Imprinted genes are a peculiar subset of the genome. Most genes are expressed from both the maternal and paternal copies, but imprinted genes carry chemical tags laid down in the egg or sperm that silence one copy entirely, leaving only the maternal or paternal version active. In the placenta, these parent-of-origin effects are thought to reflect evolutionary conflict: paternal genes tend to push for greater extraction of maternal resources, while maternal genes restrain fetal demands to preserve the mother’s ability to sustain current and future pregnancies. Phlda2, formally known as Pleckstrin Homology-Like Domain Family A Member 2, is one of the maternal army’s foot soldiers, normally expressed only from the chromosome inherited from the mother.

Researchers at Cardiff University, led by R. M. John together with colleagues including A. R. Isles, had previously shown that elevated Phlda2 expression, a state mimicking loss of imprinting in which the gene’s dosage is effectively doubled, produces placentas that secrete fewer hormones, fetuses that grow poorly, and mothers that neglect their pups after birth. Their earlier work on the paternally expressed gene Peg3 had already demonstrated that imprinted genes can act as master regulators of placental endocrine lineages and that disrupting them can produce sexually dimorphic outcomes, with males suffering more. The natural question was whether a maternally expressed gene would show the same pattern, or whether the sexes would flip.

To find out, the team crossed wildtype C57BL/6 female mice with transgenic males carrying an extra bacterial artificial chromosome copy of Phlda2, generating embryos that overexpressed the gene and thus modelled loss of imprinting. They then dissected the placenta at embryonic day 14.5, a critical window when the placenta’s endocrine machinery is being established, and collected fetal and placental weights at embryonic day 18.5, near the end of gestation.

The team used RNAscope, a high-resolution in situ hybridization technique, combined with classical histology to map the placenta’s specialized cell populations. The mouse placenta is organized into two major compartments. The labyrinth zone handles nutrient and gas exchange, while the junctional zone is the endocrine heart of the organ, packed with hormone-producing cells including the spongiotrophoblast layer, glycogen cells, and parietal trophoblast giant cells. The spongiotrophoblast in particular churns out a vast repertoire of prolactin-related hormones and pregnancy-specific glycoproteins that remodel the mother’s physiology, directing blood flow, nutrient mobilization, and even priming her brain for maternal care.

What the researchers saw was unambiguous. In placentas carrying the Phlda2 overexpression, the junctional zone lost a significant number of spongiotrophoblast cells, and the depletion was substantially more severe in female placentas than in male ones. Glycogen cells and parietal trophoblast giant cells were also reduced in number, but those losses were even-handed between the sexes. The sexual dimorphism was confined specifically to the spongiotrophoblast compartment, the very cell population responsible for the placenta’s most powerful endocrine output.

Molecular measurements backed up the cellular picture. Using reverse-transcription quantitative PCR and bulk RNA sequencing, the team found that genes encoding placental hormones normally produced by the spongiotrophoblast were downregulated in the mutant placentas, and once again the reduction was biased toward females. Gene set enrichment analysis reinforced the conclusion: the female mutant placenta showed a more profound dampening of its hormone-producing program than the male, despite both sexes carrying the identical genetic modification.

The most consequential result, however, appeared in the growth curves. At embryonic day 18.5, late in gestation, female fetuses with elevated Phlda2 expression were significantly growth restricted compared with their wildtype female littermates. Male fetuses carrying the same transgene showed no comparable reduction in weight. In other words, the genetic perturbation narrowed the female placenta’s endocrine signalling capacity, and the female fetus paid the price by growing more slowly, while the male fetus maintained its growth trajectory despite the same molecular insult.

The authors interpret this asymmetry through the lens of evolutionary theory about parental resource allocation. Phlda2 is known to be responsive to maternal adversity: when the mother experiences nutritional deficiency or other forms of prenatal stress, placental Phlda2 expression rises, and the resulting restraint on hormone production lowers the fetus’s demands on her body. The new findings suggest that this demand-dampening system is not symmetrical. Female fetuses appear to reduce their claims on the mother when Phlda2 signalling rises, effectively shrinking their own growth to spare maternal reserves. Male fetuses, by contrast, hold their ground, continuing to extract resources even under adverse conditions. Such sex-specific strategies could reflect the different reproductive payoffs that sons and daughters offer under variable environmental conditions, with parent-of-origin imprinting serving as the molecular lever that mediates the negotiation.

The translational implications are hard to ignore. In human pregnancies, elevated placental PHLDA2 has repeatedly been associated with fetal growth restriction and low birthweight, yet these clinical studies have generally not accounted for the sex of the infant. If, as the mouse data suggest, the gene’s effects on growth are sex-specific, then pooling male and female pregnancies may have obscured important patterns and muddied the interpretation of biomarker studies. The Cardiff team argues that fetal sex deserves routine consideration in research on imprinted genes and pregnancy complications, from intrauterine growth restriction to the long-term metabolic and behavioural consequences of prenatal adversity. The work, funded by the Biotechnology and Biological Sciences Research Council, adds Phlda2 to a growing list of imprinted genes, alongside Peg3, whose disruption produces sex-dimorphic placental and offspring phenotypes, and it strengthens the broader hypothesis that the placenta is not merely a passive conduit for nutrients but an active, sexually differentiated endocrine organ that negotiates the terms of maternal investment on the fetus’s behalf.

Subject of Research: Sex-specific effects of loss-of-imprinting of the maternally expressed gene Phlda2 on placental endocrine development and fetal growth in mice

Article Title: Sex-specific consequences of loss-of-imprinting of the maternally expressed gene Pleckstrin Homology-Like Domain Family A Member 2 (Phlda2) on placental development and fetal growth

Article References: Chibnall, A., Harrison, D. J., Lysikova, E., Malinoshevska, M., Stoddart, A., Perry, I. A., Christofides, S., Isles, A. R., & John, R. M. (2026). Sex-specific consequences of loss-of-imprinting of the maternally expressed gene Pleckstrin Homology-Like Domain Family A Member 2 (Phlda2) on placental development and fetal growth. Biology of Sex Differences. https://doi.org/10.1186/s13293-026-00972-z

Image Credits: AI Generated

DOI: 10.1186/s13293-026-00972-z

Keywords: genomic imprinting, Phlda2, placenta, placental hormones, fetal growth restriction, sexual dimorphism, spongiotrophoblast, junctional zone, prenatal adversity, parent-of-origin effects, Biology of Sex Differences, Cardiff University

Central precocious puberty, or CPP, occurs when the hypothalamic-pituitary-gonadal axis is activated prematurely, leading to early breast development in girls and virilization in boys before the customary age. The condition matters clinically for more than cosmetic reasons: early activation of the axis accelerates skeletal maturation, can compromise final adult height, and may carry psychological and social consequences for children whose bodies mature years ahead of their peers. Over the past decade, loss-of-function mutations in MKRN3, an imprinted gene located in the Prader–Willi syndrome region of chromosome 15, have emerged as one of the most common genetic causes of familial CPP. Yet the same genomic neighborhood presents a paradox: when MKRN3 is deleted as part of the larger deletion that causes Prader–Willi syndrome, patients typically develop hypogonadism and delayed or incomplete puberty rather than precocious puberty. Because MKRN3 is paternally expressed and maternally imprinted, its loss through mutation removes an inhibitory brake on gonadotropin-releasing hormone secretion, hastening puberty, whereas the complex multi-gene deletion in Prader–Willi syndrome disrupts hypothalamic development in ways that blunt reproductive function.

The mechanistic basis for these opposing reproductive phenotypes has remained largely unclear. To address this, the team led by Yu-Yu Jin, Xiao Wang, Lin Yang, Jian Mu, and Fei-Hong Luo performed whole-exome sequencing on 98 Chinese children with central precocious puberty. They then carried out a systematic review of previously reported MKRN3 pathogenic and likely pathogenic variants to summarize genotype–phenotype correlations. Finally, they performed genome-wide DNA methylation profiling in CPP patients carrying the MKRN3 pathogenic variant and compared their methylation patterns with those of patients with Prader–Willi syndrome, patients with idiopathic central precocious puberty, and healthy controls. This three-pronged design allowed the investigators to move from variant discovery to clinical correlation to epigenetic characterization within a single cohort framework.

The sequencing effort identified a pathogenic frameshift variant, designated c.476dupC (p.Ala159fs*15), which introduces a duplication of a single cytosine base that shifts the reading frame and generates a premature stop codon. Notably, this is the first frameshift mutation reported within the inter-C3H1 hotspot region of MKRN3 in an Asian cohort, a finding that further confirms the functional significance of this segment of the protein. The MKRN3 protein carries multiple C3H-type zinc finger domains and a RING finger domain, and pathogenic variants have previously clustered in certain regions, making variant location an important clue to functional impact. The RING finger domain confers E3 ubiquitin ligase activity, meaning the protein tags other cellular proteins for degradation or modification, while the zinc finger domains mediate binding to RNA and protein targets. Frameshift variants that truncate the protein before these domains are complete are therefore expected to eliminate function almost entirely.

The genotype–phenotype analysis revealed a striking correlation between variant severity and clinical presentation. Patients carrying severe MKRN3 variants, such as frameshift or nonsense mutations predicted to truncate the protein, exhibited significantly earlier pubertal onset than those with missense mutations, beginning at a mean age of 5.80 years compared with 7.50 years (P = 0.029). The severity of the hormonal activation also differed: children with severe variants showed significantly higher luteinizing hormone peak levels during gonadotropin-releasing hormone stimulation testing, at 34.55 versus 11.00 IU/L (P = 0.047). These findings suggest that the degree of residual MKRN3 function, or the manner in which a truncated protein is produced and degraded, influences how vigorously the reproductive axis is disinhibited. In practical terms, the two-and-a-half-year difference in mean onset age between the variant classes is clinically meaningful, since earlier activation leaves a longer window of compromised growth potential and often requires more aggressive intervention with gonadotropin-releasing hormone agonist therapy to pause pubertal progression.

The most novel component of the study was the methylation analysis. DNA methylation, the addition of methyl groups to cytosine bases across the genome, is a key epigenetic mechanism that regulates gene expression without altering the underlying DNA sequence. Epigenetic regulation is known to be central to the timing of puberty: the reactivation of gonadotropin-releasing hormone secretion at the end of childhood is thought to depend on carefully orchestrated changes in chromatin state within hypothalamic neurons, and animal studies have shown that disrupting epigenetic machinery can shift pubertal timing substantially. MKRN3 itself has been implicated in epigenetic switching through its ubiquitination of MBD3, a component of chromatin-remodeling complexes. Given that MKRN3 sits in an imprinted genomic domain governed by methylation marks, the investigators asked whether MKRN3-associated CPP carries a distinct methylation signature.

Surprisingly, the analysis revealed no differences in methylation at the MKRN3 locus itself between the study groups. However, the comparison between MKRN3-CPP patients and Prader–Willi syndrome patients identified 18,609 differentially methylated positions across the genome, indicating that the two conditions, despite sharing the same chromosomal region, are epigenetically worlds apart. Key findings included pronounced hypermethylation of IGSF10 (a difference in methylation beta values of 0.37), ZC3H18 (0.27), SH3RF3 (0.36), and PTH1R (0.28) in the MKRN3-CPP group relative to PWS, alongside hypomethylation of MAGEL2 (−0.19) and PTPA (−0.23), where the delta-beta value represents the difference in DNA methylation beta values between the groups.

Several of these differentially methylated genes have plausible links to reproductive biology. IGSF10 is well established as a gene involved in the migration of gonadotropin-releasing hormone neurons during embryonic development, and mutations in it cause delayed puberty, making its epigenetic dysregulation particularly relevant to pubertal timing disorders. PTH1R, the parathyroid hormone 1 receptor, signals through the cAMP/protein kinase A pathway and is best known for its actions on bone. MAGEL2, which lies within the Prader–Willi syndrome critical region, is required for the production of secretory granules and neuropeptides in the hypothalamus, and its loss contributes to the impaired melanocortin pathway function characteristic of PWS. PTPA, a protein phosphatase 2A activator, has been shown to physically associate with the C-terminus of GPR54, the kisspeptin receptor that is a master gatekeeper of puberty, and has roles in osteoblast differentiation. SH3RF3 has been connected to the LIN28B pathway, which itself is among the genetic loci associated with age at menarche in large genome-wide association studies. Taken together, these targets trace a coherent map of the neuroendocrine and developmental circuits that determine when and how forcefully the reproductive axis awakens.

The authors propose that this pattern of differential methylation in downstream neuroendocrine pathways could potentially explain how divergent pubertal phenotypes arise in the setting of MKRN3 deficiency. In isolated MKRN3 mutations, the loss of the gene’s inhibitory action in kisspeptin-expressing neurons, together with its known role in modulating the stability and translation of GNRH1 messenger RNA through ubiquitination of poly(A)-binding proteins, removes the pubertal brake. In Prader–Willi syndrome, the methylation landscape of genes such as MAGEL2, a paternally expressed neighbor of MKRN3, is altered in the opposite direction, and the broader deletion disrupts neuropeptide production and hypothalamic circuitry, leading to insufficient rather than excessive gonadotropin secretion. The methylation data thus suggest that the clinical contrast between the two conditions may be inscribed in the epigenetic programming of shared downstream pathways rather than in MKRN3 itself. If confirmed, this would represent a conceptual shift: the direction of the pubertal phenotype would be determined not by the presence or absence of a single gene, but by the constellation of epigenetic states across the network it regulates.

The study has limitations worth noting. The methylation profiling was performed in peripheral blood samples, which may not fully reflect methylation states in the hypothalamic neurons that actually govern puberty, a common constraint in human neuroendocrine epigenetics. Blood-based methylation signatures can be confounded by differences in cell-type composition between patient groups, and tissue specificity remains a persistent challenge for the field. The cohort of MKRN3-mutant patients available for methylation comparison was necessarily small given the rarity of the condition, and the cross-sectional design cannot establish whether the methylation differences are causes or consequences of the divergent pubertal phenotypes. The researchers also note that the data were processed using beta-mixture quantile normalization to correct probe design bias in Illumina methylation arrays, and variant interpretation followed the American College of Medical Genetics and Genomics standards, but functional validation of the identified methylation changes will require further work, ideally in neuronal models or longitudinal cohorts.

Nevertheless, the implications are considerable. For clinicians, the genotype–phenotype correlation reinforces the value of molecular testing in children with central precocious puberty, particularly those with a family history or very early onset, and suggests that children with truncating MKRN3 variants may warrant particularly close attention to the pace of pubertal progression. Because MKRN3-related CPP is inherited in a pattern determined by the imprinting of the gene, molecular diagnosis also carries implications for genetic counseling of affected families, including the identification of relatives who carry the variant but may be asymptomatic. For researchers, the identification of an epigenetic signature distinguishing MKRN3-CPP from Prader–Willi syndrome opens a line of inquiry into how ubiquitin ligases and chromatin modifiers in the imprinted 15q11-q13 domain jointly sculpt the timing of human puberty. The work was supported by the National Natural Science Foundation of China and the Science and Technology Commission of Shanghai Municipality, and the data are available from the corresponding author on request.

More broadly, the study adds to a growing body of evidence that epigenetic marks can serve as interpretable intermediates between genetic lesions and complex developmental phenotypes. Just as methylation scores are being explored as predictors of metabolic disease risk, methylation profiling of pubertal disorders may eventually help stratify patients, refine prognosis, and illuminate therapeutic targets in the neuroendocrine circuitry that launches human reproduction. In an era when sequencing routinely uncovers rare variants of uncertain significance, studies that connect variant class, clinical severity, and epigenetic state provide exactly the kind of layered evidence needed to translate genomic findings into meaningful care for children whose puberty arrives, or fails to arrive, at the wrong time.