Investigating autophagy genes expression and their possible relations with apoptosis in PBMCs of patients with thin endometrium

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In most cases, the origin of EA is not known, but factors can cause thinning of the endometrium, including contraceptive drugs, inflammatory, iatrogenic, and in some cases, hereditary factors. Autophagy has an important consequence in the proper functioning of the uterus, reproductive physiology and endometrial atrophy. Method: In this study, Real-Time PCR was used to measure the expression levels of autophagy genes ATG5, ATG7, LC3B, Beclin1, FOXO1, FOXO3a, FOXO4 and FOXO6 in 40 women with thin endometrium and 40 healthy pregnant women. In addition to, apoptosis was done by flow cytometry method. Results: Evaluation of the expression level of autophagy genes showed a significant difference in studied groups, so that the expression levels of ATG5, ATG7 and LC3B, Beclin1, FOXO1, FOXO3a, FOXO4 and FOXO6 genes were higher in the patient group. Moreover, there was a positive correlation between the expression of autophagy gene LC3B and the frequency of apoptotic cells in the studied patients. Disscusion: To further elucidate the biological pathways and processes associated with the differentially expressed autophagy genes, we conducted a KEGG pathway enrichment analysis using the EnrichR tool. Our results showed that autophagy genes with apoptosis in PBMC cells may be involved in endometrial thinning of EA patients. Autophagy Endometrium Apoptosis ATG FOXO Figures Figure 1 Figure 2 Figure 3 Figure 4 1.Introduction The achievement of human conception inevitably depends on the implantation of the embryo in the endometrium of the host. This endometrium supports the placenta during pregnancy [ 1 , 2 ]. The importance of thin endometrium in implantation failure is well known [ 3 – 5 ]. Women with thin endometrium face a lower implantation rate due to implantation failure [ 5 , 6 ]. Evaluation of the endometrium and its thickness is an important item in the success of assisted reproduction and is directly related to the increase in circulating estrogen levels [ 7 ]. In most studies, endometrial thickness less than 7 mm or 8 mm is called thin endometrium. The odds of pregnancy with this thickness is very low, but despite this, several studies have reported pregnancies with an endometrial thickness of 4 [ 8 , 9 ] between 4 and 6 [ 10 ]. One of the important problems in assisted reproduction is the distrust caused by thin endometrium, which is often associated with unsuccessful pregnancy after treatment. The cause of thin endometrium is unknown and complex, which makes it difficult to investigate the pathological mechanisms and treat these patients. Various factors cause thinning of the endometrium, the most common of which are inflammatory and iatrogenic. Some women also have a naturally thin endometrium [ 11 ]. The pathological causes of thin endometrium are: Asherman's syndrome, fibroids, previous intrauterine surgery, drugs, hormone level disorders and premature ovarian failure [ 12 – 14 ]. Recently, the key role of autophagy genes in the physiological and pathophysiological environment of human endometrium has been mentioned [ 15 , 16 ]. Autophagy is a cellular catabolic process, meaning "self-eating" [ 17 ]. Autophagy genes are induced under various cellular stress conditions and are widely involved as a regulatory pathway in many cellular processes. Stimuli such as hypoxia and starvation induce autophagy, which mainly relies on activation of AMP-activated protein kinase (AMPK) and inactivation of rapamycin complex 1 (mTORC1) [ 18 ]. ATG conserved proteins trigger autophagy [ 19 ]. Initially, in response to stimuli, the phagophore is formed near the endoplasmic reticulum (ER). Two models of ubiquitin-like conjugation are involved along the autophagosome membrane: the conjugation of Atg12 with Atg5, which is located together with Atg16 in the phagophore, and downstream, the conjugation of Atg8 with phosphoethanolamine (PE), which includes both the phagophore and the autophagosomal membrane [ 20 ]. The interaction of ATG12-ATG5 with ATG16L1 results in the formation of the multimeric ATG5-ATG12-ATG16 complex. Together with ATG8 family proteins (ATG3, ATG7, LC3 and GABARAP subfamilies), the mature autophagosome is formed. On the other hand, the Atg7-Atg5 complex plays a role in the evolution of the autophagosome membrane and the lipidation of microtubule-associated light chain 3 (LC3). LC3 includes a soluble form of LC3I and a lipid form called LC3II and is expressed as 3 isoforms (LC3A, LC3B, LC3C) in mammalian tissue, of which LC3B is related to autophagy [ 21 ]. FOXO family proteins (FOXO1, FOXO3a, FOXO4, and FOXO6) are important factors in the autophagy pathway [ 22 ]. One of the important genes in the induction of autophagy is Beclin-1, which is involved in the nucleation of autophagosomes and the activation of other autophagy-inducing factors [ 23 ]. Various reports have defined the involvement of autophagy in the establishment of endometrial homeostasis and successful pregnancy. Low estrogen levels may cause the endometrium to thin, which is called atrophic. The molecular mechanism of autophagy genes responsible for this atrophy has been reported [ 24 ]. In fact, autophagy in atrophic endometrial cells causes an increase in autophagosomes and apoptotic cell death response. The initial response to initiate this process is the inactivation of AKT (AKT serine/threonine kinase)-MTOR (mechanistic target of rapamycin kinase) signaling by ER stress. The purpose of this study is to investigate the expression level of genes involved in autophagy in women with thin endometrium compared to women with normal pregnancy and their corelations with the level of apoptosis in PBMCs of these patients. 2. Materials and Methods 2.1. Study population The present study was approved by the Research Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.REC.1401.733). 40 pregnant women with thin endometrium (mean age: 27.19 ± 4.13) who visited Valiasr Hospital from September 2021 to April 2022 were included in this study. 40 healthy pregnant women (mean age: 26.34 ± 3.74) with a history of pregnancy and prior successful delivery were considered as controls. All participants signed informed consent forms before the start of the study. The clinical characteristics and laboratory findings of the studied population are shown in Table 1 . Table 1 Primer sequences of evaluated genes. Abbreviations: ATG: Autophagy-related; Beclin1: A mammalian ortholog of yeast ATG6; LC3B: Microtubule-associated proteins 1A/1B light chain 3B; FOXO: Forkhead box transcription factors. Gene Primer Sequence (5′→3′) ATG5 Forward Reverse GCAGATGGACAGTTGCACACAC GAGGTGTTTCCAACATTGGCTCA ATG7 Forward Reverse CGTTGCCCACAGCATCATCTTC CACTGAGGTTCACCATCCTTGG Beclin1 Forward Reverse CTGGACACTCAGCTCAACGTCA CTCTAGTGCCAGCTCCTTTAGC LC3B Forward Reverse GAGAAGCAGCTTCCTGTTCTGG GTGTCCGTTCACCAACAGGAAG FOXO1 Forward Reverse CTACGAGTGGATGGTCAAGAGC CCAGTTCCTTCATTCTGCACACG FOXO3a Forward Reverse TCTACGAGTGGATGGTGCGTTG CTCTTGCCAGTTCCCTCATTCTG FOXO4 Forward Reverse ACGAGTGGATGGTCCGTACTGT CCTTGATGAACTTGCTGTGCAGG FOXO6 Forward Reverse CCTGCGCATCAAGGGCAAG GCACTCGGGGAGCTGTCGTC β-actin Forward Reverse CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT 2.2. Isolation of peripheral blood mononuclear cells (PBMCs) After collecting 10 ml of blood samples from patients and healthy groups under sterile conditions and anticoagulant drugs (EDTA-K2), isolation of peripheral blood mononuclear cells (PBMCs) was done using 1.077 g/ml Ficoll gradient centrifugation (Biosera, UK) at 450 g for 25 min. After separating PBMCs using Ficoll and gradient centrifugation, the cells were divided into two sections: one section to evaluate the frequency of apoptotic cells by flow cytometry, and the other section to extract RNA and examine gene expression. 2.3. Apoptosis assay In order to detect apoptosis, flow cytometry method was used. Apoptosis in this method is detected by primary staining of cells with annexin V solution and propidium iodide and then flow cytometric analysis. In summary, normal cells are hydrophobic in nature because they express phosphatidylserine on the inner membrane (cytoplasmic side), and when cells undergo apoptosis, the inner membrane becomes the outer membrane, thereby exposing phosphatidylserine.. Exposed phosphatidylserine is recognized by Annexin V and propidium iodide stains necrotic cells, which have a leaky DNA content that helps distinguish between apoptotic and necrotic cells. After 48 hours of cell culture, 50 microliters of annexin binding buffer was added to 3 x 10 5 cells. Then the cells were labeled with 5 µl of annexinV-FITC. After 15 minutes of incubation at room temperature in the dark, 200 µl of binding buffer was added. Finally, PI was added at a final concentration of 50 µg/ml. Annexin V-FITC apoptosis detection kit was purchased from (BD Biosciences, CA, USA). 2.4. Real-time PCR The expression level of ATG5, ATG7, Beclin1, LC3B, FOXO1, FOXO3a, FOXO4 and FOXO6 genes in patients with thin endometrium and healthy pregnant women was evaluated by Real Time-PCR. After isolation of PBMCs, RNX-PLUS solution (Sina Clone, Tehran, Iran) was used for RNA extraction. In the following, using the Revert Aid Reverse Transcriptase kit (Thermo Fisher, Waltham, MA, USA), complementary DNA synthesis (cDNA) was performed according to the manufacturer's instructions. Finally, gene specific primers and SYBR Green in a Roche Light Cycler ® 96 Instrument were used to measure gene expression. In Table 2 , the sequence of specific primers designed in the assay with OLIGO v. 7.56 is displayed. Data were assesseed with 2-ΔΔCT. Table 2 Clinical characteristics of the studied population Characteristics Control (Mean ± SD) N = 40 Thin endometrium (Mean ± SD) N = 40 p value Maternal age (years) 26.34 ± 3.74 27.19 ± 4.13 NS BMI (kg/m2) 25.82 ± 3.31 26.94 ± 3.69 NS Systolic blood pressure (mmHg) 113.5 ± 11.87 117.8 ± 14.52 NS Diastolic blood pressure (mmHg) 73.55 ± 7.38 75.14 ± 6.94 NS Fasting Blood Sugar (mg/dl) 101.4 ± 14.88 105.2 ± 11.35 NS Triglyceride (mg/dl) 132.8 ± 28.5 133.4 ± 25.32 NS Cholesterol (mg/dl) 157.8 ± 21.36 161.4 ± 24.17 NS HDL- Cholesterol (mg/dl) 53.24 ± 6.16 52.18 ± 6.72 NS LDL-Cholesterol (mg/dl) 105.7 ± 17.81 108.3 ± 16.46 NS 2.5. Statistical analysis GraphPad Prism 7 (GraphPad Software; San Diego, CA, USA) was used to analyze the study data. Unpaired t-test was used to evaluate the frequency of apoptotic cells and examine the expression of autophagy genes in the studied groups. The Pearson r test was used to evaluate the correlation between the expression of autophagy and apoptosis genes. P < 0.05 was considered as statistically significant. 2.6.Bioinformatics Analysis To further elucidate the biological pathways and processes associated with the differentially expressed autophagy genes identified in the study, we conducted a bioinformatics analysis using the EnrichR tool [ 25 ]. The list of differentially expressed autophagy genes, including ATG5, ATG7, LC3B, Beclin1, FOXO1, FOXO3a, FOXO4, and FOXO6, was submitted to the EnrichR web-based platform ( https://maayanlab.cloud/Enrichr/ ). We performed an enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) 2021 database[ 26 ] to identify significantly overrepresented biological pathways associated with the input genes. The KEGG pathway enrichment analysis in EnrichR provides a combined score, which is calculated by multiplying the log of the p-value obtained from the Fisher's exact test with the z-score of the deviation from the expected rank. A combined score greater than 2 was considered as the threshold for statistically significant enrichment [ 25 ]. 3. Results 3.1. Laboratory findings of the study population The general characteristics of the studied groups are given in Table 1 . The average age of the patient and control groups was 27.19 ± 4.13 and 26.34 ± 3.74 years, respectively. There was no significant difference in common laboratory findings between patients and control group. 3.2. Flow cytometry and apoptosis assay Detection of cell apoptosis in isolated PBMCs was performed using flow cytometry and Annexin V-PI staining. The results after flow cytometry analysis showed a significant difference in the apoptosis of patients and control cells. The percentage of apoptotic cells in women with thin endometrium was significantly higher compared to healthy pregnant women (p = 0.0002, Fig. 1 A ). Figure 1 B shows the percentage of apoptotic cells in unstained, control and patient samples. Patient samples contain a higher percentage of apoptotic cells. 3.3. Gene expression The expression of autophagy genes in women with thin endometrium and healthy subjects was evaluated by Real-time PCR (Table 2 ). A significant difference was observed in the expression of autophagy genes in the patients and controls. 3.3.1. Increased expression level of ATG genes in women with thin endometrium Our results showed a significant expression level of Atg5 (0.0004), Atg7 (0.0035), Beclin1 (0.0002) and LC3B (0.0005) genes in women with thin endometrium compared to healthy pregnant women. (Fig. 2 ; Table 3 ). The expression level of these genes in women with thin endometrium was higher than healthy pregnant women. Table 3 Molecular changes in thin endometrium women vs healthy fertile women. Real-Time PCR (Fold Change) Target Control (Mean ± SD) N = 40 Thin endometrium (Mean ± SD) N = 40 p value ATG5 1.000 ± 0.1129 1.855 ± 1.383 0.0004 ATG7 1.000 ± 0.1004 1.573 ± 1.161 0.0035 Beclin1 1.000 ± 0.09249 1.666 ± 1.037 0.0002 LC3B 1.000 ± 0.1015 1.856 ± 1.420 0.0005 FOXO1 1.000 ± 0.1038 2.043 ± 1.275 < 0.0001 FOXO3a 1.000 ± 0.1199 2.275 ± 1.671 < 0.0001 FOXO4 1.000 ± 0.08839 2.767 ± 1.809 < 0.0001 FOXO6 1.000 ± 0.1172 1.981 ± 1.556 0.0003 Flow cytometry Apoptosis (%) 8.735 ± 2.084 10.87 ± 2.689 0.0002 Data are presented as mean ± SD. p < 0.05 considered as significant. Abbreviations: ATG: Autophagy-related; Beclin1: A mammalian ortholog of yeast ATG6; LC3B: Microtubule-associated proteins 1A/1B light chain 3B; FOXO: Forkhead box transcription factors. 3.3.2. Increased expression levels of FOXO family genes in women with thin endometrium Our results showed a significant expression levels of FOXO1 (< 0.0001), FOXO3a (< 0.0001), FOXO4 (< 0.0001) and FOXO6 (0.0003) genes in women with thin endometrium compared to healthy pregnant women (Fig. 3 ; Table 3 ). The expression level of these genes in women with thin endometrium was higher than healthy pregnant women. 3.4. Correlation assay The correlation analysis between the percentage of apoptosis of PBMC cells and the expression of ATG5, LC3B, Beclin1 and FOXO1 genes in patients with thin endometrium was performed. According to the reported data, about the L3CB gene, the obtained correlation is positive and significant (Fig. 4 , p = 0.0339, r = 0.3362). 3.5.Bioinformatics Analysis To further elucidate the biological pathways and processes associated with the differentially expressed autophagy genes, we conducted a KEGG pathway enrichment analysis using the EnrichR tool (Table 4 ). Table 4 The results of Bioinformatics Analysis. Term P-value Adjusted P-value Odds Ratio Combined Score Genes Shigellosis 1.50E-06 3.16E-05 81.61157 1094.192 FOXO6;FOXO4;FOXO1;ATG5 FoxO signaling pathway 1.50E-05 1.58E-04 93.1125 1034.156 FOXO6;FOXO4;FOXO1 Ferroptosis 1.14E-04 7.97E-04 170.5385 1548.508 ATG7;ATG5 Longevity regulating pathway 7.07E-04 0.003711 66.30667 481.0329 FOXO1;ATG5 Autophagy 0.001269 0.005332 49.02963 326.9852 ATG7;ATG5 Mitophagy 0.026883 0.076562 42.48401 153.6333 ATG5 RIG-I-like receptor signaling pathway 0.027664 0.076562 41.24845 147.9839 ATG5 Prostate cancer 0.038154 0.076562 29.60714 96.70056 FOXO1 AGE-RAGE signaling pathway in diabetic complications 0.039314 0.076562 28.70563 92.89679 FOXO1 Glucagon signaling pathway 0.042014 0.076562 26.80054 84.95102 FOXO1 Insulin resistance 0.042399 0.076562 26.54873 83.91052 FOXO1 AMPK signaling pathway 0.047012 0.076562 23.85714 72.93978 FOXO1 Thyroid hormone signaling pathway 0.047395 0.076562 23.65714 72.13611 FOXO1 Insulin signaling pathway 0.053513 0.080269 20.85714 61.06622 FOXO1 Cellular senescence 0.060733 0.085026 18.28295 51.21548 FOXO1 NOD-like receptor signaling pathway 0.070159 0.086661 15.72381 41.77794 ATG5 Neutrophil extracellular trap formation 0.073158 0.086661 15.04863 39.35412 ATG7 Transcriptional misregulation in cancer 0.074281 0.086661 14.81002 38.50463 FOXO1 Ras signaling pathway 0.089133 0.098516 12.22078 29.54525 FOXO4 Human papillomavirus infection 0.125 0.13125 8.511688 17.69958 FOXO1 Pathways in cancer 0.193707 0.193707 5.245822 8.610534 FOXO1 The KEGG pathway enrichment analysis revealed several significantly enriched pathways (combined score > 2) related to the differentially expressed autophagy genes in the thin endometrium group. The top-ranked pathways included "Shigellosis" (combined score = 1094.192), "FoxO signaling pathway" (combined score = 1034.156), and "Ferroptosis" (combined score = 1548.508). The "Shigellosis" pathway was significantly enriched, involving the FOXO1, FOXO4, FOXO6, and ATG5 genes. The involvement of FOXO transcription and the autophagy gene ATG5 in this pathway suggests a potential link between bacterial infection, inflammation, and autophagy dysregulation in the endometrium[ 27 , 28 ] .Shigellosis, caused by the Shigella bacteria, is known to induce host cell autophagy as a defense mechanism[ 29 ]. However, the pathogen can also hijack the autophagy machinery to promote its own survival and replication[ 30 ]. The dysregulation of this pathway may contribute to an imbalance in the endometrial immune response, potentially leading to chronic inflammation and impaired tissue homeostasis, which are hallmarks of endometrial atrophy[ 31 ] . The "FoxO signaling pathway" was also among the top enriched pathways, highlighting the important role of FOXO transcription factors (FOXO1, FOXO4, FOXO6) in the regulation of autophagy and other cellular processes. The FOXO transcription factors play a critical role in the regulation of autophagy, cell cycle, and cell survival[ 32 ].Dysregulation of the FoxO signaling pathway may lead to altered endometrial cell proliferation, differentiation, and apoptosis[ 33 ], which are essential for maintaining endometrial thickness and function .Impaired FoxO signaling can disrupt the delicate balance between cell renewal and cell death[ 34 ], potentially contributing to the development of endometrial atrophy. Furthermore, the "Ferroptosis" pathway, which is a form of regulated cell death[ 35 ], was significantly enriched and involved the ATG7 and ATG5 genes. Ferroptosis is a form of regulated cell death characterized by the accumulation of lipid peroxides and iron-dependent oxidative stress.The involvement of the autophagy genes ATG7 and ATG5 in this pathway suggests a potential link between dysregulated autophagy and increased ferroptosis in the endometrium .Elevated ferroptosis, coupled with impaired autophagy, may lead to excessive cell death and failure to maintain the endometrial tissue, ultimately contributing to endometrial atrophy. Other significantly enriched pathways included "Longevity regulating pathway," "Autophagy," and "Mitophagy", further highlights the critical role of autophagy-related genes in endometrial homeostasis and function .Dysregulation of these pathways may impair cellular processes, such as energy metabolism, mitochondrial quality control, and cellular senescence, which are essential for endometrial tissue maintenance and regeneration . 4. Discussion One of the important issues in pregnancy is the thickness of the endometrium. The best chance of a full-term pregnancy is associated with a proportionally thick endometrium. This optimal thickness allows the embryo to implant properly and provide the required nutrition. Therefore, the evaluation of endometrial thickness in the process of in vitro fertilization (IVF) is performed as an index of endometrial acceptance [ 36 ]. Several reports have described low pregnancy rates with thin endometrium [ 37 , 38 ]. A challenging debate among doctors is the appropriate thickness of the endometrium for conception. Generally, a thickness of less than 7 mm in ultrasound is considered less than the determined limit for in vitro fertilization and is associated with a decrease in the probability of pregnancy. Autophagy has a key role and diverse functions in the endometrium. New evidence obtained supports the main role of autophagy in preserving endometrial physiology (endometrial cyclic regeneration, menstruation and implantation, and decidualization during pregnancy) and endometrial pathology (hyperplasia, endometriosis, endometrial cancer, and atrophic endometrium) [ 39 – 42 ]. According to the results obtained from the studies, autophagy causes cell death by destroying the basic components of cells, or it can even be caused by apoptosis-inducing stimuli. In two studies conducted by Choi et al [ 42 ] it was found that autophagy causes cell death by inducing apoptosis in granulosa and luteal cells [ 43 , 44 ]. Based on the above reports, in the first stage, we evaluated the frequency of apoptotic cells in patients and control groups. Our results showed a significant increase in the frequency of apoptotic cells in patients with thin endometrium compared to the control group. Choi et al reported[ 42 ] that autophagy is important in the endometrial cell cycle through apoptosis. In the second stage, we assessed the expression level of autophagy related genes including: Atg5, Atg7, Beclin1 (Atg6), LC3B (Atg8), FOXO1, FOXO3a, FOXO4, FOXCO6 .Our results showed a significant difference in the expression of these genes in patients with thin endometrium compared to healthy pregnant women. So the expression level of these genes in patients was higher than in controls. The KEGG pathway enrichment analysis revealed that several significantly enriched pathways are associated with differentially expressed autophagy genes in the thin endometrium group, including "Shigellosis," "FoxO signaling pathway," and "Ferroptosis." The "Shigellosis" pathway, involving FOXO1, FOXO4, FOXO6, and ATG5, suggests a link between bacterial infection, inflammation, and autophagy dysregulation, potentially contributing to chronic inflammation and endometrial atrophy. The "FoxO signaling pathway" highlights the role of FOXO transcription factors in regulating autophagy and cellular processes, where dysregulation may impact endometrial cell function and thickness[ 45 ]. The "Ferroptosis" pathway indicates a connection between dysregulated autophagy and increased cell death in the endometrium[ 46 ], implicating autophagy genes ATG7 and ATG5. Other enriched pathways like "Longevity regulating pathway," "Autophagy," and "Mitophagy" underscore the importance of autophagy-related genes in endometrial homeostasis, suggesting that dysregulation could impair essential cellular processes and contribute to endometrial atrophy. These findings provide a basis for exploring targeted therapies to restore endometrial health. In a study, Liu and colleagues [ 47 ] showed that the survival of the embryo after the transfer operation is affected by the thickness of the endometrium, and the survival rate decreased with the decrease in thickness. The role of autophagy in endometrial atrophy has not been thoroughly investigated. Currently, there is no study that identifies the role of autophagy in endometrial atrophy. However, several studies have addressed the role of autophagy genes in endometrium-related diseases. Feng et al showed increased expression of the autophagy adapter SQSTM1 in a mouse model of endometrial hyperplasia [ 41 ]. Many researches have pointed out the uncoordinated activation of autophagy and the therapeutic potential of anticancer drugs to modulate them in endometrial cancer (EC). For example, in endometrial adenocarcinoma, increased expression of Beclin1 has been reported, which is associated with poor prognosis such as high tumor rate and myometrial invasion [ 48 ]. In contrast, another study reported a decrease in Beclin1 expression in the stage of neoplastic transformation [ 49 ]. Considering the relationship between cell death and autophagy genes, in the last step, we examined the correlation between the expression of autophagy genes and cell death in patients. Contrary to our expectation, the expression of only one gene was positively correlated with cell death of PBMCs. In terms of correlation, L3CB gene was positive and significant. It can be concluded that maybe autophagy is closely related to apoptosis in patients with thin endometrium. Therefore, targeting apoptotic pathways in autophagy mechanisms may be a promising strategy for the treatment of these patients. Abbreviations EA: Endometrial atrophy ATG: Autophagy-related LC3B: Microtubule-Associated Protein 1 Light Chain 3 beta FOXO: Forkhead Box O AMPK: AMP-activated protein kinase mTORC: Rapamycin complex 1 ER: Endoplasmic reticulum PE: Phosphoethanolamine LC3: lipidation of microtubule-associated light chain 3 PBMCs: Peripheral blood mononuclear cells KEGG: Kyoto Encyclopedia of Genes and Genomes IVF: In vitro fertilization EC: Endometrial cancer Declarations Acknowledgment The present study was approved by the Ethics Committee of the Deputy Director of the Immunology Research Center (IR.TBZMED.REC.1401.733). Funding This work was supported by Tabriz University of Medical Sciences, Tabriz, Iran [grant number: 70489]. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Data availability Data will be made available on request. References Madani, J., et al., Fetus, as an allograft, evades the maternal immunity. 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Li, H., et al., Autophagy-dependent ferroptosis is involved in the development of endometriosis. Gynecological Endocrinology, 2023. 39 (1): p. 2242962. Liu, K., et al., The impact of a thin endometrial lining on fresh and frozen–thaw IVF outcomes: an analysis of over 40 000 embryo transfers. Human Reproduction, 2018. 33 (10): p. 1883–1888. Giatromanolaki, A., et al., High Beclin 1 expression defines a poor prognosis in endometrial adenocarcinomas. Gynecologic oncology, 2011. 123 (1): p. 147–151. Zhao, J.-H., et al., Expression and clinical significance of Beclin1 and PTEN in endometrial carcinoma. Ai Zheng= Aizheng= Chinese Journal of Cancer, 2006. 25 (6): p. 753–757. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7704238","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528001193,"identity":"690fb96b-07d7-41c3-ae10-92a7b287cb65","order_by":0,"name":"Lida-Aslanian kalkhoran","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lida-Aslanian","middleName":"","lastName":"kalkhoran","suffix":""},{"id":528001194,"identity":"16a2402f-1a69-4ec3-b70f-26778b35065b","order_by":1,"name":"Mohammad Sadegh Soltani-Zangbar","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Sadegh","lastName":"Soltani-Zangbar","suffix":""},{"id":528001195,"identity":"4d52ef00-fda0-475f-9003-5e0aa83af0d8","order_by":2,"name":"Ali Aghebati-Maleki","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Aghebati-Maleki","suffix":""},{"id":528001196,"identity":"23de9136-0bea-42b1-954c-418b2c08574a","order_by":3,"name":"Mahya AhmadpourYoushanlui","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahya","middleName":"","lastName":"AhmadpourYoushanlui","suffix":""},{"id":528001197,"identity":"7aae2ce1-73af-41db-bc8a-d1050181fe4a","order_by":4,"name":"Mehdi Yousefi","email":"","orcid":"","institution":"Tabriz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Yousefi","suffix":""},{"id":528001198,"identity":"aee17779-e831-4e89-b076-94375072b861","order_by":5,"name":"leili Aghebati-Maleki","email":"data:image/png;base64,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","orcid":"","institution":"Tabriz University of Medical 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02:43:21","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114250,"visible":true,"origin":"","legend":"","description":"","filename":"6b5173d322c24aeaa61c77104a2b76bd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/cd91bd9acc5ce6a43a76c645.xml"},{"id":93544064,"identity":"34bed4a7-e399-4768-aa19-786a4de569f7","added_by":"auto","created_at":"2025-10-15 02:51:21","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126055,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/72aca5d801d730939b5d993c.html"},{"id":93543533,"identity":"dd67e982-4013-4fa7-a468-d6e52f43d4e2","added_by":"auto","created_at":"2025-10-15 02:43:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3607253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell apoptosis by flow cytometry technique in patients with thin endometrium and healthy controls. A) \u003c/strong\u003eComparison of cell apoptosis between patients with thin endometrium and healthy controls. Compared to the healthy control group, patients with thin endometrium showed a higher percentage of apoptosis. \u003cstrong\u003eB) \u003c/strong\u003eDot plots showing the frequency of apoptotic cells in unstained, healthy and patients groups, respectively.\u003c/p\u003e","description":"","filename":"Fig.1.Apoptosis.png","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/317a839ebe17f08114e82571.png"},{"id":93543534,"identity":"83f7641f-9761-4fa0-8fdf-ff2497cda8b9","added_by":"auto","created_at":"2025-10-15 02:43:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2713763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of ATG family genes in patients with thin endometrium compared to healthy pregnant women as a control group. \u003c/strong\u003eAccording to the figure shown, women with thin endometrium have significant expression of Atg5, Atg7, beclin1 and LC3B genes compared to healthy pregnant women. p\u0026lt;0.05 was considered as significancy. (Case No: 40 and controls No:40).\u003c/p\u003e","description":"","filename":"Fig.2.ATGsGeneExpression.png","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/e88aa235a7f680c75ee0d875.png"},{"id":93544062,"identity":"c4fe00d3-d7db-41b1-88cc-78c8add7184e","added_by":"auto","created_at":"2025-10-15 02:51:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2765645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of FOXO family genes in patients with thin endometrium compared to healthy pregnant women as a control group. \u003c/strong\u003eAccording to the figure shown, women with thin endometrium have significant expression of FOXO1, FOXO3a, FOXO4 and FOXO6 genes compared to healthy pregnant women. p\u0026lt;0.05 was considered as significancy. (Case No: 40 and controls No:40).\u003c/p\u003e","description":"","filename":"Fig.3.FOXOsgeneexpression.png","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/16c16219443b99a33f4780f2.png"},{"id":93543539,"identity":"ba625c6d-9f75-44f3-8bf5-3a9ff983ec3f","added_by":"auto","created_at":"2025-10-15 02:43:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4329737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between expression levels of autophagy genes and apoptosis frequency in PBMC cells of patients. \u003c/strong\u003eAccording to the representative graphs, a positive correlation was seen between LC3B expression and apoptosis rate of PBMCs in thin endometrium subjects (p=0.0339). p\u0026lt;0.05 was considered as significancy. (Case No: 40).\u003c/p\u003e","description":"","filename":"Fig.4.CorrelationAssay.png","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/eed0f5b84188e9b90e9b040a.png"},{"id":101398294,"identity":"aa6ebcd3-6d51-425e-aeb7-b9babe990654","added_by":"auto","created_at":"2026-01-29 09:40:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13453666,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7704238/v1/048d6a4f-7c3a-47dc-9ace-d64b6316f26e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating autophagy genes expression and their possible relations with apoptosis in PBMCs of patients with thin endometrium","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eThe achievement of human conception inevitably depends on the implantation of the embryo in the endometrium of the host. This endometrium supports the placenta during pregnancy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The importance of thin endometrium in implantation failure is well known [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Women with thin endometrium face a lower implantation rate due to implantation failure [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Evaluation of the endometrium and its thickness is an important item in the success of assisted reproduction and is directly related to the increase in circulating estrogen levels [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In most studies, endometrial thickness less than 7 mm or 8 mm is called thin endometrium. The odds of pregnancy with this thickness is very low, but despite this, several studies have reported pregnancies with an endometrial thickness of 4 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] between 4 and 6 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. One of the important problems in assisted reproduction is the distrust caused by thin endometrium, which is often associated with unsuccessful pregnancy after treatment.\u003c/p\u003e\u003cp\u003eThe cause of thin endometrium is unknown and complex, which makes it difficult to investigate the pathological mechanisms and treat these patients. Various factors cause thinning of the endometrium, the most common of which are inflammatory and iatrogenic. Some women also have a naturally thin endometrium [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The pathological causes of thin endometrium are: Asherman's syndrome, fibroids, previous intrauterine surgery, drugs, hormone level disorders and premature ovarian failure [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecently, the key role of autophagy genes in the physiological and pathophysiological environment of human endometrium has been mentioned [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Autophagy is a cellular catabolic process, meaning \"self-eating\" [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Autophagy genes are induced under various cellular stress conditions and are widely involved as a regulatory pathway in many cellular processes. Stimuli such as hypoxia and starvation induce autophagy, which mainly relies on activation of AMP-activated protein kinase (AMPK) and inactivation of rapamycin complex 1 (mTORC1) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. ATG conserved proteins trigger autophagy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Initially, in response to stimuli, the phagophore is formed near the endoplasmic reticulum (ER). Two models of ubiquitin-like conjugation are involved along the autophagosome membrane: the conjugation of Atg12 with Atg5, which is located together with Atg16 in the phagophore, and downstream, the conjugation of Atg8 with phosphoethanolamine (PE), which includes both the phagophore and the autophagosomal membrane [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The interaction of ATG12-ATG5 with ATG16L1 results in the formation of the multimeric ATG5-ATG12-ATG16 complex. Together with ATG8 family proteins (ATG3, ATG7, LC3 and GABARAP subfamilies), the mature autophagosome is formed. On the other hand, the Atg7-Atg5 complex plays a role in the evolution of the autophagosome membrane and the lipidation of microtubule-associated light chain 3 (LC3). LC3 includes a soluble form of LC3I and a lipid form called LC3II and is expressed as 3 isoforms (LC3A, LC3B, LC3C) in mammalian tissue, of which LC3B is related to autophagy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. FOXO family proteins (FOXO1, FOXO3a, FOXO4, and FOXO6) are important factors in the autophagy pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. One of the important genes in the induction of autophagy is Beclin-1, which is involved in the nucleation of autophagosomes and the activation of other autophagy-inducing factors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVarious reports have defined the involvement of autophagy in the establishment of endometrial homeostasis and successful pregnancy. Low estrogen levels may cause the endometrium to thin, which is called atrophic. The molecular mechanism of autophagy genes responsible for this atrophy has been reported [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In fact, autophagy in atrophic endometrial cells causes an increase in autophagosomes and apoptotic cell death response. The initial response to initiate this process is the inactivation of AKT (AKT serine/threonine kinase)-MTOR (mechanistic target of rapamycin kinase) signaling by ER stress. The purpose of this study is to investigate the expression level of genes involved in autophagy in women with thin endometrium compared to women with normal pregnancy and their corelations with the level of apoptosis in PBMCs of these patients.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study population\u003c/h2\u003e\u003cp\u003e The present study was approved by the Research Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.REC.1401.733). 40 pregnant women with thin endometrium (mean age: 27.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13) who visited Valiasr Hospital from September 2021 to April 2022 were included in this study. 40 healthy pregnant women (mean age: 26.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74) with a history of pregnancy and prior successful delivery were considered as controls. All participants signed informed consent forms before the start of the study. The clinical characteristics and laboratory findings of the studied population are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences of evaluated genes. Abbreviations: ATG: Autophagy-related; Beclin1: A mammalian ortholog of yeast ATG6; LC3B: Microtubule-associated proteins 1A/1B light chain 3B; FOXO: Forkhead box transcription factors.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequence (5\u0026prime;\u0026rarr;3\u0026prime;)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATG5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGCAGATGGACAGTTGCACACAC\u003c/p\u003e\u003cp\u003eGAGGTGTTTCCAACATTGGCTCA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATG7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCGTTGCCCACAGCATCATCTTC\u003c/p\u003e\u003cp\u003eCACTGAGGTTCACCATCCTTGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBeclin1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTGGACACTCAGCTCAACGTCA\u003c/p\u003e\u003cp\u003eCTCTAGTGCCAGCTCCTTTAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLC3B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGAGAAGCAGCTTCCTGTTCTGG\u003c/p\u003e\u003cp\u003eGTGTCCGTTCACCAACAGGAAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTACGAGTGGATGGTCAAGAGC\u003c/p\u003e\u003cp\u003eCCAGTTCCTTCATTCTGCACACG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFOXO3a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTCTACGAGTGGATGGTGCGTTG\u003c/p\u003e\u003cp\u003eCTCTTGCCAGTTCCCTCATTCTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFOXO4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACGAGTGGATGGTCCGTACTGT\u003c/p\u003e\u003cp\u003eCCTTGATGAACTTGCTGTGCAGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFOXO6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCTGCGCATCAAGGGCAAG\u003c/p\u003e\u003cp\u003eGCACTCGGGGAGCTGTCGTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eβ-actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCACCATTGGCAATGAGCGGTTC\u003c/p\u003e\u003cp\u003eAGGTCTTTGCGGATGTCCACGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Isolation of peripheral blood mononuclear cells (PBMCs)\u003c/h2\u003e\u003cp\u003eAfter collecting 10 ml of blood samples from patients and healthy groups under sterile conditions and anticoagulant drugs (EDTA-K2), isolation of peripheral blood mononuclear cells (PBMCs) was done using 1.077 g/ml Ficoll gradient centrifugation (Biosera, UK) at 450 g for 25 min. After separating PBMCs using Ficoll and gradient centrifugation, the cells were divided into two sections: one section to evaluate the frequency of apoptotic cells by flow cytometry, and the other section to extract RNA and examine gene expression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Apoptosis assay\u003c/h2\u003e\u003cp\u003eIn order to detect apoptosis, flow cytometry method was used. Apoptosis in this method is detected by primary staining of cells with annexin V solution and propidium iodide and then flow cytometric analysis. In summary, normal cells are hydrophobic in nature because they express phosphatidylserine on the inner membrane (cytoplasmic side), and when cells undergo apoptosis, the inner membrane becomes the outer membrane, thereby exposing phosphatidylserine.. Exposed phosphatidylserine is recognized by Annexin V and propidium iodide stains necrotic cells, which have a leaky DNA content that helps distinguish between apoptotic and necrotic cells. After 48 hours of cell culture, 50 microliters of annexin binding buffer was added to 3 x 10\u003csup\u003e5\u003c/sup\u003e cells. Then the cells were labeled with 5 \u0026micro;l of annexinV-FITC. After 15 minutes of incubation at room temperature in the dark, 200 \u0026micro;l of binding buffer was added. Finally, PI was added at a final concentration of 50 \u0026micro;g/ml. Annexin V-FITC apoptosis detection kit was purchased from (BD Biosciences, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Real-time PCR\u003c/h2\u003e\u003cp\u003eThe expression level of ATG5, ATG7, Beclin1, LC3B, FOXO1, FOXO3a, FOXO4 and FOXO6 genes in patients with thin endometrium and healthy pregnant women was evaluated by Real Time-PCR. After isolation of PBMCs, RNX-PLUS solution (Sina Clone, Tehran, Iran) was used for RNA extraction. In the following, using the Revert Aid Reverse Transcriptase kit (Thermo Fisher, Waltham, MA, USA), complementary DNA synthesis (cDNA) was performed according to the manufacturer's instructions. Finally, gene specific primers and SYBR Green in a Roche Light Cycler \u0026reg; 96 Instrument were used to measure gene expression. In Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the sequence of specific primers designed in the assay with OLIGO v. 7.56 is displayed. Data were assesseed with 2-ΔΔCT.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical characteristics of the studied population\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) N\u0026thinsp;=\u0026thinsp;40\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThin endometrium (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) N\u0026thinsp;=\u0026thinsp;40\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMaternal age (years)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e26.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e27.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBMI (kg/m2)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e25.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e26.94\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSystolic blood pressure (mmHg)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e113.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e117.8\u0026thinsp;\u0026plusmn;\u0026thinsp;14.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDiastolic blood pressure (mmHg)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e73.55\u0026thinsp;\u0026plusmn;\u0026thinsp;7.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e75.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFasting Blood Sugar (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e101.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e105.2\u0026thinsp;\u0026plusmn;\u0026thinsp;11.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTriglyceride (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e132.8\u0026thinsp;\u0026plusmn;\u0026thinsp;28.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e133.4\u0026thinsp;\u0026plusmn;\u0026thinsp;25.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCholesterol (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e157.8\u0026thinsp;\u0026plusmn;\u0026thinsp;21.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e161.4\u0026thinsp;\u0026plusmn;\u0026thinsp;24.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHDL- Cholesterol (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e53.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e52.18\u0026thinsp;\u0026plusmn;\u0026thinsp;6.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLDL-Cholesterol (mg/dl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e105.7\u0026thinsp;\u0026plusmn;\u0026thinsp;17.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e108.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e\u003cp\u003eGraphPad Prism 7 (GraphPad Software; San Diego, CA, USA) was used to analyze the study data. Unpaired t-test was used to evaluate the frequency of apoptotic cells and examine the expression of autophagy genes in the studied groups. The Pearson r test was used to evaluate the correlation between the expression of autophagy and apoptosis genes. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6.Bioinformatics Analysis\u003c/h2\u003e\u003cp\u003eTo further elucidate the biological pathways and processes associated with the differentially expressed autophagy genes identified in the study, we conducted a bioinformatics analysis using the EnrichR tool [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe list of differentially expressed autophagy genes, including ATG5, ATG7, LC3B, Beclin1, FOXO1, FOXO3a, FOXO4, and FOXO6, was submitted to the EnrichR web-based platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://maayanlab.cloud/Enrichr/\u003c/span\u003e\u003cspan address=\"https://maayanlab.cloud/Enrichr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We performed an enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) 2021 database[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] to identify significantly overrepresented biological pathways associated with the input genes.\u003c/p\u003e\u003cp\u003eThe KEGG pathway enrichment analysis in EnrichR provides a combined score, which is calculated by multiplying the log of the p-value obtained from the Fisher's exact test with the z-score of the deviation from the expected rank. A combined score greater than 2 was considered as the threshold for statistically significant enrichment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Laboratory findings of the study population\u003c/h2\u003e\u003cp\u003eThe general characteristics of the studied groups are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The average age of the patient and control groups was 27.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13 and 26.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74 years, respectively. There was no significant difference in common laboratory findings between patients and control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Flow cytometry and apoptosis assay\u003c/h2\u003e\u003cp\u003eDetection of cell apoptosis in isolated PBMCs was performed using flow cytometry and Annexin V-PI staining. The results after flow cytometry analysis showed a significant difference in the apoptosis of patients and control cells. The percentage of apoptotic cells in women with thin endometrium was significantly higher compared to healthy pregnant women (p\u0026thinsp;=\u0026thinsp;0.0002, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA ). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the percentage of apoptotic cells in unstained, control and patient samples. Patient samples contain a higher percentage of apoptotic cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Gene expression\u003c/h2\u003e\u003cp\u003eThe expression of autophagy genes in women with thin endometrium and healthy subjects was evaluated by Real-time PCR (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A significant difference was observed in the expression of autophagy genes in the patients and controls.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Increased expression level of ATG genes in women with thin endometrium\u003c/h2\u003e\u003cp\u003eOur results showed a significant expression level of Atg5 (0.0004), Atg7 (0.0035), Beclin1 (0.0002) and LC3B (0.0005) genes in women with thin endometrium compared to healthy pregnant women. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e ; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The expression level of these genes in women with thin endometrium was higher than healthy pregnant women.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMolecular changes in thin endometrium women vs healthy fertile women.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eReal-Time PCR (Fold Change)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTarget\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) N\u0026thinsp;=\u0026thinsp;40\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eThin endometrium (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) N\u0026thinsp;=\u0026thinsp;40\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eATG5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.855\u0026thinsp;\u0026plusmn;\u0026thinsp;1.383\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eATG7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.573\u0026thinsp;\u0026plusmn;\u0026thinsp;1.161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0035\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBeclin1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.666\u0026thinsp;\u0026plusmn;\u0026thinsp;1.037\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLC3B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.856\u0026thinsp;\u0026plusmn;\u0026thinsp;1.420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0005\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFOXO1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.043\u0026thinsp;\u0026plusmn;\u0026thinsp;1.275\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFOXO3a\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1199\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.275\u0026thinsp;\u0026plusmn;\u0026thinsp;1.671\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFOXO4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.767\u0026thinsp;\u0026plusmn;\u0026thinsp;1.809\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFOXO6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.981\u0026thinsp;\u0026plusmn;\u0026thinsp;1.556\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eFlow cytometry\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eApoptosis\u003c/b\u003e (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.735\u0026thinsp;\u0026plusmn;\u0026thinsp;2.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.689\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered as significant.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: ATG: Autophagy-related; Beclin1: A mammalian ortholog of yeast ATG6; LC3B: Microtubule-associated proteins 1A/1B light chain 3B; FOXO: Forkhead box transcription factors.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Increased expression levels of FOXO family genes in women with thin endometrium\u003c/h2\u003e\u003cp\u003eOur results showed a significant expression levels of FOXO1 (\u0026lt;\u0026thinsp;0.0001), FOXO3a (\u0026lt;\u0026thinsp;0.0001), FOXO4 (\u0026lt;\u0026thinsp;0.0001) and FOXO6 (0.0003) genes in women with thin endometrium compared to healthy pregnant women (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e ; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The expression level of these genes in women with thin endometrium was higher than healthy pregnant women.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Correlation assay\u003c/h2\u003e\u003cp\u003eThe correlation analysis between the percentage of apoptosis of PBMC cells and the expression of ATG5, LC3B, Beclin1 and FOXO1 genes in patients with thin endometrium was performed. According to the reported data, about the L3CB gene, the obtained correlation is positive and significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, p\u0026thinsp;=\u0026thinsp;0.0339, r\u0026thinsp;=\u0026thinsp;0.3362).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5.Bioinformatics Analysis\u003c/h2\u003e\u003cp\u003eTo further elucidate the biological pathways and processes associated with the differentially expressed autophagy genes, we conducted a KEGG pathway enrichment analysis using the EnrichR tool (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe results of Bioinformatics Analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTerm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAdjusted P-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOdds Ratio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCombined Score\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShigellosis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.50E-06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.16E-05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e81.61157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1094.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO6;FOXO4;FOXO1;ATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFoxO signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.50E-05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.58E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e93.1125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1034.156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO6;FOXO4;FOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFerroptosis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.14E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.97E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e170.5385\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1548.508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eATG7;ATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLongevity regulating pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.07E-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.003711\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e66.30667\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e481.0329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1;ATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAutophagy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.001269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.005332\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e49.02963\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e326.9852\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eATG7;ATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMitophagy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.026883\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e42.48401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e153.6333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRIG-I-like receptor signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.027664\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e41.24845\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e147.9839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProstate cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.038154\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29.60714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e96.70056\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAGE-RAGE signaling pathway in diabetic complications\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.039314\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28.70563\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e92.89679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlucagon signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.042014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.80054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e84.95102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInsulin resistance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.042399\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.54873\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e83.91052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAMPK signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.047012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.85714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e72.93978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThyroid hormone signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.047395\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.076562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.65714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e72.13611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInsulin signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.053513\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.080269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20.85714\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61.06622\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellular senescence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.060733\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.085026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18.28295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.21548\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNOD-like receptor signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.070159\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.086661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15.72381\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e41.77794\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eATG5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNeutrophil extracellular trap formation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.073158\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.086661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15.04863\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e39.35412\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eATG7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTranscriptional misregulation in cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.074281\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.086661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.81002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e38.50463\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRas signaling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.089133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.098516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.22078\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e29.54525\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHuman papillomavirus infection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.13125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.511688\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17.69958\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePathways in cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.193707\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.193707\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.245822\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.610534\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFOXO1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe KEGG pathway enrichment analysis revealed several significantly enriched pathways (combined score\u0026thinsp;\u0026gt;\u0026thinsp;2) related to the differentially expressed autophagy genes in the thin endometrium group. The top-ranked pathways included \"Shigellosis\" (combined score\u0026thinsp;=\u0026thinsp;1094.192), \"FoxO signaling pathway\" (combined score\u0026thinsp;=\u0026thinsp;1034.156), and \"Ferroptosis\" (combined score\u0026thinsp;=\u0026thinsp;1548.508).\u003c/p\u003e\u003cp\u003eThe \"Shigellosis\" pathway was significantly enriched, involving the FOXO1, FOXO4, FOXO6, and ATG5 genes. The involvement of FOXO transcription and the autophagy gene ATG5 in this pathway suggests a potential link between bacterial infection, inflammation, and autophagy dysregulation in the endometrium[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] .Shigellosis, caused by the Shigella bacteria, is known to induce host cell autophagy as a defense mechanism[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the pathogen can also hijack the autophagy machinery to promote its own survival and replication[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The dysregulation of this pathway may contribute to an imbalance in the endometrial immune response, potentially leading to chronic inflammation and impaired tissue homeostasis, which are hallmarks of endometrial atrophy[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eThe \"FoxO signaling pathway\" was also among the top enriched pathways, highlighting the important role of FOXO transcription factors (FOXO1, FOXO4, FOXO6) in the regulation of autophagy and other cellular processes. The FOXO transcription factors play a critical role in the regulation of autophagy, cell cycle, and cell survival[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].Dysregulation of the FoxO signaling pathway may lead to altered endometrial cell proliferation, differentiation, and apoptosis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which are essential for maintaining endometrial thickness and function .Impaired FoxO signaling can disrupt the delicate balance between cell renewal and cell death[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], potentially contributing to the development of endometrial atrophy.\u003c/p\u003e\u003cp\u003eFurthermore, the \"Ferroptosis\" pathway, which is a form of regulated cell death[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], was significantly enriched and involved the ATG7 and ATG5 genes. Ferroptosis is a form of regulated cell death characterized by the accumulation of lipid peroxides and iron-dependent oxidative stress.The involvement of the autophagy genes ATG7 and ATG5 in this pathway suggests a potential link between dysregulated autophagy and increased ferroptosis in the endometrium .Elevated ferroptosis, coupled with impaired autophagy, may lead to excessive cell death and failure to maintain the endometrial tissue, ultimately contributing to endometrial atrophy.\u003c/p\u003e\u003cp\u003eOther significantly enriched pathways included \"Longevity regulating pathway,\" \"Autophagy,\" and \"Mitophagy\", further highlights the critical role of autophagy-related genes in endometrial homeostasis and function .Dysregulation of these pathways may impair cellular processes, such as energy metabolism, mitochondrial quality control, and cellular senescence, which are essential for endometrial tissue maintenance and regeneration .\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOne of the important issues in pregnancy is the thickness of the endometrium. The best chance of a full-term pregnancy is associated with a proportionally thick endometrium. This optimal thickness allows the embryo to implant properly and provide the required nutrition. Therefore, the evaluation of endometrial thickness in the process of in vitro fertilization (IVF) is performed as an index of endometrial acceptance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Several reports have described low pregnancy rates with thin endometrium [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A challenging debate among doctors is the appropriate thickness of the endometrium for conception. Generally, a thickness of less than 7 mm in ultrasound is considered less than the determined limit for in vitro fertilization and is associated with a decrease in the probability of pregnancy.\u003c/p\u003e\u003cp\u003eAutophagy has a key role and diverse functions in the endometrium. New evidence obtained supports the main role of autophagy in preserving endometrial physiology (endometrial cyclic regeneration, menstruation and implantation, and decidualization during pregnancy) and endometrial pathology (hyperplasia, endometriosis, endometrial cancer, and atrophic endometrium) [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. According to the results obtained from the studies, autophagy causes cell death by destroying the basic components of cells, or it can even be caused by apoptosis-inducing stimuli. In two studies conducted by Choi et al [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] it was found that autophagy causes cell death by inducing apoptosis in granulosa and luteal cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBased on the above reports, in the first stage, we evaluated the frequency of apoptotic cells in patients and control groups. Our results showed a significant increase in the frequency of apoptotic cells in patients with thin endometrium compared to the control group. Choi et al reported[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] that autophagy is important in the endometrial cell cycle through apoptosis.\u003c/p\u003e\u003cp\u003eIn the second stage, we assessed the expression level of autophagy related genes including: Atg5, Atg7, Beclin1 (Atg6), LC3B (Atg8), FOXO1, FOXO3a, FOXO4, FOXCO6 .Our results showed a significant difference in the expression of these genes in patients with thin endometrium compared to healthy pregnant women. So the expression level of these genes in patients was higher than in controls. The KEGG pathway enrichment analysis revealed that several significantly enriched pathways are associated with differentially expressed autophagy genes in the thin endometrium group, including \"Shigellosis,\" \"FoxO signaling pathway,\" and \"Ferroptosis.\" The \"Shigellosis\" pathway, involving FOXO1, FOXO4, FOXO6, and ATG5, suggests a link between bacterial infection, inflammation, and autophagy dysregulation, potentially contributing to chronic inflammation and endometrial atrophy. The \"FoxO signaling pathway\" highlights the role of FOXO transcription factors in regulating autophagy and cellular processes, where dysregulation may impact endometrial cell function and thickness[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The \"Ferroptosis\" pathway indicates a connection between dysregulated autophagy and increased cell death in the endometrium[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], implicating autophagy genes ATG7 and ATG5. Other enriched pathways like \"Longevity regulating pathway,\" \"Autophagy,\" and \"Mitophagy\" underscore the importance of autophagy-related genes in endometrial homeostasis, suggesting that dysregulation could impair essential cellular processes and contribute to endometrial atrophy. These findings provide a basis for exploring targeted therapies to restore endometrial health.\u003c/p\u003e\u003cp\u003eIn a study, Liu and colleagues [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] showed that the survival of the embryo after the transfer operation is affected by the thickness of the endometrium, and the survival rate decreased with the decrease in thickness. The role of autophagy in endometrial atrophy has not been thoroughly investigated. Currently, there is no study that identifies the role of autophagy in endometrial atrophy. However, several studies have addressed the role of autophagy genes in endometrium-related diseases.\u003c/p\u003e\u003cp\u003eFeng et al showed increased expression of the autophagy adapter SQSTM1 in a mouse model of endometrial hyperplasia [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Many researches have pointed out the uncoordinated activation of autophagy and the therapeutic potential of anticancer drugs to modulate them in endometrial cancer (EC). For example, in endometrial adenocarcinoma, increased expression of Beclin1 has been reported, which is associated with poor prognosis such as high tumor rate and myometrial invasion [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In contrast, another study reported a decrease in Beclin1 expression in the stage of neoplastic transformation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsidering the relationship between cell death and autophagy genes, in the last step, we examined the correlation between the expression of autophagy genes and cell death in patients. Contrary to our expectation, the expression of only one gene was positively correlated with cell death of PBMCs. In terms of correlation, L3CB gene was positive and significant. It can be concluded that maybe autophagy is closely related to apoptosis in patients with thin endometrium. Therefore, targeting apoptotic pathways in autophagy mechanisms may be a promising strategy for the treatment of these patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eEA:\u003c/strong\u003e Endometrial atrophy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATG:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eAutophagy-related\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC3B:\u003c/strong\u003e Microtubule-Associated Protein 1 Light Chain 3 beta\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFOXO:\u0026nbsp;\u003c/strong\u003eForkhead Box O\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAMPK:\u0026nbsp;\u003c/strong\u003eAMP-activated protein kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emTORC:\u0026nbsp;\u003c/strong\u003eRapamycin complex 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eER:\u0026nbsp;\u003c/strong\u003eEndoplasmic reticulum\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePE:\u0026nbsp;\u003c/strong\u003ePhosphoethanolamine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC3:\u0026nbsp;\u003c/strong\u003elipidation of microtubule-associated light chain 3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBMCs:\u0026nbsp;\u003c/strong\u003ePeripheral blood mononuclear cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKEGG:\u003c/strong\u003e Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIVF:\u0026nbsp;\u003c/strong\u003eIn vitro fertilization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEC:\u0026nbsp;\u003c/strong\u003eEndometrial cancer\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Ethics Committee of the Deputy Director of the Immunology Research Center\u0026nbsp;(IR.TBZMED.REC.1401.733).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Tabriz University of Medical Sciences, Tabriz, Iran [grant number:\u0026nbsp;70489].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMadani, J., et al., \u003cem\u003eFetus, as an allograft, evades the maternal immunity.\u003c/em\u003e Transpl Immunol, 2022. \u003cstrong\u003e75\u003c/strong\u003e: p. 101728.\u003c/li\u003e\n\u003cli\u003eSarkesh, A., et al., \u003cem\u003eThe immunomodulatory effect of intradermal allogeneic PBMC therapy in patients with recurrent spontaneous abortion.\u003c/em\u003e J Reprod Immunol, 2023. \u003cstrong\u003e156\u003c/strong\u003e: p. 103818.\u003c/li\u003e\n\u003cli\u003eAbdalla, H., et al., \u003cem\u003eEndometrial thickness: a predictor of implantation in ovum recipients?\u003c/em\u003e Human Reproduction, 1994. \u003cstrong\u003e9\u003c/strong\u003e(2): p. 363\u0026ndash;365.\u003c/li\u003e\n\u003cli\u003eGonen, Y., et al., \u003cem\u003eEndometrial thickness and growth during ovarian stimulation: a possible predictor of implantation in in vitro fertilization.\u003c/em\u003e Fertility and sterility, 1989. \u003cstrong\u003e52\u003c/strong\u003e(3): p. 446\u0026ndash;450.\u003c/li\u003e\n\u003cli\u003eRichter, K.S., et al., \u003cem\u003eRelationship between endometrial thickness and embryo implantation, based on 1,294 cycles of in vitro fertilization with transfer of two blastocyst-stage embryos.\u003c/em\u003e Fertility and sterility, 2007. \u003cstrong\u003e87\u003c/strong\u003e(1): p. 53\u0026ndash;59.\u003c/li\u003e\n\u003cli\u003eAlam, V., et al., \u003cem\u003eA prospective study of echographic endometrial characteristics and pregnancy rates during hormonal replacement cycles.\u003c/em\u003e Journal of assisted reproduction and genetics, 1993. \u003cstrong\u003e10\u003c/strong\u003e: p. 215\u0026ndash;219.\u003c/li\u003e\n\u003cli\u003eHershko-Klement, A. and R. 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Germeyer, \u003cem\u003eDecreased autophagy impairs decidualization of human endometrial stromal cells.\u003c/em\u003e 2019.\u003c/li\u003e\n\u003cli\u003eLebovitz, C.B., et al., \u003cem\u003eCross-cancer profiling of molecular alterations within the human autophagy interaction network.\u003c/em\u003e Autophagy, 2015. \u003cstrong\u003e11\u003c/strong\u003e(9): p. 1668\u0026ndash;1687.\u003c/li\u003e\n\u003cli\u003eFeng, L., et al., \u003cem\u003eTamoxifen activates Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia.\u003c/em\u003e Theranostics, 2017. \u003cstrong\u003e7\u003c/strong\u003e(7): p. 1890.\u003c/li\u003e\n\u003cli\u003eChoi, J., et al., \u003cem\u003eThe role of autophagy in human endometrium.\u003c/em\u003e Biology of reproduction, 2012. \u003cstrong\u003e86\u003c/strong\u003e(3): p. 70, 1\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eChoi, J.Y., et al., \u003cem\u003eThe role of autophagy in follicular development and atresia in rat granulosa cells.\u003c/em\u003e Fertility and sterility, 2010. \u003cstrong\u003e93\u003c/strong\u003e(8): p. 2532\u0026ndash;2537.\u003c/li\u003e\n\u003cli\u003eChoi, J., et al., \u003cem\u003eThe role of autophagy in corpus luteum regression in the rat.\u003c/em\u003e Biology of reproduction, 2011. \u003cstrong\u003e85\u003c/strong\u003e(3): p. 465\u0026ndash;472.\u003c/li\u003e\n\u003cli\u003eLu, J., et al., \u003cem\u003eFOXO1 is a critical switch molecule for autophagy and apoptosis of sow endometrial epithelial cells caused by oxidative stress.\u003c/em\u003e Oxidative Medicine and Cellular Longevity, 2021. \u003cstrong\u003e2021\u003c/strong\u003e(1): p. 1172273.\u003c/li\u003e\n\u003cli\u003eLi, H., et al., \u003cem\u003eAutophagy-dependent ferroptosis is involved in the development of endometriosis.\u003c/em\u003e Gynecological Endocrinology, 2023. \u003cstrong\u003e39\u003c/strong\u003e(1): p. 2242962.\u003c/li\u003e\n\u003cli\u003eLiu, K., et al., \u003cem\u003eThe impact of a thin endometrial lining on fresh and frozen\u0026ndash;thaw IVF outcomes: an analysis of over 40 000 embryo transfers.\u003c/em\u003e Human Reproduction, 2018. \u003cstrong\u003e33\u003c/strong\u003e(10): p. 1883\u0026ndash;1888.\u003c/li\u003e\n\u003cli\u003eGiatromanolaki, A., et al., \u003cem\u003eHigh Beclin 1 expression defines a poor prognosis in endometrial adenocarcinomas.\u003c/em\u003e Gynecologic oncology, 2011. \u003cstrong\u003e123\u003c/strong\u003e(1): p. 147\u0026ndash;151.\u003c/li\u003e\n\u003cli\u003eZhao, J.-H., et al., \u003cem\u003eExpression and clinical significance of Beclin1 and PTEN in endometrial carcinoma.\u003c/em\u003e Ai Zheng= Aizheng= Chinese Journal of Cancer, 2006. \u003cstrong\u003e25\u003c/strong\u003e(6): p. 753\u0026ndash;757.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autophagy, Endometrium, Apoptosis, ATG, FOXO","lastPublishedDoi":"10.21203/rs.3.rs-7704238/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7704238/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction: Endometrial atrophy (EA) is a disease in which the endometrium becomes very thin, so that its thickness never reaches more than 5 mm. In most cases, the origin of EA is not known, but factors can cause thinning of the endometrium, including contraceptive drugs, inflammatory, iatrogenic, and in some cases, hereditary factors. Autophagy has an important consequence in the proper functioning of the uterus, reproductive physiology and endometrial atrophy.\u003c/p\u003e\u003cp\u003eMethod: In this study, Real-Time PCR was used to measure the expression levels of autophagy genes ATG5, ATG7, LC3B, Beclin1, FOXO1, FOXO3a, FOXO4 and FOXO6 in 40 women with thin endometrium and 40 healthy pregnant women. In addition to, apoptosis was done by flow cytometry method.\u003c/p\u003e\u003cp\u003eResults: Evaluation of the expression level of autophagy genes showed a significant difference in studied groups, so that the expression levels of ATG5, ATG7 and LC3B, Beclin1, FOXO1, FOXO3a, FOXO4 and FOXO6 genes were higher in the patient group. Moreover, there was a positive correlation between the expression of autophagy gene LC3B and the frequency of apoptotic cells in the studied patients.\u003c/p\u003e\u003cp\u003eDisscusion: To further elucidate the biological pathways and processes associated with the differentially expressed autophagy genes, we conducted a KEGG pathway enrichment analysis using the EnrichR tool. Our results showed that autophagy genes with apoptosis in PBMC cells may be involved in endometrial thinning of EA patients.\u003c/p\u003e","manuscriptTitle":"Investigating autophagy genes expression and their possible relations with apoptosis in PBMCs of patients with thin endometrium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 02:43:15","doi":"10.21203/rs.3.rs-7704238/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4039dd5-821b-4e1f-abd4-3248ec6f71f8","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-28T13:43:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 02:43:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7704238","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7704238","identity":"rs-7704238","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}