Enhanced glycolysis in the myometrium with ectopic endometrium of patients with adenomyosis: a preliminary study

Abstract

Objectives

The objective of this study was to investigate the glycolytic activity of adenomyosis, which is characterized by malignant biological behaviors including abnormal cell proliferation, migration, invasion, cell regulation, and epithelial-mesenchymal transition.

Methods

From January 2021 to August 2022, a total of 15 patients who underwent total hysterectomy for adenomyosis and 14 patients who had non-endometrial diseases, specifically with cervical squamous intraepithelial neoplasia and uterine myoma, were included in this study. Myometrium with ectopic endometrium from patients with adenomyosis while normal myometrium from patients in the control group were collected. All samples were confirmed by a histopathological examination. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS), real-time quantitative PCR, NAD+/NADH assay kit as well as the glucose and lactate assay kits.

Results

Endometrial stroma and glands could be observed within the myometrium of patients in the adenomyosis group. We found that the mRNA expressions of HK1, PFKFB3, glyceraldehyde-3-phospate dehydrogenase (GAPDH), PKM2, and PDHA as well as the protein expressions of PFKFB3 were elevated in ectopic endometrial tissues of the adenomyosis group as compared to normal myometrium of the control group. The level of fructose 1,6-diphosphate was increased while NAD + and NAD+/NADH ratio were decreased compared with the control group. Besides, increased glucose consumption and lactate production were observed in myometrium with ectopic endometrium.

Conclusions

We concluded that altered glycolytic phenotype of the myometrium with ectopic endometrium in women with adenomyosis may contribute the development of adenomyosis.

Keywords:

• Adenomyosis
• myometrium with ectopic endometrium
• glycolysis
• glycolytic enzymes
• lactate

Introduction

Adenomyosis is a chronic gynecological disease that frequently characterized by abnormal uterine bleeding, dysmenorrhea, dyspareunia, and infertility, negatively affecting fertility, pregnancy outcomes, and the quality of life of adenomyosis patients. Patients with adenomyosis or endometriosis have an increased risk for developing various cancers, such as endometrial and ovarian cancers, suggesting that certain cellular functions may be disrupted in patients with adenomyosis and endometriosis [1,2]. Although the pathology of adenomyosis is histologically benign, it is characterized by malignant biological behaviors, such as abnormal cell proliferation, migration, invasion, cell regulation, and epithelial-mesenchymal transition [3,4], which may be related to metabolic reprogramming.

It has been hypothesized that shifts in mitochondrial bioenergetic pathways from oxidative phosphorylation (OXPHOS) to glycolysis could be involved in estrogen-induced tumorigenesis [5]. Glycolysis is closely related to biological behaviors such as proliferation, epithelial-mesenchymal transition, angiogenesis, metastasis, and immunosuppression [6,7]. Most cancer cells rely on aerobic glycolysis for energy supply rather than mitochondrial OXPHOS [8]. Glycolysis could cause a high metabolic rate of glucose in tumor cells and acidification in the microenvironment due to lactate accumulation. Lactate is an end-product of glycolysis, which has a marked effect on immune evasion, angiogenesis, and chemoresistance [9,10]. Ectopic endometriotic lactate induces M2 macrophage polarization, cell invasion, and angiogenesis, which are crucial steps in the development of endometriosis and adenomyosis [11,12].

Upregulated expression of key glycolytic enzymes can significantly promote aerobic glycolysis. Pyruvate kinase isozyme M2 isoform (PKM2) is one of the critical enzymes in the glycolytic pathway and plays an essential role in maintaining the metabolic homeostasis of tumor cells. It has been shown that the mRNA expression of PKM2 in adenomyosis in eutopic endometrial tissues during both the secretory and proliferative phases are higher than those in normal endometrial tissues [13]. The invasion of the endometrial basalis of adenomyosis may be related to the increased expression of PKM2, suggesting that glycolytic function of adenomyosis cells is more active than that of normal endometrial tissues [13]. The proteomic result from a previous study revealed that the factors related to endometriosis glycolysis were upregulated, while those related to TCA cycle and OXPHOS ere downregulated, suggesting that endometriosis tissues preferred glycolytic metabolism over mitochondrial OXPHOS to generate energy for cell growth [2]. These malignant biological behaviors may lead to hypoxia in ectopic endothelial tissues, while the hypoxic microenvironment induces cellular glycolysis and promotes ectopic endothelial implantation, growth, neointima formation, and epithelial-mesenchymal transformation.

Ectopic endometrium is critical to the development of adenomyosis. Understanding the physiology of ectopic endometrium in patients with adenomyosis can help understand the pathology of adenomyosis. To the best of our knowledge, this study is the first report on the glycolytic function of myometrium with ectopic endometrium in women with adenomyosis.

Methods

Patient selection and tissue collection

This study was approved by the ethical committee of the Third Affiliated Hospital of Guangzhou University of Chinese Medicine (No. 2020025). From January 2021 to August 2022, women with adenomyosis (adenomyosis group), aged 20–50 years, without endometriosis or uterine fibroids and had undergone hysterectomy were included. The gold standard to make the definitive diagnosis of adenomyosis has always been considered histological examination of hysterectomy specimens [14]. Additionally, 14 patients with uterine myoma but without adenomyosis or endometriosis were selected as matched myometrial tissue controls. All samples underwent histopathological examination for confirmation. In total, the study included 15 patients with adenomyosis and 14 control patients. The clinical characteristics of the participants are shown in Supplementary Table 1.

All patients in the adenomyosis group were diagnosed as having benign invasion of the endometrial stroma and glands into the myometrium [15]. Patients with squamous cervical intraepithelial neoplasia and uterine myoma were included in the control group. Patients in both groups were non-menopausal and had not received endocrine therapy within three months prior to the visit. Exclusion criteria included patients who did not meet the criteria for adenomyosis, cervical squamous intraepithelial neoplasia, or uterine myoma; those with other gynecological conditions, such as endometrial cancer, endometrial polyps, or cervical cancer with FIGO stage II or higher; and those who were menopausal. All patients gave informed consent. The myometrium with ectopic endometrium from adenomyosis patients and the normal myometrium from control patients were collected for further study.

Histological analysis

Hematoxylin and eosin (H&E) staining was performed to determine whether the endometrium had invaded the myometrium. The ectopic lesion from patients with adenomyosis and the myometrium from patients without adenomyosis were fixed in 4% paraformaldehyde for at least 24 h, followed by paraffin embedding and continuous sectioning at 4 μm sections. The sections were stained with H&E according to standard procedures for observation.

Quantitative real time-polymerase chain reaction (qRT-PCR) analysis

Total RNA was isolated from the myometrium with ectopic endometrium from patients with adenomyosis and normal myometrium from controls using Nucleozol reagent according to the manufacturer’s instructions. Nano-drop measurements of RNA samples were processed to determine the quality and quantity of the RNA, and cDNA was synthesized from the purified total RNA using cDNA synthesis kit (Vazyme, China). Real-time polymerase chain reaction (RT-PCR) was performed using SYBR green RT-PCR kit (Vazyme, China) and custom-designed primers with the CFX96 TouchTM real-time PCR detection system (Bio-Rad, Hercules, CA). β-actin was used as an internal control for mRNAs. Relative gene expression was determined using the 2^−ΔΔCT method. The primer sequences are shown in Supplementary Table 2.

Protein extraction and Western blot analysis

The tissues were lysed using Radio Immunoprecipitation Assay (RIPA) buffer (phenylmethanesulfonylfluoride (PMSF)/RIPA: 1/100) and homogenized with a pellet pestle mixer. Protein was collected after centrifugation at 14,000 g for 10 min at 4 °C, and the concentration was determined through the Bicinchoninic Acid Assay (BCA) method. The protein from each group was prepared for SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat dry milk for 2 h and subsequently probed with the following primary antibodies: HK1 (2024s, CST, Danvers, MA), 6-phosphofructo-2-kinase/fructose 2,6-biphosphatase 3 (PFKFB3, ab181861, Abcam, Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab181602, Abcam), PKM2 (ab150377, Abcam), PDHA (ab168379, Abcam), and β-Actin (af7018, affinity, Jiangsu, China) in a blocking buffer overnight at 4 °C. This was followed by incubation with a horseradish-peroxidase (HRP)-conjugated secondary antibody (Southern Biotech, Birmingham, AL). Finally, the signals of protein bands were developed by enhanced chemiluminescence (ECL). The relative protein expression was calculated by the gray value of target protein/gray value of reference protein (β-actin).

Metabolomics

The samples were accurately weighed into a 2 mL centrifuge tube and 1000 µL of extraction medium (75% 9:1 methanol:chloroform, 25% H₂O) was added, and subsequently ground at 50 Hz for 60 s, sonicated for 30 min, incubated on ice for 30 min, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was transferred to a centrifuge tube, concentrated and dried. Subsequently, 2-amino-3-(2-chloro-phenyl)-propionic acid was added to dissolve the sample, the supernatant was filtered through a 0.22 μm membrane and transferred to the vial for liquid chromatography-mass spectrometry (LC-MS) detection as previously described [16,17].

The LC analysis was performed on a Vanquish UHPLC System (Thermo Fisher Scientific, Waltham, MA). Chromatography was carried out with an ACQUITY UPLC^® HSS T3 (150 × 2.1 mm, 1.8 µm) (Waters, Milford, MA). The column was maintained at 40 °C. The flow rate and injection volume were set at 0.25 mL/min and 2 μL, respectively. For LC-ESI (+)-MS analysis, the mobile phases consisted of (C) 0.1% formic acid in acetonitrile (v/v) and (D) 0.1% formic acid in water (v/v). Separation was conducted under the following gradient: 0–1 min, 2% C; 1–9 min, 2–50% C; 9–12 min, 50–98% C; 12–13.5 min, 98% C; 13.5–14 min, 982% C; 14–20 min, 2% C. For LC-ESI (-)-MS analysis, the analytes were subjected to solvent systems consisting of (A) acetonitrile and (B) ammonium formate (5 mM). Separation was conducted under the following gradient: 0–1 min, 2%A; 1–9 min, 2–50% A; 9–12 min, 50–98% A; 12–13.5 min, 98% A; 13.5–14 min, 98–2% A; 14–17 min, 2% A [16].

Mass spectrometric detection of metabolites was performed on Q Exactive (Thermo Fisher Scientific) with an ESI ion source. Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition was used. The parameters were as follows: sheath gas pressure, 30 arb; aux gas flow, 10 arb; spray voltage, 3.50 and −2.50 kV for ESI (+) and ESI (–), respectively; capillary temperature, 325 °C; MS1 range, m/z 81–1000; MS1 resolving power, 70,000 FWHM; number of data-dependent scans per cycle, 10; MS/MS resolving power, 17,500 FWHM; normalized collision energy, 30%; dynamic exclusion time, automatic [17].

The NAD+/NADH ratio in myometrium with ectopic endometrium in patients with adenomyosis

NADH levels as well as the ratio of NAD + and NADH were determined using NAD+/NADH assay kit with WST-8 method (Beyotime, Nantong, China) according to the manufacturer’s instructions. Briefly, tissues were prepared with NAD+/NADH extraction solution and the supernatant was retained after homogenization and centrifugation. To measure total NAD+/NADH, 20 μL of the prepared tissues suspension was added to a 96-well plate. To measure NADH, the prepared tissues suspension was incubated at 60 °C for 30 min and 20 μL was added to a 96-well plate. Subsequently, 90 μL of alcohol dehydrogenase was added and incubated at 37 °C for 10 min. Finally, 10 μL of chromogenic solution was added to the plate and the mixture was incubated at 37 °C for 30 min. The standard curve was generated and measured at the same time as the samples. The absorbance values were measured at 450 nm and analyzed using a plate reader. The NAD+/NADH ratio was calculated using the standard curves for NAD + and NADH.

Glucose measurement

The tissues were weighed and mechanically homogenized with saline, centrifuged at 2500 rpm for 10 min, and the supernatant was removed for measurement. The glucose assay kit (Nanjing Jiancheng, Jiangsu, China) was used according to the instructions, and the optical density (OD) of the enzyme marker was measured at 505 nm. The tissue glucose level was calculated as: glucose level (mmol/L) = (OD of sample wells – OD of blank wells)/(OD of standard wells – OD of blank wells) * standard concentration (5.55 mmol/L) * dilution ratio.

Lactate measurement

The tissues were weighed and mechanically homogenized with saline, and centrifuged at 2500 rpm for 10 min. The supernatant was measured using a lactate assay kit (Nanjing Jiancheng, Jiangsu, China). The OD value was measured at 530 nm on an enzyme marker according to the instructions of the lactate assay kit. The tissue lactate level was calculated as: lactate content (mmol/L) = (OD of sample wells – OD of blank wells)/(OD of standard wells – OD of blank wells) * standard concentration (3 mmol/L).

Statistical analysis

The Shapiro–Wilk test was used to test the normal distribution of continuous variables. Normally distributed data were expressed as mean ± standard deviation (SD) and differences between groups were analyzed by two independent samples t-test. Skewed data were expressed as median (interquartile range) and differences between groups were analyzed by two Mann–Whitney U tests. SPSS version 25.0 (SPSS, Chicago, IL) was used to analyze the data and GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA) was used for graphing. A p value less than 0.05 was considered statistically significant.

Results

Clinical characteristics of patients

Baseline characteristics are listed in Table 1. The levels of carbohydrate antigen 125 (CA125) were significantly higher in the adenomyosis group than in the control group (p < 0.001, Table 1).

Table 1. Clinical characteristics of patients with adenomyosis.

	Control, N = 14	Adenomyosis, N = 15	p Value
Age, mean (SD), years	47.50 (3.06)	46.13 (4.05)	0.317
Uterine volume, median (IQR), cm^3	363.49 (133.81– 673.59)	523.60 (378.29 to 1016.96)	0.116
Gravidity, median (IQR)	3.00 (2.75–4.00)	4.00 (2.00–5.00)	0.421
Parity, median (IQR),	2.00 (2.00–3.00)	2.00 (1.00–2.00)	0.146
Abortions times, median (IQR),	1.00 (0.00–2.00)	2.00 (0.00–4.00)	0.134
CA125, median (IQR), U/mL	15.01 (12.60–24.64)	49.56 (31.79–78.87)	<0.001
BMI, mean (SD) kg/m^2	22.01 (2.35)	22.08 (2.40)	0.935
Patients with anemia, No. (%)	11 (78.57)	14 (93.33)	0.119

Values are expressed as mean (SD) or median (25th–75th percentile). The comparisons between data were performed using the independent samples t-test for normally distributed data or the Mann–Whitney U test for skewed data. A p value less than 0.05 was considered statistically significant.

Morphology

As shown in Figure 1, endometrial stroma and glands could be observed within the myometrium of patients in the adenomyosis group, whereas the myometrium of the patients in the control group subjects exhibited regularly arranged smooth muscle tissue devoid of endometrial stroma and glands.

Figure 1. Morphology of the myometrium in women with adenomyosis. Black arrows refer to myometrium with ectopic endometrium.

Glycolysis-related enzymes upregulated in myometrium with ectopic endometrium in patients with adenomyosis

In this study, the mRNA expression of GLUT1, HK1, HK2, PFKFB3, GAPDH, PKM2, PDHA, and LDHA were detected to assess the activity of glycolysis in myometrium with ectopic endometrium in women with adenomyosis.

As shown in Figure 2(A), the gene expressions of HK1, PFKFB3, GAPDH, PKM2, and PDHA of myometrium with ectopic endometrium were elevated in the adenomyosis group compared with the control group (p < 0.001, p < 0.05, p < 0.01, p < 0.05, and p < 0.001, respectively, Figure 2(A)). The expression of GLUT1 and HK2 mRNA in the patients’ myometrium with ectopic endometrium in the adenomyosis group showed an upward trend when compared to the myometrium of the control group (p > 0.05, Figure 2(A)).

Figure 2. Glycolysis-related enzymes of myometrium with ectopic endometrium from women with adenomyosis. (A) The mRNA expression of GLUT1, HK1, HK2, PFKFB3, GAPDH, PKM2, PDHA, and LDHA of myometrium with ectopic endometrium from women with adenomyosis (n = 15) and normal myometrium from controls (n = 14). (B) The protein expression of HK1, PFKFB3, GAPDH, PKM2, and PDHA of myometrium with ectopic endometrium from women with adenomyosis (n = 6) and normal myometrium from controls (n = 6). Values are expressed as mean (SD) or median (25th–75th percentile). The comparisons between data were performed using the Mann–Whitney U test for skewed data. A p value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. GLUT1: glucose transporter 1; HK1: Hexokinase 1; HK2: Hexokinase 2; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; GAPDH: glyceraldehyde-3-phospate dehydrogenase; PKM2: pyruvate kinase M; PDHA: pyruvate dehydrogenase E1 alpha subunit; LDHA: lactate dehydrogenase A.

In addition, the protein expressions of HK1, PFKFB3, GAPDH, PKM2, and PDHA were observed. We found that the protein expression of PFKFB3 was significantly increased in myometrium with ectopic endometrium in the adenomyosis group when compared to the myometrium of the control group (p < 0.01, Figure 2(B)). The protein expressions of HK1, GAPDH, and PKM2 were also increased in myometrium with ectopic endometrium in women with adenomyosis without statistical difference (p > 0.05, Figure 2(B)). These results suggested that the factors regulating glycolysis of myometrium with ectopic endometrium from women with adenomyosis were aberrantly expressed when compared with the normal myometrium of the control group.

Metabolomics of glycolytic metabolites

As shown in Figure 3, there were six metabolites related to glycolysis were detected by metabolomics, namely glucose 6-phosphate, fructose 1,6-diphosphate, nicotinamide adenine dinucleotide oxidized form (NAD+), dihydroxyacetone phosphate, glycerol 3-phosphate, and phosphoenolpyruvate. Compared with the normal myometrium of control patients, a significant increase in the concentration of fructose 1,6-diphosphate (p < 0.01, Figure 3(C)) and a decrease in NAD+ (p < 0.05, Figure 3(F)) were observed in the myometrium with ectopic endometrium of adenomyosis, indicating enhanced glycolytic activity. Furthermore, we also found an increase of NADH and a decrease in the ratio of NAD + and NADH (p < 0.05 for both, Figure 3(I,J). Glycerol 3-phosphate was also decreased in myometrium with ectopic endometrium in women with adenomyosis, although the group difference was not significant by Mann–Whitney U test (p > 0.05, Figure 3(D)).

Figure 3. Metabolites of myometrium with ectopic endometrium in women with adenomyosis. The levels of glucose 6-phosphate (B) fructose 1,6-bisphosphate (C), glycerol 3-phosphate (D), dihydroxyacetone phosphate (E), NAD+ (F), phosphoenolpyruvate (G) were determined by metabonomics (n = 6/group). And the levels of glucose (A), lactate (H), NADH (I), and NAD+/NADH ratio (J) of myometrium with ectopic endometrium from women with adenomyosis (n = 15) and normal myometrium from controls (n = 14) were determined by specific assay kits. Values are expressed as mean (SD) or median (25th–75th percentile). The comparisons between data were performed using the independent samples t-test for normally distributed data or the Mann–Whitney U test for skewed data. A p value less than 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. NAD+: nicotinamide adenine dinucleotide oxidized form.

Glucose consumption and lactate production in myometrium with ectopic endometrium in adenomyosis

The decreased glucose concentration of myometrium with ectopic endometrium in women with adenomyosis was observed when compared with normal myometrium of women without adenomyosis (p < 0.001, Figure 3(A)), indicating increased glucose consumption in myometrium with ectopic endometrium of women with adenomyosis. Lactate concentration of myometrium with ectopic endometrium tissue was significantly increased in the adenomyosis group when compared with the control group (p < 0.01, Figure 3(H)), indicating increased lactate production in myometrium with ectopic endometrium from women with adenomyosis, which was consistent with the upregulated expression of glycolysis-related enzymes.

Discussion

Adenomyosis is a common estrogen-dependent disorder that is considered an important risk factor for the development and progression of estrogen-dependent endometrial cancer. Despite the recent improvements in diagnostic tools, there is still a lack of common definition, classification and knowledge of the disease. Nowadays, attention has started to focus on the role of metabolic abnormalities in the development of endometrial hyperplasia. To our knowledge, this study was the first to compare the expression of key glycolytic enzymes between the myometrium with ectopic endometrium of adenomyosis patients and normal myometrium of non-adenomyosis patients.

It is known that cells produce ATP mainly through metabolic pathways, including glycolysis in the cytoplasm, the pentose phosphate pathway, the tricarboxylic acid cycle in the mitochondria, and OXPHOS [18]. Most tumor cells mainly use glycolysis to obtain energy regardless of the presence or absence of oxygen. Each stage of glycolysis is primarily catalyzed by a specific enzyme, so that the expression of glycolysis-related metabolic enzymes may indirectly reflect the glycolytic activity [19]. We profiled the expression of GLUT1, HK1, HK2, PFKFB3, GAPDH, PKM2, PDHA, and LDHA in the endometrium by quantitative PCR analysis. Glycolysis begins with the uptake of glucose through GLUT family carriers, followed by the synthesis of glucose through crucial enzymes that play an essential role in cellular energy metabolism. The data showed that the expression of the GLUT1 gene tended to increase in the endometrium of women with adenomyosis. Consistent with previous studies, our PCR data showed that the upregulation of PKM2 mRNA was observed in adenomyosis patients. Moreover, we also found that the expression of HK1, PFKFB3, GAPDH, and PDHA mRNA were upregulated in myometrium with ectopic endometrium in women with adenomyosis than in normal myometrium in women without adenomyosis, indicating increased glycolysis in myometrium with ectopic endometrium in women with adenomyosis.

Our study showed a significant increase in 1,6-diphosphate fructose in ectopic endothelium from women with adenomyosis compared with normal myometrium from women without adenomyosis, which is consistent with the upregulated expressions of PFKFB3 mRNA and protein. PFKFB3 is one of the key enzymes in glycolysis, converting fructose-6-bisphosphate to fructose-2,6-bisphosphate, which is the most effective transactivator of the rate-limiting enzyme phosphofructokinase 1 (PFK1)[20]. Upregulated PFKFB3 in myometrium with ectopic endometrium of adenomyosis patients promotes the conversion of fructose-6-phosphate to fructose-2,6-diphosphate, which further activates PFK1 and then catalyzes fructose-6- bisphosphate to fructose-1,6-diphosphate. It was shown that the glycolytic process in myometrium with ectopic endometrium of women with adenomyosis may be regulated through PFKFB3.

Nicotinamide adenine dinucleotide (reduced: NADH or oxidized: NAD+) is a key cofactor for electron transfer in metabolism. Reduction–oxidation (redox) reactions catalyzed by various NAD(H)-dependent dehydrogenases are essential for biochemical processes such as glycolysis and mitochondrial metabolism [21]. In our study, we found that the level of NAD + was decreased in the myometrium with ectopic endometrium of women with adenomyosis. During glycolysis biosynthesis, NAD + serves as an electron acceptor for reactions catalyzed by GAPDH, and is thereby reduced to NADH [22]. Consequently, the NADH/NAD + ratio in cancer cells is usually very high [21]. A significant decrease in NAD + level and NAD+/NADH ratio was observed in the myometrium with ectopic endometrium of women with adenomyosis, which was consistent with the increasing level of GAPDH mRNA, indicating that more NAD + converted to NADH and H + and in turn participated in the glycolytic process. However, the detailed mechanism of regulation of endometrial cell survival and proliferation through glycolytic enzymes and their downstream targets in adenomyosis patients needs to be further investigated.

The two most abundant circulating carbon metabolites in the bloodstream are glucose and lactate, which they can be interconverted in glycolysis and gluconeogenesis [23]. Glucose is a primary energy source and is used for maintaining cellular energy balance through the process of aerobic glycolysis and mitochondrial OXPHOS [21]. Pyruvate can be converted into lactate quickly by LDHA, which is the final product of glycolysis and has been shown to play an important role in cell invasion, angiogenesis and wound healing, ischemic tissue damage, memory formation, and cancer growth and metastasis [24,25]. The lactate concentration in tumor tissues is significantly higher than that in normal tissues, and the increased serum concentration of lactate is associated with metastasis and rapid growth of tumors [26]. In our study, the increase in glucose consumption and lactate production were consistent with the aberrant regulation of glycolytic enzyme activities and changes in fructose 1,6-diphosphate fructose and NAD+. Enhanced glycolysis function was found in ectopic endothelium in adenomyosis, which may lead to endometrial invasion into myometrium and promote the development of adenomyosis.

Our study is not without limitations. One potential weakness is the limited sample size, which may have induced selection bias and confounding bias in the statistical analysis. Additionally, the expression and location effects of glycolytic enzymes in patients with adenomyosis patients were not conducted using immunohistochemistry.

Conclusion

In conclusion, our findings suggested the possible involvement of glycolysis in the pathogenesis and development of adenomyosis. This study may provide new insight into the diagnosis and treatment of adenomyosis. In our forthcoming study, our team intends to examine the efficacy of inhibitor of the glycolytic pathway in adenomyosis in vivo or in vitro.

Authors’ contributions

Shiya Huang, Xueqian Huang, and Xiaohui Wen performed most of the experiments; Shiya Huang and Xueqian Huang drafted the manuscript. Xuehong Liu, Linling Xie, Yishu Wang, and Shanjia Liu contributed to the data analysis. Kunyin Li and Yongge Guan designed the study. Hongxia Ma and Kunyin Li sought the funding. All authors read and approved the final manuscript.

Acknowledgments

We are grateful to everyone involved in conducting this study, analyzing the data, and writing the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data supporting the findings in this study are available from the corresponding author on reasonable request.

Additional information

Funding

This work was supported by Special Projects in Key Areas for General Universities in Guangdong Province (No. 2021ZDZX2015) and National Natural Science Foundation of China (No. 818751487).