant proteins showed the same trend as the proportion of normal cells detected by immunofluorescence.

Fig. 3.

HeLa cells were transfected with constructs engineered to express FLAG-tagged TUBB8 (wild-type and mutant). a The results of immunostaining with antibodies targeting the FLAG epitope to detect expression of the transgene (green) and antibody to α-tubulin to detect the endogenous microtubule network (red). Scale bar = 5 µm. b Quantitative analysis of the microtubule phenotypes shown in a. Approximately 200 transfected cells expressing either wild-type or mutant TUBB8 were examined in three separate experiments. Bars indicate standard deviations. *P < 0.05, **P < 0.01. c The expression level of WT, p.H28Y, p.R306Sfs*21, p.T429M, and p.H28Y & p.R306Sfs*21 TUBB8 protein in Hela cells detected by Western Blot. d Quantitative analysis of expression level of wild-type and mutant TUBB8 shown in c. Bars indicate standard deviations. N = 3 independent transfections. ***P < 0.001; ns, no significance

Discussion

Here, we identified a new compound heterozygous variant and a benign heterozygous variant in TUBB8 in four female patients from two families. All the three sisters harboring the compound heterozygous c.915_916delCC and c.82C > T variant were infertile, two of whom had undergone failed IVF/ICSI attempts due to early embryonic development arrest. The infertile patient with the heterozygous c.1286C > T variant obtained high-quality embryos, failed in the first FET cycle, but conceived from the second FET cycle. These results expand the mutational and phenotypic spectrum of TUBB8.

According to the clinical information of 11 compound heterozygous mutations reported in 11 patients (Tables 3 and 4), compound heterozygous mutations in TUBB8 mainly cause compound phenotypes including meiotic arrest, poor fertilization, complete cleavage failure, and embryonic arrest but not total MI arrest. Consistent with these reports, the compound heterozygous mutations reported here mainly caused early embryonic developmental arrest.

Table 3.

Overview of the 11 reported compound heterozygous variants in TUBB8

cDNA alteration	Amino acid alteration	Inheritance pattern	Phenotypes
			MA	PF	CCF	EA	NA
c.580G > A/c.1245G > A (18)	p.E194K/p.M415I	De novo/de novo	√			√
c.322G > A/c.426dupG (16)	p.E108K/p.T143Dfs*12	Inherited-pa/inherited-ma		√	√
c.1286C > T/c.1301_1327del (16)	p.T429M/p.434-442del	Inherited-ma (both are from her mother)					√
c.1103 T > C/c.382dup (19)	p.I368T/p.D128Gfs*27	Inherited-ma/inherited-pa	√	√
c.1103 T > C/c.80_100del (19)	p.I368T/p.E27_A33del	Unknown/inherited-ma	√		√
c.82C > T/c.148_154delins (19)	p.H28Y/p.Y50_N52delins	Inherited-pa/inherited-ma	√
c.400C > T/c.353A > G (19)	p.Q134*/p.D118G	Inherited-ma/inherited-pa	√		√
c.82C > T/c.398 T > C (19)	p.H28Y/p.F133S	Unknown				√
c.722G > C/c.1190_1192dup (17)	p.R241P/p.W397dup	Inherited-ma/inherited-pa	√			√
c.269dupT/c.426dupG (17)	p.I91Hfs*35/p.T143Dfs*12	Unknown/inherited-pa	√	√	√
c.322G > A/c.966dupC (17)	p.E108K/p.M323Hfs*6	Inherited-pa/inherited-ma			√

Abbreviation: MA, oocyte meiotic arrest; PF, poor fertilization; CCF, complete cleavage failure; EA, embryonic arrest; NA, not available

Table 4.

Clinical characteristics of the eleven reported patients with compound heterozygous mutations in TUBB8

cDNA alteration	Age (years)	Duration of infertility (years)	Previous IVF/ICSI cycles	Total no. of oocyte retrieved	GV oocyte	MI oocyte	PB1 oocyte	Oocyte with abnormal morphology	Immature oocyte (unknown stage)	Fertilized oocyte	No. of cleavage embryos	Embryos arrested at early stage	Usable embryos	Outcome of ET
c.580G > A/c.1245G > A (18)	28	2	2	19	0	0	3	0	16	3	3	3	0
c.322G > A/c.426dupG (16)	39	12	2	27	1	4	20	2	0	2	0	/	0
c.1286C > T/c.1301_1327del (16)	Had no clinical information
c.1103 T > C/c.382 dup (19)	30	4	2	13	0	9	2	0	2	0	0	0	0
c.1103 T > C/c.80_100del (19)	26	/	1	10	0	9	1	0	0	1	0	0	0
c.82C > T/c.148_154delins (19)	35	9	4	31	0	0	11	0	20	11	8	/	2	Failure
c.400C > T/c.353A > G (19)	31	7	3	51	0	1	0	44	50	44	0	/	0
c.82C > T/c.398 T > C (19)	33	11	1	12	0	2	8	2	0	3	1	1	0
c.722G > C/c.1190_1192dup (17)	34	9	2	36	16	15	4	0	0	4	3	3	0
c.269dupT/c.426dupG (17)	24	4	1	30	0	9	9	8	4	2	0	/	0
c.322G > A/c.966dupC (17)	28	4	1	15	2	0	13	0	0	11	0	/	0

The TUBB8 c.82C > T mutation has been found in two other reported compound heterozygous mutations, c.82C > T/c.148_154delins and c.82C > T/c.398 T > C. In the c.82C > T/c.148_154delins mutation, c.82C > T was from the patient’s father, and c.148_154delins was from her mother. The inheritance pattern of the c.82C > T/c.398 T > C mutation is unknown. Since both parents in family 2 were deceased and only the c.915_916delCC mutation was detected in the brother, we presumed the inheritance pattern of the c.915_916delCC/c.82C > T mutations in our three patients was compound heterozygosity. According to our results, although the expression of p.His28Tyr is lower than that of wile-type, it did not result in abnormal microtubule networks in vitro, and there is no report of a heterozygous c.82C > T mutation causing female infertility, so the pathogenicity of c.82C > T needs further study to confirm.

The TUBB8 c.915_916delCC mutation is a novel mutation that clearly disturbed the cytoplasmic microtubule network in cultured cells, and its effect was even worse than c.82C > T and c.915_916delCC co-transfection. p.Arg306Serfs*21 shows a diffuse mottled stain throughout the cytoplasm, suggesting that it cannot form functional microtubules with α-tubulin. The productive folding of α-tubulin and β-tubulin and their integration into heterodimers require the interaction of newly synthesized polypeptides with several molecular chaperones. These include prefoldin; the cytosolic chaperonin, CCT; and five tubulin specific chaperones termed TBCA-E that function in concert as a GTP-dependent heterodimer assembly nanomachine [8]. Western blot analysis confirmed that p.R306Sfs*21 plasmid produced a truncated and trace protein, which implied the p.Arg306Serfs*21 variant resulted in a severely impaired protein function following a loss-of-function mechanism. We predict that it was degraded due to the change of three-dimensional structure that cannot combine with molecular chaperones, like another truncated TUBB8 protein, T143Dfs*12 [8]. Consistent with previous studies, the loss-of-function variants in TUBB8 usually affect female fertility in a homozygous or compound heterozygous pattern, implying the haploinsufficiency mechanism, while the missense variants mostly present as heterozygous pattern, implying the dominant-negative mechanism of TUBB8 variant-related phenotypes.

Our expanded analysis of family 1 showed that the patient’s grandma, father, and younger aunt were all fertile, even when the c.1286C > T heterozygous variant was present, i.e., a heterozygous c.1286C > T mutation did not cause infertility in this family. Furthermore, immunofluorescence analysis confirmed that the c.1286C > T variant did not cause significant microtubule network abnormality in vitro. Extended pedigree segregation and mutational effect assays are indispensable for determining the true pathogenicity of mutants when patients acquire usable embryos, especially high-quality embryos. Therefore, we reported to the patient in family 1 that the TUBB8 c.1286C > T variant does not affect fertility and advised that her oocytes were suitable for assisted reproductive treatment.

Few TUBB8 variants have been examined in functional assays for classification according to the American College of Medical Genetics and Genomics (ACMG) guidelines, making them suitable as genetic markers for counseling [23]. For patients with likely pathogenic mutations, oocyte donation may be the most feasible infertility treatment approach at present, while the pathogenicity of other TUBB8 variants undergoes further definitive evaluation. When other variants in TUBB8 or other OOMD genes (PADI6 [24], WEE2 [25], PATL2 [26], PANX1 [27], TRIP13 [28], LHX8 [29], CDC20 [30], ZP1 [31], ZP2 [32], ZP3 [33], BTG4 [34], TLE6[35], NLRP2 [36], NLRP5 [37], KHDC3L [38], and REC114 [39]) of uncertain significance are detected in female infertile patients who fail ART, receptive endometrium and synchronized development between the embryo and the endometrium should both be taken into account together with use of high-quality embryos to optimize outcomes [40].

In conclusion, here we present a novel compound heterozygous mutation in TUBB8 that causes familial female infertility and another benign heterozygous mutation, expanding the known mutational spectrum in TUBB8 associated with early embryonic development arrest.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

JZ, project development, data analysis, and manuscript writing; SL, data collection or management and manuscript editing; FH and TS, project development; RX, prepared Fig. 2; DW, B L, and DL, data collection and analysis; XS and JC, data collection or management; HH, data collection or management, data analysis, manuscript writing, and revision process. All authors read and approved the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (81501248), the Science and Technology Innovation Program of Hunan Province (2021RC3031), the Natural Science Foundation of Hunan Province (2022JJ30066), the Scientific Research Program of Hunan Provincial Health Commission (20220503347), and the Open Research Program of Key Laboratory of Regenerative Biology of Chinese Academy of Sciences (KLRB202010).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Institute Review Board of the Department of Reproductive Medicine, Central South University. Informed consent was obtained from all individual participants included in the study.

Consent for publication

Patients signed informed consent regarding publishing their data and photographs.

Competing interests

The authors declare no competing interests.