Human reproduction begins with gametogenesis. High-quality sperm-oocyte union is followed by degradation of a large amount of maternal mRNA and protein in the oocyte. The embryonic genome is activated and the embryo begins to divide, passing through four-cell, eight-cell up to blastocyst. Failure of any step in this process results in infertility (Sang et al., 2021). The oocyte-to-embryo transition (OTE) process is initiated with the resumption of oocyte meiosis. This OTE process involves the initiation of degradation and translational activation of numerous unstable maternal mRNAs stored in the cytoplasm (Svoboda et al., 2015; Sha et al., 2019). Maternal factors have an important role in the OTE process. If OTE is faulty, even treatment with in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles may make it difficult to obtain normally developed blastocysts.

The subcortical maternal complex (SCMC) is a key maternal factor that is distributed in the oocyte and embryonic cortex. The SCMC has been identified with well-defined components such as peptidylarginine deiminase, type VI (PADI6), maternal antigen that embryos require (MATER), factor located in oocytes permitting embryonic development (FLOPED), transducin-like enhancer of split 6 (TLE6), and KHDC3 (KH domain containing 3), among others(Bebbere et al., 2021). In clinical practice, early embryonic arrest is common and is one of the main reasons for failed IVF/ICSI attempts (Gardner and Lane, 1997). However, the exact cause of this genetic phenotype is not known. Novel mutations in NLRP5, TLE6 and KHDC3L have been reported in the literature to cause preimplantation embryonic arrest in human (Alazami et al., 2015; Cao et al., 2018; Wang et al., 2018; Mu et al., 2019; Lin et al., 2020; Maddirevula et al., 2020; Xu et al., 2020; Sang et al., 2021; Zheng et al., 2021).

Female Padi6 ^−/− mouse is infertile and developmental arrest occurs at the 2-cell stage. The mechanism of 2-cell arrest in Padi6 ^−/− embryos may be due to failure to fully activate the embryonic genome. In Padi6 ^−/− 2-cell embryos, the level of ribosomal components is reduced and de novo protein synthesis is dysregulated (Yurttas et al., 2008). Similarly, early embryonic developmental arrest was also found in patients with PADI6 mutations (Xu et al., 2016). Phosphorylated RNA polymerase II and genes associated with embryonic genome activation were significantly reduced in embryos of patients with PADI6 mutations. This phenotype is consistent with the presence of impaired embryonic genome activation in Padi6 ^−/− mice (Xu et al., 2016). In addition to the early embryonic arrest phenotype, PADI6 mutations in human also result in recurrent hydatidiform moles (Qian et al., 2018; Dong et al., 2022).

In this study, we identified by whole exome sequencing a homozygous variants [NM_207421.4, c.394C>T(p.R132C)] in the middle protein-arginine deiminase (PAD) domain in PADI6 gene.PADI6 mutant embryos have impaired embryonic development and fail to develop beyond the 4-cell stage. The effect of this new mutation on the PADI6 protein was then further confirmed by bioinformatic analysis.

PADI6 (peptidylarginine deiminase, type VI), an important maternal gene, is exceptionally abundantly expressed in meiotic maturation of oocytes (Figures 1D, E) (Xiong et al., 2022). By re-analyzing single-cell transcriptome sequencing data during human, marmoset and mouse pre-implantation embryonic development, we found that PADI6 expression pattern was conserved across species (Figure 1C) (Boroviak et al., 2018). PADI6 is predominantly distributed in the subcortical region in oocyte and zygote, and is a major member of the subcortical maternal complex (SCMC) (Bebbere et al., 2021). The formation of specific fibrous structures, cytoplasmic lattices (CPLs), in the oocyte is inseparable from PADI6 (Esposito et al., 2007). Oocyte cytoplasmic lattices (CPLs) presumably provide ribosomal reservoirs for early embryos. In Padi6-KO oocytes, the ribosomes rRNAs associated with CPLs are significantly reduced. PADI6 can interact with MSY2 to preserve ribosomes and mRNAs in an inactive form. A simple but powerful mechanism is provided to ensure the successful activation of the embryonic genome by simultaneous release of mRNA and ribosomes (Yu et al., 2001; Yu et al., 2002; Liu et al., 2017).

In our findings, it was observed that approximately 40% of the immature oocytes obtained from the patient during the three retrieval cycles failed to reach maturity and subsequently degenerated. This observation is consistent with the role of PADI6 as a major component of the subcortical maternal complex (SCMC) in regulating oocyte meiosis. While there have been studies on the impact of PADI6 on oocyte meiosis in mice, further exploration is required to understand its role in human oocytes, given the limited availability of human samples for research purposes.

To date, a total of 14 mutant loci have been reported for the human PADI6 gene. Interestingly, twelve of these loci are found within the PAD_ C-terminal domain (12/14), while the novel loci we have newly identified are distinct, relatively rare, and primarily situated in the PAD_ Middle domain. These newly identified loci, being in close proximity to the N-terminal region of the PADI6 protein, have the potential to exert earlier effects on the protein’s functionality. Specifically, the mutation at the identified locus leads to a substitution of arginine with cysteine at position 132 in the PADI6 protein. The fundamental difference between cysteine and arginine lies in their respective R-groups. Arginine possesses a basic R-group, while cysteine exhibits a polar and neutral R-group. Cysteine is nearly insoluble in water, whereas arginine is readily soluble. Furthermore, our structural prediction analysis indicates that this amino acid mutation further impacts the spatial conformation of the protein.

Patients with PADI6 mutation showed early embryonic arrest, zygotic cleavage failure, recurrent hydatidiform moles and other different phenotypes (Alazami et al., 2015; Wang et al., 2018; Mu et al., 2019; Lin et al., 2020; Maddirevula et al., 2020; Xu et al., 2020; Sang et al., 2021; Zheng et al., 2021; Dong et al., 2022). The different phenotypes of patients with PADI6 mutation are presumed to be related to the location of the PADI6 mutation (Qian et al., 2018). If one aims to rescue this phenotype, it could be attempted to repair the mutation using base editing gene-editing technology (McAuley et al., 2023). Additionally, supplementation of PADI6-mRNA could be explored as a potential rescue strategy. However, it is important to consider ethical considerations and engage in further discussions regarding the feasibility and implications of these ideas.

Mutations in two other members of the SCMC complex, NLRP7 and KHDC3L, contribute to 60% of the clinically recurrent hydatidiform moles (Murdoch et al., 2006; Parry et al., 2011). Immunofluorescence showed co-localization of PADI6 and NLRP7 (Qian et al., 2018). Although most mutated PADI6 proteins cause early embryonic developmental disorders, two variants (c.1793A>G, p.(Asn598Ser) and c.2045G > A, p.(Arg682Gln) caused recurrent hydatidiform moles. This result suggests that part of the function of PADI6 is still in play and that there may be some interaction between PADI6 and NLRP7 (Qian et al., 2018). In addition, in hydatidiform moles, PADI6 can regulate trophoblast cell migration-invasion through Hippo/YAP1 Pathway(Huang et al., 2021).

During Oocyte-to-Embryo transition (OTE) process, mouse embryonic genome activation starts from 2-cell stage and in humans from 4-cell stage (Eckersley-Maslin et al., 2018). In Padi6_KO mouse, two-cell embryos present impaired de novo protein synthesis and impaired embryonic genomic activation (Yurttas et al., 2008). Similarly, the majority of human embryos with PADI6 mutations are arrest in four-cells, also suggesting abnormalities in human embryonic genome activation. It is interesting to follow whether new methods can be used to rescue the process of embryonic genome activation malfunction.

Although we observed the significant role of PADI6^R132C mutations in early embryonic development, this study does have some limitations. Firstly, the sample size is relatively small, and it would be necessary to identify the same mutation site with similar phenotypes in multiple reproductive centers. Secondly, due to the inherent limitations of the exome sequencing analysis process and the continuous refinement of the pathogenic variants database, our study may have missed the discovery of some other pathogenic variants in different genes (Richards et al., 2015; Kalia et al., 2017).

To conclude, our study identified a new mutated site of PADI6 especially at the PAD_middle domain, which enriches the understanding of PADI6 mutations causing early embryonic division arrest. Our findings improve the understanding of the genetic basis of female infertility.