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Molecular evidence for the participation of the second polar body in dispermic fertilization and its implications

Zhiwen Shi , Lingbo Wang , Yu Xiong , Xiaoling Zhong , Huanqiang Zhao , Bingbing Wu , Chengtao Li , Jinsong Li , Taosheng Huang , Lingqian Wu , Wenhao Zhou , Xiaotian Li , Hongyan Wang

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Vita > Correspondence > DOI: 10.15302/vita.2026.06.0045
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Molecular evidence for the participation of the second polar body in dispermic fertilization and its implications

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Dear Editor,
Accurate identification of the origin of 46, XX/46, XY chimerism — whether arising from classical tetragametic fusion, sesquizygotic twinning, or dispermic fertilization of the second polar body (PB2) — remains technically challenging1,2. Because the haploid oocyte and its PB2 arise from homologous recombination (HR) following DNA replication, they share up to 99.9% sequence identity3. The residual < 0.1% divergence is usually indistinguishable from short-read sequencing errors or the microheterogeneity induced by HR. Consequently, cryptic maternal contributions from the PB2 are easily misattributed to the parthenogenetic endoreplication of a single oocyte. Here, by applying the third-generation long-read sequencing to distinguish maternal haplotypes in a single-molecule resolution, our genetic analysis results strongly suggest that PB2 can participate in chimeric fertilization, providing compelling molecular evidence to distinguish PB2-derived fertilization from parthenogenetic replication.
To demonstrate the possible involvement of PB2 in chimerism, we initially analyzed a rare case of naturally conceived, opposite-sex monochorionic twins who are both chimeric as revealed by the karyotype results. Short tandem repeat (STR) profiling confirmed the parent−child relationship in this chimeric-twin family (Supplementary Table S1). To precisely quantify the maternal genetic contributions, we utilized a high-density single-nucleotide polymorphism (SNP) array (Illumina, OmniZhongHua-8). We specifically focused on informative “candidate SNPs”, defined as genomic loci where the paternal genotype is uniformly homozygous (e.g., ApAp or BpBp) and the maternal genotype is heterozygous (AmBm). In the offspring, any successfully inherited maternal heterozygous alleles at these candidate loci are defined as “offset SNPs”, which uniquely trace the divergence of the maternal genomes. High-density SNP array genotyping identified ~35,000 maternal offset SNPs being inherited by the twins. The ratio of these offset SNPs to total candidate SNPs fluctuated around ~50%, which matched our internal validation controls comprising artificially mixed 1:1 DNA from dizygotic twins (Supplementary Table S2a). This ~50% genomic divergence between maternal contributions confirmed classic tetragametic chimerism4 (derived from two distinct oocytes and two distinct sperm) with no evidence of PB2 involvement (Supplementary Fig. S1).
To specifically investigate the elusive PB2-mediated fertilization mechanism, we subsequently analyzed a 12-year-old naturally conceived proband with 46, XX/46, XY ovotesticular disorder of sex development (DSD). STR-based parentage testing identified 5 informative paternal STR alleles co-presented in the proband’s genome, confirming fertilization by two distinct sperm. In stark contrast, none of the 8 informative maternal STR loci were shared across the proband’s genome, indicating that the maternal genome transmitted to the ovotesticular DSD did not originate from two distinct oocytes (Supplementary Table S3). High-resolution SNP array analysis detected only 147 maternal heterozygous offset SNPs among 123,000 candidate loci in the chimera (Fig. 1a), corresponding to a minuscule ~0.1% maternal sequence divergence in the ovotesticular DSD proband. To evaluate the authenticity of HR or of reading errors, the only two possible causes of ~0.1% maternal sequence divergence, we performed identical arrays on artificial 1:1 DNA mixtures from three pairs of confirmed monozygotic twins. These controls yielded an average of 28 offset SNPs — reflecting systematic array artifacts — suggesting that conventional short-read data alone are incapable of catching longer fragments to reflect the HR over technical noise (Supplementary Table S2b).
To obtain direct molecular evidence, we conducted third-generation Pacific Biosciences (PacBio) long-read sequencing of the proband’s DNA (average depth 120×, read length 15 kb), alongside PCR-free whole-genome short-read sequencing of both parents (50×) to enable precise SNP phasing. Suppose the chimera was derived from both the oocyte and PB2, HR events would have two distinct maternal haplotypes that simultaneously coexisted at certain loci. We interrogated adjacent SNP pairs where the father was homozygous (e.g., AA or BB at locus 1 and CC or DD at locus 2) and the mother was heterozygous (e.g., AB at locus 1 and CD at locus 2) (Fig. 1b). Strikingly, on chromosome 9q34.11 and chromosome 6p21.32, the offspring displayed three distinct haplotypes: one paternal wild-type haplotype and two distinct maternal haplotypes matching those of the mother (Fig. 1c, d). Similar loci were additionally identified at another 11 sites across the entire genome (Supplementary Fig. S2). Crucially, all 13 of these loci were independently validated using short-read PCR-free WGS performed on the patient's sample. The coexistence of dual intact maternal haplotypes within the proband’s genome — despite a global maternal sequence identity of 99.9% — constitutes robust, single-molecule evidence strongly supporting the hypothesis that both the oocyte and the PB2 were independently fertilized by two distinct sperm to form this sex-chimeric embryo. Detailed bioinformatic parameters and exact read-level support, excluding alignment artifacts, are provided in Supplementary information.
To systematically validate the PB2-mediated fertilization model, we evaluated alternative mechanisms capable of generating 46, XX/46, XY chimerism by contrasting the predicted genomic signatures with our data (Supplementary Table S4). Classic tetragametic chimerism involving two distinct oocytes is excluded by the minuscule ~0.1% maternal sequence divergence, which starkly contrasts with the ~50% divergence expected from two independent oocytes. In contrast, sesquizygotic twinning, referred to as a single haploid oocyte fertilized by two sperm via parthenogenesis2, dictates an identical maternal genome (0% divergence) and exactly one maternal haplotype across all lineages. Two distinct maternal haplotypes at multiple HR loci detected by our long-read sequencing unequivocally exclude sesquizygosity, as well as parthenogenetic endoreplication of a single haploid oocyte. Furthermore, the possibility that an XX/XY chimera arises from a diploid zygote via post-zygotic genome-wide endoreplication and subsequent mitotic error directly contradicts our observation of multiple paternal STR alleles. Finally, the observed ~0.1% microheterogeneity rules out potential technical artifacts from cell-free maternal DNA admixture or sample contamination. Such confounding factors would indiscriminately introduce a complete alternative maternal haploid genome, thereby yielding a ~50% maternal offset SNP profile rather than the minimal divergence detected here. Therefore, the currently detected combination of dispermic paternal STRs, ~0.1% maternal SNP divergence, and the coexisting maternal haplotype in the three-haplotype long-read signature strongly supports the PB2-participation model.
As a preliminary and hypothesis-generating exploration of dispermic embryonic development in vivo, we injected two sperm heads into mouse oocytes while simultaneously blocking artificial PB2 extrusion using cytochalasin B (CB) (Supplementary Fig. S3a). Among 697 reconstructed embryos transferred, three pups were successfully born (Supplementary Fig. S3b), and one resulting pup exhibited heterogeneous Y:X ratios indicative of dispermic chimerism (Supplementary Fig. S3c). As a control, natural PB2 extrusion with double sperm injection yielded one chimeric pup from 256 embryos, which can be inferred as a result of spontaneous parthenogenetic duplication coupled with fertilization (Fig. 1e). It is imperative to interpret these animal model results with scientific caution. Given that the retention of the PB2 via CB is not guaranteed in every treated oocyte, it is inherently challenging to definitively attribute the resulting chimeric phenotype solely to the retention and fertilization of the PB2; alternative atypical mechanisms, such as parthenogenetic endoreplication (as seen in the control group), cannot be entirely ruled out. Therefore, while our preliminary animal results conceptually suggest that a retained PB2 may participate in chimeric development, the incomplete penetrance and technical complexities of CB blockade exactly underscore why robust support for this mechanism relies predominantly on the high-resolution, single-molecule genomic evidence presented above. Direct visualization of PB2 retention (e.g., live imaging) and high-resolution molecular analysis of individual blastomeres could likely provide more definitive evidence in future studies.
Identifying cryptic chimerism carries profound implications for clinical diagnostics and forensic parentage testing5,6. Because chimeras formed by same-sex gametes (e.g., two sperm or two oocytes) are often morphologically normal and exhibit identical sex chromosome compositions, conventional karyotyping fails to detect them. We propose a high-sensitivity strategy utilizing multi-tissue STR profiling (e.g., hair follicles, buccal swabs, urine, alongside blood). Because meiotic recombination ensures that each gamete carries a unique STR profile, the detection of ≥ 3 alleles at maternal or paternal loci across divergent tissues serves as a robust indicator of cryptic chimerism, which recently helped correct a misdiagnosed false-negative maternity test in our practice. Furthermore, it was reported that > 80% of reported constitutional XX/XY chimerism cases in the literature occurred following assisted reproductive technology (ART)7. Given this strong association, in vitro manipulation may inadvertently increase the risk of embryonic fusion, highlighting the necessity for targeted cryptic chimerism screening in ART populations.
While the single-molecule haplotype data from the proband are compelling, we explicitly acknowledge the inherent limitation of drawing generalized biological conclusions from a single human case (n = 1). Proposing PB2 fertilization as a natural developmental mechanism represents an extraordinary biological claim that demands cautious interpretation. To further substantiate these findings and determine the broader prevalence of PB2-mediated chimerism, additional evidence — such as retrospective multi-omics analyses of other clinical cases of cryptic chimerism and independent cohort validations — will be essential.
In conclusion, our study leverages long-read sequencing to traverse the blind spots of conventional genomics. The genetic analysis results strongly suggest that PB2 can participate in chimeric fertilization, establishing a rigorous molecular framework for identifying atypical gametogenesis and solving complex reproductive and forensic anomalies.

DATA AVAILABILITY

All data are available from the corresponding author on reasonable request at the Genome Sequence Archive database (https://ngdc.cncb.ac.cn/ with the accession number PRJCA063280).

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RIGHTS & PERMISSIONS

The Author(s) 2026. Published by Higher Education Press. This is an Open Access article distributed under the terms of the CC BY license (https://creativecommons.org/licenses/by/4.0/).

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Shi, Z. et al.  Molecular evidence for the participation of the second polar body in dispermic fertilization and its implications  Vita https://doi.org/10.15302/vita.2026.06.0045 ()
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