A growing number of women have pregnancies at an advanced maternal age (AMA), typically defined as 35 years or older (Cooke and andDavidge, 2019), which is associated with numerous pregnancy complications (Marozio et al., 2019; Norwitz, 2007), such as preeclampsia (PE), severe intrauterine growth restriction (IUGR), miscarriage, and stillbirth. Accumulative evidence suggests that these adverse pregnancy outcomes are implicated in aberrant placental aging of AMA (Xiong et al., 2021; Chen et al., 2021). Placenta aging is characterized with progressive decrease of function at the cellular, tissue, and organ level, contributing to a reduced adaptability to stress and an increased vulnerability to disease and mortality (Fedarko, 2011).

Pregnancy is a state of increased oxidative stress (Belo et al., 2004;Burton and and Jauniaux, 2004), which particularly focuses in the human placenta supra physiologically. The AMA placentas show more fragile response to oxidative stress than those young (Liao et al., 2019; Pomatto and and Davies, 2018). As a common inducer of aging, oxidative stress could lead to a series of events, including damnification to cellular lipids, proteins, DNA and ultimately result in aging (Hernandez-Segura et al., 2017). Oxidative stress of the gestation is of great correlation with the pathomechanism of adverse pregnancy outcomes. More precisely, placental trophoblasts from pregnancies complicated by abortion, PE and IUGR exhibit higher levels of oxidative stress and aging biomarkers than those from normal pregnancies (Huppertz, 2008; Sultana et al., 2017). And AMA is a high-risk factor of these complications.

Aging is an aftereffect of the interaction between environmental and genetic factors, and some genes play a vital role in regulating aging, including Yes-associated protein (YAP), which was originally identified as a key transcription factor in Hippo pathway of controlling organ size, tissue regeneration, cell fate decisions (Russell and Camargo, 2022). In recent years, YAP as the main effector of cellular mechanical signals, its attenuated function was reported linked to the decline of structure and function in aging tissues and organs (Sladitschek-Martens et al., 2022), implying that YAP may participate in regulation of senescence and resisting oxidative damage. However, the role of YAP in placenta aging and oxidative damage remained unknown in the AMA pregnancies, while it was reported the reduction of YAP in Series of pregnancy complications (Liao et al., 2022). In the present study, we aimed to explore the relationship among aging, oxidative damage and YAP in placenta trophoblast, providing valuable directions for healthcare during AMA pregnancy.

Growing number of evidence has shown that pregnancy complications are inclined to happen in AMA pregnancies compared with the young. These adverse outcomes of AMA pregnancy were connected to placenta of aging (Woods et al., 2017; Li et al., 2017). Cellular senescence is an inevitable experience in the life cycle of all living organisms, which results from progressive DNA damage. An obvious characteristic of aging is gradual decline in physiological functions occurring at the cellular, tissue, and organic level but more susceptible to disease and mortality, which is compatible with a high rate of pregnancy complications in AMA pregnancy. In senescent cells and tissues, the senescence-associated β-galactosidase (SA-β-Gal) has increased activity and is the most common and representative biomarker. In addition, increased levels of p53, p21 have been also involved with senescence and considered to be important biomarkers. The present study revealed these biomarkers above were of higher level in the human AMA placenta, which is consistent with previous results (Xiong et al., 2021; Chen et al., 2021). That was to say, AMA placenta has an apparent senescent phenotype.

p53, p21, and p16, which are related to the cell cycle, all express in placental trophoblast cells and villous stromal cells. p21 can act as a downstream of p53, and they consistently mediate irreversible DNA damage, contributing to the stability and maintenance of cellular senescence, potentially without increasing p16 (De Oliveira, 2006). During the physiological aging of placenta, gradual rise in p53, p21, and p16 (Cindrova-Davies et al., 2018) suggested that they may be necessary for the maturation of placenta. These senescence markers may facilitate the secretion of senescence-associated secretory phenotypes (SASP), promoting the initiation of labor. Unlike the programmed senescence characteristics observed in embryos, abnormal senescence in placenta shared common features with DNA damage-induced senescence and exhibited coordinated activation of the p53-p21 regulatory pathway (Chuprin et al., 2013). In placenta with PE or post-term pregnancy, premature increase in p53 and p21 may lead to placental dysfunction, resulting in adverse pregnancy outcomes (Cox and Redman, 2017; Sultana et al., 2018), but in these PE placentas, p16 showed no statistically significant increase (Cox and Redman, 2017), which was consistent with our observations in AMA placenta, suggesting that pathological placental senescence may be induced in a p16-independent manner. In fact, cellular senescence can be induced in a p53-or p16-independent way (De Oliveira, 2006; Ho et al., 2011). However, it remains plausible that p16 accumulates swiftly during placental senescence, maintaining elevated levels in late-gestation placenta, potentially rendering rise in p16 less discernible in pathological senescence. p53, p21, and p16 occupy pivotal positions and serve crucial functions within placental tissue. They collectively sustain normal proliferation, differentiation, and apoptosis processes in placental cells through intricate regulatory frameworks, guaranteeing optimal placental development and functionality.

YAP, previously known as the chief effector of cellular mechanosignalling, was reported to be involved in regulation aging independent of Hippo pathway. Our results exhibited reduction of YAP and enhanced DNA damage in the AMA placenta, as was observed in the skin and blood vessels of aged adults in other study. Prior to our study, YAP has been reported to decline in those placenta trophoblasts from pregnancies complicated by PE and IUGR (Liao et al., 2022). Not surprisingly, PE and IUGR placentas also showed a senescent phenotype. These facts implied that YAP expression possessed critical relevance in the placenta aging. DNA damage is a commonality during the development of the abnormal senescence phenotype (Lopez-Otin et al., 2023), which is determined by either endogenous or exogenous factors. Among the endogenous causes, oxidative stress is the dominant reason of injury. It has been realized that with increasing age, the accumulation of oxidized substances in the cells of the body tissue increases (Bruno et al., 2017; Sasaki et al., 2019), and the cells will suffer from oxidative damage. With these circumstances, there is apparent relationship among aging, YAP and oxidative damage. In fact, YAP transcription could be activated by nuclear (factor erythroid 2)-like 2 (NRF-2) for antioxidant defense and resisting senescence (Wu et al., 2013). Yet, the understanding of this relationship in placenta trophoblast, particularly in the context of AMA, is very limited.

As was reported in previous studies, oxidative stress contributed the senescence phenotype in species of cells (Parrinello et al., 2003; Chen et al., 2004), with no exception in HTR8/SVneo cell through our study. DNA damage induced by oxidative stress activated tumor suppressor protein p53 and its downstream p21 before SA-β-Gal staining getting positive. It was nothing strange that p53, along with p21, as important regulatory proteins of the cell cycle (Campisi and d'Adda di Fagagna, 2007; Coppe et al., 2008), responsed rapidly as self-protective mechanism once cells suffered adverse stimulation, to prevent the continued delivery of damaged genetic material, so their response preceded other senescence markers and persisted in senescent cells. Once activated, p53 exerts its functions in stimulating the transcription of apoptosis-related genes, thereby activating the apoptotic cascade, or activating p21 transcription to inhibit cells at G1 phase and repairing DNA damage (Ljungman, 2000). p16 can maintain cell cycle homeostasis and prevent hyper proliferation as placenta developed. It mediates senescence via the retinoblastoma (Rb) pathway by inhibiting the activity of cyclin-dependent kinases, ultimately resulting in G1 cell cycle arrest (Kuilman et al., 2010). As cells encounter stimulation, they may undergo senescence by activating either the p53-p21 or p16 pathway. If senescence is triggered by the activation of the p53-p21 pathway, cells had the potential to re-enter cell cycle once p53 inhibition lifted. Conversely, cells that undergo senescence exclusively through the p16-pRB pathway were unable to resume proliferation, even after inhibition of p53, pRB, or p16 (Beausejour et al., 2003). In this experiment, p16 did not exhibit a clear trend of change, which may be related to the stimulation and the duration of H₂O₂ exposure.

Interestingly, YAP of HTR8/SVneo cells showed a conspicuous decline soon after the oxidation challenge. Since YAP promoted cell proliferation, it was understandable that it attenuated as the correspondence to harmful stimulation, thus blocking the transmission of impaired genetic material. Our findings were consistent with a report that the oxidative stress inhibited YAP and downregulated eIF2αP-ATF4 signaling, resulting cell senescence and death in fibrosarcoma cell (Wang et al., 2024; Wu et al., 2015), implying that oxidative stress was the hazards factor of YAP decline. Besides, we found that the intracellular reactive oxidative species (ROS) (Supplementary Figure S6) and cell apoptosis (Supplementary Figure S7) existed even several days after the H₂O₂ attack, which illustrated the harmful effect would not clear away as the harmful strike vanished, highlighting the importance of avoiding harmful stimuli during pregnancy.

To further investigate the effects of YAP, we modulated YAP expression in HTR8/SVneo cells, which were originally obtained from a human first-trimester placenta and immortalized through gene modification, and found that YAP deficiency promoted premature aging, as reflected by the positive SA-β-Gal staining, along with improved expression of p53, p21 and p16. It happened that there was a similar case that depletion of YAP accelerated the premature aging in nucleus pulposus cell through stimulation of p53/p21, and lysosomal activity (Xie et al., 2013). These facts prompted that DNA damage likely occurred inside the YAP-knockdown cells since senescence resulted from DNA damage. Indeed, we detected the 8-OHdG in these YAP-deficiency HTR8/SVneo cells, the enhanced fluorescence intensity of 8-OHdG implying the improvement of DNA oxidative damage. 8-OHdG is one of the predominant forms of oxidative DNA lesions, which often gets significantly higher in those placentas of pregnancy complication (Takagi et al., 2004; Fujimaki et al., 2011; Kimura et al., 2013), including IUGR and PE. Furthermore, YAP was reported to decline in these complications (Liao et al., 2022), which may be a reason why 8-OHdG rises in these complications, but it needed further research for a more reliable conclusion.

In this study, we found that sh-YAP HTR8/SVneo cells exhibit an impairment in invasion ability, and it has already been well established that trophoblast invasiveness is critical in spiral artery remodeling (Lyall et al., 2013), which is of particular importance for normal placentation. In agreed with this result, the decreased invasion ability of low-YAP trophoblasts is involved in the pathophysiology of preeclampsia. Similar conclusions have been confirmed in another studies, indicating that reduced YAP levels may affect placental development by regulating trophoblast invasion and apoptosis, promoting pregnancy complications (Sun et al., 2018). Overexpression of YAP enhances the invasion of BeWo cells, HTR-8/SVneo cells, and JAR cells (Liu et al., 2020), which involves the interplay among Notch and WNT signaling pathways and YAP (Lin et al., 2023). Additionally, YAP is also regulated by microRNAs. For instance, miR-326 has been documented to target PAX8, leading to inhibition of YAP/TAZ-mediated transcriptional activity and consequently suppressing trophoblast proliferation, invasion, and migration (Zang et al., 2022). These facts distinctly suggested that besides for its role in regulating aging, YAP also positively regulates trophoblast biological functions.

It was deeply interesting that whether addition of YAP could attenuate DNA damage and ameliorate the senescence phenotype. In our study, the extra supplement of YAP at least partly compromised the senescence of HTR8/SVneo cells after oxidative stress strike, presented by the diminution in SA-β-Gal staining and decline of p53 expression. In fact, it has been reported that activation of YAP could promote nucleotide metabolism and decrease senescent phenotype (Santinon et al., 2018). Accordingly, 8-OHdG in the cells diminished, with the meaning of improvement of DNA oxidative damage, which implied that YAP played an essential role in antioxidant defense. In correspondence with our results, some antioxidant genes, containing MnSOD and catalase, can be upregulated by YAP-mediated FOXO1 activation in cardiomyocytes (Shao et al., 2014). We observed that p21 did not behave similarly as p53, which may be because of other involved p21 regulation factors such as growth factors, cytokines, or glucocorticoids (Londero et al., 2016). The specific mechanism that YAP ameliorated the DNA oxidative damage and senescence was still unclear, but at least it did not work by depressing ROS (Supplementary Figure S8).

YAP occupies a pivotal position in numerous biological processes within organisms, including but not limited to immune regulation, cell stemness and organ size control. To explore the underlying mechanisms by which YAP deficiency induces placental trophoblast senescence, RNA sequencing was performed. We conducted GSEA analysis on our RNA-seq data. The results revealed numerous DEGs in HTR8/SVneo cells that are potentially regulated by YAP and multiple DNA damage related pathways were enriched, including the DNA damage telomere stress induced senescence, DNA double-strand break pathways and inhibition of DNA recombination at telomere. Notably, many of these DEGs are related to histone expression, which are components of nucleosomes and play crucial roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability (Liu et al., 2023). This implies that YAP participants in maintaining histone function, stability of DNA and chromosomal. In fact, histones and their post-translational modifications can regulate cellular senescence. Phosphorylated H3T11 histone alleviates vascular endothelial cell senescence, whereas lactylated H4 histone accelerates smooth muscle cell senescence (Wu et al., 2023; Li et al., 2024; Dubey et al., 2024). It is noteworthy that DNA damage serves as a common pathway in stress-induced senescence (Lopez-Otin et al., 2023). And several signaling pathways related to DNA damage-induced senescence were enriched in our RNA-seq analysis. Therefore, YAP potentially modulates the damage response of genetic material through its regulation of histones, ultimately triggering senescence phenotypes. However, further research is required to fully comprehend the regulatory interactions among YAP, histones, and DNA damage.

As essential transcriptional co-activator, YAP transcends mere regulation of placental senescence. The intricate equilibrium between the proliferation and differentiation of trophoblast is indispensable for placental development (Xiao et al., 2020). In the early stages of human placenta, the YAP-TEAD4 complex holds a central position in activating genes linked to cell cycle and stemness (Lin et al., 2023). Simultaneously, they suppress genes associated with cell fusion, facilitating the expansion of cytotrophoblasts (CTB) (Meinhardt et al., 2020). YAP functions as key regulator of cell proliferation, survival, placental differentiation, and migration. Although not mention the role of YAP in regulating trophoblast senescence in above studies, it is not difficult to find that once YAP is impaired, trophoblast cells exhibit typical characteristics of senescence, such as impaired cell proliferation, decreased cellular stemness, and compromised invasive capability (cell cycle arrest, weakened cellular stemness, and functional decline). So these functions of YAP are compatible with its ability in regulating senescence.

Besides, YAP take important part in preserving nuclear envelope integrity, partly by controlling the transcription of lamin B1 and ACTR2 directly (Sladitschek-Martens et al., 2022), which is indispensable for the construction of peri-nuclear actin cap (Vergnes et al., 2004). The actin cap surrounds the top surface of the nucleus, keeping the smooth expansion of the nuclear envelope and avoiding its distortion (Khatau et al., 2009; Kim et al., 2017). The integrity of nuclear envelope is crucial for protecting DNA from damage (Perez-Hernandez et al., 2022). In other words, impaired YAP disrupts the integrity of the nuclear envelope, rendering the DNA within the nucleus more vulnerable to oxidative damage from ROS. On the other hand, YAP may help resist oxidative stress through regulating antioxidant genes (Wu et al., 2013). Downregulation of YAP may lead to decreased synthesis of antioxidant enzymes, resulting in weakened ROS clearance and enhanced oxidative damage (Yeung et al., 2019). Thus, we hypothesize that the decline in YAP may be the cause of increased oxidative damage in AMA pregnancies. Other reports have referred to the possibility that lack of YAP may lead to adverse pregnancy outcomes by increasing oxidative damage. Bisphenol A (BPA) exposure during pregnancy would diminish YAP in trophoblast, causing damage to these cells and ultimately contributing to fetal growth restriction (Sun et al., 2024). And maternal vitamin D deficiency was associated with placental hypoplasia and intrauterine growth restriction, which may also be linked to reduced YAP levels (Wang et al., 2022). Consequently, pregnant women should exercise caution to avoid exposure to harmful substances like BPA and ensure adequate vitamin D intake to maintain optimal YAP levels, thereby lowering the risk of potential adverse pregnancy outcomes. This was particularly important for AMA pregnancy.

In this study, a significant senescence phenotype in term placentas from AMA pregnancy was accompanied by decreased YAP. In other study, YAP was found declining in skin and blood vessels with age (Sladitschek-Martens et al., 2022). As the placenta develops and matures, it is reasonable to assume that the importance of YAP’s role in maintaining stemness and promoting invasion diminish, resulting in its decrease. Concurrently, other genes that promote cell fusion and senescence become activated. That is to say, the decline in YAP may be a consequence of placental senescence. But the relationship between YAP and placenta senescence is seriously complex, as it can be caused by various factors, including maternal age, health status, lifestyle, and underlying diseases.

Nevertheless, our study is not without limitations. The HTR8/SVneo cell line, derived from first-trimester human placenta and immortalized with Simian Virus 40 large T antigen cDNA. Though it is used in studies of extravillous trophoblast (EVT) (Su et al., 2019) and part of trophoblast senescence (Xiong et al., 2021), it does not fully share the characteristics of all primary trophoblast, especially for those from term placenta. This was a non-negligible limitation in our work. We emphasize that the use of HTR8/SVneo cells as a model system provides valuable insights, but these findings should be interpreted with caution when extrapolated to in vivo conditions in placenta tissue near term. In future research, we aim to explore cell models that better mimic term placental trophoblast for more accurate and comprehensive findings.

In conclusion, our study preliminarily demonstrates that placental aging in AMA preganancy is associated with decreased YAP levels and increased oxidative damage, revealing the important role of YAP in protecting placental trophoblast from oxidative damage. Additionally, it suggests that YAP may serve as a potential intervention target for improving placental development and perinatal outcomes in AMA pregnancy. Nonetheless, there are limitations in this study, and further in-depth research is still required.