RPL is a painful and profound condition that affects the quality of life of women and their partners. A decline in quality of life may be related to decreased physical ability, loss of vitality, and mental health damage. A previous study found that RPL is also associated with gestational diabetes mellitus, low birth weight, placental abruption, and perinatal death [4]. However, reliable molecular markers for predicting and treating RPL are lacking. Therefore, it is crucial to explore the molecular mechanisms and key genes related to the occurrence and development of RPL and to identify specific biomarkers. Research has shown that autophagy plays an important role in embryonic development and is closely related to adverse pregnancy outcomes, such as miscarriage [11, 12]. This study analyzed and identified autophagy-related genes that are differentially expressed in patients with RPL through bioinformatics analysis, which may provide a basis for revealing the molecular mechanisms of RPL and developing new targeted therapies.
The entire genome expression profile of recurrent miscarriages was retrieved from the GEO database, and autophagy-related genes were obtained from the human autophagy database. We identified and analyzed the DEGs between the control and RPL groups. A total of 517 DEGs were identified, with the top five upregulated genes (IRGQ, NCSTN, IRF2BP1, TSPAN14, and PCGF1) and the top five downregulated genes (ZNF90, PSIP1, SNRPE, LOC148709, and C2orf69) showing significant differences in expression between the RPL and control groups. Following functional and pathway enrichment analysis, we found that the expression of β-alanine metabolism and protein export pathways were significantly reduced in the RPL group, while the expression of proximal tubule bicarbonate reclamation and α-linolenic acid metabolism-related pathways was significantly increased, indicating the potential role of these DEGs in the progression of RPL. Taurine is one of the most abundant amino acids in the male reproductive system and has multiple functions, such as antioxidant defense, anti-apoptosis, and improvement of spermatogenesis [24]. β-Alanine is a taurine transporter (TAUT) antagonist with a high affinity for TAUT, which competitively binds to TAUT and effectively inhibits the cellular uptake of taurine [25]. TAUT has a positive impact on early embryonic development by regulating sperm morphology, and its deficiency may lead to abnormal sperm morphology. A study showed that inhibiting cellular taurine uptake by β-alanine and TAUT knockdown resulted in increased sperm abnormalities, which may lead to abnormal embryonic development and RPL [26]. Our study found that β-alanine metabolism significantly decreased in the RPL group, which is consistent with the above research results, further indicating that β-alanine metabolism is related to the occurrence and development of RPL. However, its specific molecular mechanism remains to be elucidated. At the same time, the impact of pathways, such as protein export, proximal tubule bicarbonate reclamation, and α-linolenic acid metabolism pathways on RPL is currently unclear. It needs further exploration, providing new ideas and directions for in-depth research on the pathogenesis of RPL.
Studies have found that chronic inflammatory placental disorders, such as villitis of unknown etiology, chronic histiocytic intervillositis, and massive perivillous fibrin deposition, are significantly associated with adverse obstetric outcomes, including severe fetal growth restriction, late intrauterine fetal death, and RPL. The hallmark of these diseases is the destructive infiltration of maternal immune cells into the placental architecture and intervillous space [27, 28]. Proper vascular development and maintenance at the maternal-embryo interface are crucial for successful pregnancies, as they are regulated by various cell types, with uterine natural killer (uNK) cells playing an important role. Abnormal angiogenesis and uNK cell count/function may lead to reproductive failure, particularly in patients with RPL, and recurrent implantation failure after in vitro fertilization-embryo transfer [29]. In early pregnancy, the main immune cells present in the decidua, uNK (CD56brightCD16) cells, have receptors that bind to ligands on invasive extravillous trophoblast cells. Therefore, NK cells have become a core element of immunological research in women with RPL [7]. Immune infiltration analysis revealed that the degree of infiltration of two types of immune cells (CD56bright natural killer cells and monocytes) was significantly higher in the RPL group than in the control group. Correlation tests between hub genes and the corresponding immune cells showed a significant correlation between FAM115A, RGS11, TRIM59, and monocytes.
Transient receptor potential (TRP) channels form a large family of cation channels involved in multiple physiological functions and are expressed in almost all cell types. They play important roles in Ca2+ absorption, vasorelaxation, cell death, mechanical transduction, and sensory conduction. FAM115A, a protein with an unknown function, consists of 921 amino acids (NP 055534) and is also known as TRP channel-associated factor 1 (TCAF1) [30]. The TCAF1 protein can directly interact with TRPM8 (member 8 of the transient receptor potential cation channel subfamily M member 8), promoting the opening and migration of TRPM8 channels and acting as a thermal sensor in peripheral somatosensory neurons [31, 32]. GSVA further analyzed key genes related to signaling pathways and found that two hallmark signaling pathways were significantly upregulated in patients with RPL (HALLMARK_BILE_ACID_METABOLISM and HALLMARK_KRAS_
SIGNALING_DN), whereas FAM115A was associated with many pathways, including HALLMARK_XENOBIOTIC_METABOLISM. The hub gene interaction analysis created a PPI network, including six hub genes with interaction relationships (TCAF1/FAM115A, RGS11, TRIM59, CENPK, GLRX, and P2RY14) and 20 genes related to hub genes. We conducted an interaction analysis between the hub mRNA and its RBP to construct an RBP-mRNA network, including five mRNAs (RGS11, TRIM59, CENPK, GLRX, and P2RY14) and 48 RBPs. These genes and mRNA may provide new insights into the mechanisms of RPL and new biomarkers for its diagnosis and treatment.
Many centromeric proteins form a network called the constitutive centromere-associated network (CCAN) that binds to the centromere throughout the cell cycle. CENP-K and CENP-A are the two subcomplexes of CCAN that interact with and depend on each other [33]. Anti-centromere antibodies may be among the antinuclear antibodies most closely related to abnormal oocyte maturation and embryo cleavage. In vitro co-culture experiments of mouse embryos with polyclonal anti-centromere protein A (CENP-A) have revealed that embryos cultured with anti-CENP-A antibodies showed significant growth impairment, and embryos may be direct targets of anti-CENP-A antibodies, which is consistent with our research direction [34].
WGCNA and Pearson correlation analyses were conducted to assess the gene sets of DEGs and autophagy-related modules. Six autophagy-related hub genes (FAM115A, RGS11, TRIM59, CENPK, GLRX, and P2RY14) were identified. The validation of the ROC curves indicated that the hub gene had discriminative ability as a potential biomarker for RPL. However, the six autophagy-related hub genes identified through our comprehensive bioinformatics analysis currently do not have sufficient evidence, and in-depth experimental research is needed to further explore the relevant mechanisms. These autophagy-related hub genes have not been previously studied in RPL, providing new directions and ideas for future research. Nevertheless, our study had some limitations, such as the lack of histological verification and comparison of key autophagy genes in RPL and the lack of research on the molecular mechanisms by which hub genes regulate the development of RPL. Based on the results obtained in this study, we hope to collect relevant tissues from patients with clinical RPL, conduct further experimental research to verify the expression levels of autophagy-related genes, and explore the underlying molecular mechanisms.