In this study, using WES and bioinformatics analyses, we identified biallelic TLE6 variants in 4 from a total of 28 infertile women with PEL, accounting for 14.29% of the cohort. Moreover, the three novel TLE6 frameshift variants in the present study were all LOF variants that cause protein truncation as well as impaired function. By immunofluorescence staining, we found that the biallelic variants in TLE6 impaired the stability of the TLE6 and resulted in its degradation in oocytes.
TLE6, also known as Groucho family member 6 (GRG6), is part of the SCMC that is necessary for mammalian embryonic development [20–21]. Research has shown that high levels of cAMP in the oocyte maintain an increase in cAMP-dependent protein kinase (PKA) activity, which causes meiotic prophase I arrest. Following the luteinizing hormone surge during ovulation, PKA activity reduces due to a decrease in cAMP levels, leading to a resumption of meiosis, while PKA activity increases throughout the process of meiosis from the time of germinal vesicle breakdown (GVBD) until the MII arrest [22]. TLE6 is a substrate of PKA during mouse oocyte maturation. Inhibition of PKA activity can lead to delays in GVBD dynamics, abnormal spindle and chromatin structures, and a reduced ability of oocytes to undergo MII [22]. Therefore, it has been speculated that the PKA-dependent phosphorylation of TLE6 during GVBD may be relevant to oocyte maturation and subsequent embryonic development. In mouse models, knockout of the Tle6 gene has no effect on folliculogenesis, oogenesis, and ovulation, but instead results in embryonic development arrest at the two-cell stage, and some embryos showed significant fragmentation at embryonic day 2.5 and embryonic day 3.5, eventually leading to female mice infertility [12–13]. Further studies indicate that the SCMC might regulate spindle assembly by controlling formation of the F-actin cytoskeleton to ensure symmetric division of mouse zygotes, while the absence of TLE6 affects the formation of F-actin cytoskeleton due to destruction of the integrity and function of the SCMC, and thus, results in asymmetric cleavage as well as early embryonic arrest [13].
Alazami et al. first identified a homozygous TLE6 variant c.1529C > A (p.S510Y) in affected individuals from two Saudi families, which resulted in the earliest known human PEL phenotype, including fertilization failure and early cleavage failure. Their further research showed that the TLE6 variant not only caused a significant reduction in the PKA-mediated TLE6 phosphorylation but also impaired its binding to other component proteins of the SCMC [14]. Wang et al. found the TLE6 variant c.1133delC (p.A378Efs*75) being responsible for embryonic developmental arrest on day 3, similar to the phenotype of Tle6Null mice [23]. Furthermore, another study also found that three patients carrying biallelic TLE6 variants had fertilization failure and early embryonic arrest in several IVF/ICSI attempts. Two of them obtained a very low number of low-quality embryos but failed to establish pregnancy [24]. Recently, using time-lapse imaging, Zheng et al. found that the TLE6 missense variant c.1564G > C (p.Asp522His) is associated with direct cleavage (zygotes directly cleaved into more than two blastomeres) [25].
Phenotypes of infertile women harboring biallelic TLE6 variants in this study are similar to the previously reported clinical cases [14, 23–25]. These four patients underwent a total of nine IVF/ICSI attempts in which the number of retrieved oocytes and frequency of oocyte maturity were not clearly abnormal; however, the fertilization rate and blastocyst development rate were very low. The overwhelming majority of the embryos had developmental arrest on the third day, so that none of these patients obtained high-quality blastocysts or established pregnancy successfully. One of the patients (II-4 in family 1) got pregnant successfully at the first attempt using donated oocytes. Therefore, these biallelic TLE6 variants are responsible for PEL, which cannot be rescued by IVF or ICSI. In contrast, subject II-1 in family 1, who had a heterozygous variant in TLE6, had normal fertility and two healthy children. Furthermore, in our cohort of control women pursuing IVF/ICSI due to male infertility, we also found that a subject harboring a heterozygous missense variant in TLE6 established pregnancy successfully via sperm donation. These clinical cases combined with Sanger sequencing can help further understand the inheritance pattern of the TLE6 mutant gene.
In addition, we used light microscopy for five consecutive days to observe the development and morphology of the embryos from one patient (II-4 in family 1) who carried the homozygous TLE6 frameshift variant c.1631_1632delCA (p.Pro544Argfs*5), and found that three embryos had a high percentage of fragmentation during culture, whereas the other five embryos were arrested on the third day, showing similar phenotype in embryogenesis between infertile women carrying TLE6 variants and Tle6Null female mice. Thus, combined with previous reports, we speculated that the TLE6 missense/frameshift variants lead to embryo fragmentation by disrupting the F-actin and spindle dynamics. In addition, our study is the first to assess the expression levels of the TLE6 protein in the oocytes of affected individuals. Immunofluorescence staining showed that the biallelic variants in TLE6 in the present study resulted in the degradation of TLE6 in the oocytes of affected individuals. From this, we speculate that TLE6 protein degradation might then affect the stability and function of the SCMC, eventually leading to preimplantation embryonic lethality.
There are a few limitations associated with the current study. First, the exact molecular mechanism of PEL could not be completely elucidated owing to the paucity of human oocytes and embryos. It will be worthwhile to study the molecular mechanism using knock-in mice for each variant in future. Second, sample size of affected women was limited in the present study, so the incidence of TLE6 variants in patients with PEL requires further research.
In conclusion, this study extends the spectrum of variants in TLE6 and shows that biallelic TLE6 variants are responsible for preimplantation embryonic lethality, which cannot be rescued by IVF or ICSI. Thus, oocyte donation may be the best option for a successful pregnancy in patients with primary infertility who harbor biallelic TLE6 variants.