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Mutations in the tubulin beta 8 class VIII (TUBB8) gene have been proven to cause oocyte maturation arrest. The aim of this study was to describe newly discovered mutations in TUBB8 and to investigate the prevalence of TUBB8 mutations in our cohort. Nine women with oocyte maturation arrest and 100 fertile female controls were recruited. Sanger sequencing of the coding regions of TUBB8 revealed a heterozygous variant c.535G > A (p.V179M) in two unrelated affected individuals and a heterozygous variant c.5G > T (p.R2M) in one affected individual. These TUBB8 variants were inherited from the unaffected fathers and were absent in 100 fertile female control individuals. In total, 33.33% (3/9) of the affected individuals in our cohort obtained a clear genetic diagnosis through sequencing of the TUBB8 gene. These two novel variants extend the spectrum of TUBB8 mutations and this study confirmed that TUBB8 mutations occur in a high proportion of infertile women with oocyte maturation arrest.
). Morphologically, oocytes at prophase I have an intact nucleus, termed the germinal vesicle (GV), and after GV breakdown the oocytes progress to metaphase I (MI). Following extrusion of a polar body and asymmetric division, MI is soon completed and the oocyte is again arrested at MII until fertilization (
). TUBB8 encodes a protein that is the primary beta-tubulin subunit of the oocyte and early embryo. TUBB8 heterozygous mutations, either paternally inherited or de novo, have dominant-negative effects that disrupt microtubule behaviour and oocyte meiotic spindle assembly, and thus arrest oocyte maturation (
). Using immunostaining analysis, oocytes harbouring these TUBB8 mutations can show one of three distinct phenotypes: MI oocytes with an impaired spindle; MI oocytes that are dysfunctional but have a visible although morphologically defective spindle; or oocytes with MII morphology that either lack an identifiable spindle or contain an impaired spindle (
). Through standard or polarizing light microscopy, oocytes with certain mutations (e.g. p.I210V, p.T238M, p. E27_A33del, p.T143Dfs*12) were observed to have a small proportion of morphologically identifiable MII oocytes with an extruding first polar body. These morphologically MII oocytes can be fertilized and cleave, but these embryos later become developmentally arrested (
). Overall, different TUBB8 mutations can result in an extensive variety of oocyte phenotypes with respect to spindle morphology and extrusion of the first polar body.
In the present study, we aimed to investigate the prevalence of both known and novel TUBB8 mutations in our cohort of infertile women with oocyte maturation arrest. We confirmed that TUBB8 gene mutations occur in a high proportion of infertile women with oocyte maturation arrest. We identified two novel mutations and discussed the possible effects on the function of the TUBB8 protein. These new findings broaden the mutation spectrum of TUBB8.
Materials and methods
Patient and control individuals
Nine affected individuals diagnosed with oocyte maturation arrest were recruited from the Center for Reproductive Medicine, Anhui Provincial Hospital. All of them had a normal karyotype (46, XX). DNA samples were obtained from all nine affected individuals and all of their available family members. One hundred DNA samples of unrelated, anonymous, fertile women from the same population were used as controls. Written informed consent was obtained from every participant. This genetic investigation was approved by the biomedical research ethics committee of Anhui Medical University on 1 March 2014 (reference number 20140022).
Genomic DNA was extracted from peripheral blood samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
All the coding regions of TUBB8 were amplified by PCR. Primers are shown in Table 1. PCR conditions were as follows: pre-denaturing at 94°C for 5 min, followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 30 s and extending at 72°C for 1 min, and final elongation at 72°C for 5 min. The PCR products were directly Sanger sequenced in both forward and reverse directions, using an ABI 3100 DNA analyser (Applied Biosystems, Foster City, CA, USA).
Table 1Genomic PCR primers used to amplify TUBB8 exons for Sanger sequencing.
) were used to search the allele frequencies of the variants.
Attempted intracytoplasmic sperm injection (ICSI) in patient II-1 from family 1 resulted in oocytes that were all in MI (Figure 1A); none of which were fertilized (Table 2). In patient II-2 from family 2, oocytes failed to mature (Figure 1A) even after extended culture in vitro and fertilization after ICSI failed. These two unrelated affected individuals were found to be heterozygous for a missense variant NM_177987.2 (TUBB8): c.535G > A (p.Val179Met) (Figure 2A). Both of these two individuals were from non-consanguineous families. In both cases, the variant was paternally inherited.
Table 2Oocyte and embryo characteristics of IVF and ICSI attempts for three affected individuals.
Duration of infertility (years)
Previous IVF/ICSI cycles
Total no. of oocytes retrieved
Stages of oocytes
Fertilization and embryo development
Family 1, Patient II-1
24 in MI
Family 2, Patient II-2
46 in MI
Family 3, Patient II-1
21 in MI
Abnormal fertilization in three oocytes (after IVF) but with no cleavage
ICSI, intracytoplasmic sperm injection; MI, metaphase I.
In patient II-1 from family 3, attempted IVF was performed and all oocytes were found to be in MI (Figure 1A) during preparation for early rescue ICSI. Three oocytes (two with one pronucleus and one with three pronuclei) had extruded one or two polar bodies after IVF on day 1 (Figure 1B) but no cleavage was observed on day 3 (Table 2). This patient was also from a non-consanguineous family and was found to be heterozygous for a paternally inherited missense variant NM_177987.2 (TUBB8): c.5G > T (p.Arg2Met) (Figure 2A).
The amino acids in positions p.V179 and p.R2 are highly conserved based on analysis with MultiAlin (Figure 2B). The two novel variants were predicted to be highly damaging to the function of the TUBB8 protein, by SIFT and PolyPhen 2 (Table 3).
Table 3Effects of TUBB8 mutations, predicted with in-silico tools.
To exclude the possibility that the identified variants may be common in the studied population, we performed Sanger sequencing of TUBB8 in 100 unrelated fertile women. The above two variants were absent in all of the 100 control DNA samples and not recorded in dbSNP (v142). In addition, inspection of the ExAC browser identified that c.535G > A has a low allele frequency (1/120158), indicating that c.535G > A was found in a single individual with unknown gender and only in the heterozygous state. The c.5G > T variant was not found in the ExAC browser.
The genetic analysis of a cohort of nine infertile women with oocyte maturation defects allowed us to define the prevalence of TUBB8 mutations in that cohort. All four exons of TUBB8 can be amplified and Sanger sequenced using only two pairs of primers, thus it is quite feasible and not expensive for genetic diagnosis. In total, three out of nine unrelated affected individuals (33.33%) were genetically diagnosed with heterozygous TUBB8 mutations. A previous study showed that 29.17% (7/24) of infertile women with an oocyte maturation defect had TUBB8 mutations (
). The difference may be due to a bias in patient recruitment or a limited number of patients analysed in all studies to date. Nevertheless, our study independently confirms that a high proportion of defects of oocyte maturation in infertile women are indeed caused by molecular defects of TUBB8. It is recommended that TUBB8 should be preferentially analysed as part of the genetic diagnosis of infertile women with oocyte maturation arrest.
Two previous studies identified 16 different mutations in TUBB8, including 13 heterozygous mutations (p.R2K, p.S176L, p.I210V, p.V229A, p.T238M, p.V255M, p.R262Q, p.R262W, p.T285P, p.M300I, p.N348S, p.M363T and p.D417N) and two homozygous mutations (p.E27_A33del and p.T143Dfs*12) (
) (Table 4). Here, we report two additional novel mutations of TUBB8, which further expand the mutation spectrum of TUBB8. One heterozygous mutation (p.V179M) was identified in two unrelated patients and the other heterozygous mutation (p.R2M) was found in an additional patient. The amino acid positions of these two mutations were highly conserved and these two mutations were predicted to be deleterious with in-silico tools. The p.V179 residue lies in the beta:T5 loop and is located at a longitudinal interface between assembled alpha/beta-tubulin heterodimer; its substitution may disrupt the longitudinal interactions, thus affecting microtubule assembly. The p.R2 residue is located at the alpha/beta-tubulin interface within the heterodimer and the mutation p.R2M could influence dimer assembly and stability (
). Immunostaining analysis of oocytes from the affected individuals may help to evaluate whether these two newly discovered TUBB8 mutations cause oocyte maturation arrest through the same pathogenic mechanism. However, no oocytes of the affected individuals in this study were available for experimental purposes.
Previous studies showed that some of the oocytes (with p.I210V, p.T238M or p.N348S mutations) that were morphologically recognizable as MII can be fertilized and even cleave, but these embryos later become developmentally arrested (Table 4) (
). In the routine use of clinical ICSI, only morphological MII oocytes identified by light microscopy are selected for fertilization. In this study, oocytes with the p.R2M mutation were only found to be in MI, thus the procedure of early rescue ICSI was not performed; three out of 21 oocytes became abnormally fertilized after IVF on day 1, but no cleavage was observed on day 3. After oocyte denudation and additional incubation with spermatozoa overnight, some oocytes can be further matured and inseminated (
). In this way, it is possible that a small proportion of oocytes with p.R2M could extrude the first polar body (PB1). This phenotype was reported in cases with some TUBB8 mutations in a previous study (
). Even with an impaired spindle, these in-vitro maturated MII oocytes still have an opportunity to be fertilized by spermatozoa in an incubator overnight, but a polynucleated cytoplasm could be observed due to the blocked extrusion of the second polar body. A single pronucleus could also be formed due to a decreased ability to induce sperm decondensation in these oocytes with incomplete cytoplasmic maturation (
), the proportion of these morphologically mature oocytes of total retrieved oocytes may vary in different conditions (e.g. triggering final follicular maturation with human chorionic gonadotrophin (HCG), gonadotrophin-releasing hormone agonists (GnRHa), or both, and the timing of triggering (
)). Our study assumed that a small proportion of oocytes with some TUBB8 mutations could extrude the PB1 after in-vitro maturation (not only after the normal routine of oocyte retrieval) and could be fertilized by IVF, but that the ensuing embryos would become arrested at an early stage of development.
The fathers in two unrelated families that carried the TUBB8 c.535G > A mutation were fertile. The ExAC browser revealed that c.535G > A had a low allele frequency (1/120158). In fact, it is possible that the prevalence of fertile males with heterozygous TUBB8 mutations could be underestimated. This could be one of the underlying factors for the high proportion of TUBB8 mutations in infertile women with oocyte maturation arrest.
There were 66.67% (6/9) of the affected individuals in our cohort with no TUBB8 mutations. This indicates that human oocyte maturation arrest can also be caused by defects of other genes that are yet to be implicated in this disorder. However, our data in combination with two previous studies (
) suggest that between 29.17% and 37.20% of patients with oocyte maturation arrest may obtain a genetic diagnosis through genetic screening of the TUBB8 gene. The presence of a TUBB8 mutation will probably confirm the diagnosis of oocyte maturation arrest and assign a poor prognosis for the success of assisted reproductive treatment.
We would like to thank all families and controls for participating in this study. This study was supported by the National Natural Science Foundation of China ( 81370757 to Y.L.) and the Central Guided Local Development of Science and Technology Special Fund (2016080802D114 to X.T.).
A method and server for predicting damaging missense mutation.
Dr Lingli Huang received her MD in 2008 and PhD in 2012 from Central South University. She works at the Reproductive Medicine Center in Anhui Provincial Hospital Affiliated to Anhui Medical University. Her main focus is on reproductive endocrinology and genetic diseases, follicle monitoring, IVF puncture, embryo transfer and genetic counselling.
Two new heterozygous TUBB8 gene mutations, p.R2M and p.V179M, were identified in three infertile women out of nine women with oocyte maturation arrest, expanding the spectrum of TUBB8 mutations and confirming TUBB8 mutations in a high proportion of infertile women with this condition.
Published online: June 13, 2017
Received in revised form:
Declaration: The authors report no financial or commercial conflicts of interest.