Serum miR-329-3p as a potential biomarker for poor ovarian response in an in vitro fertilization
Article information
Abstract
Objective
Several miRNAs have been identified as differentially expressed in patients with poor ovarian response (POR) compared to those with normal responses. This study aims to assess the potential of serum miR-329-3p as a biomarker for diagnosing POR.
Methods
We conducted a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to confirm the target genes of miR-329-3p. KGN cells were transfected with both miR-329-3p mimic and inhibitor to assess the differential expression of these target genes. In accordance with the Bologna criteria, we enrolled 16 control patients and 16 patients with POR. We collected patient samples, including serum from day 2 and the human chorionic gonadotropin (hCG) day, as well as granulosa and cumulus cells, to validate the expression of miR-329-3p using quantitative real-time polymerase chain reaction.
Results
KEGG pathway analysis revealed that miR-329-3p targeted adenylyl cyclase 9 (ADCY9) and protein kinase A subunit beta (PRKACB), both of which are involved in ovarian steroidogenesis. In KGN cells treated with a miR-329-3p mimic, ADCY9 and PRKACB expression levels were significantly reduced (p<0.05). Elevated levels of miR-329-3p suppressed aromatase expression and 17β-estradiol production by modulating ADCY9 and PRKACB in KGN cells. These effects were also observed in POR patients. Follicle-stimulating hormone receptor (FSHR) expression was diminished in the granulosa cells of POR patients. On day 2, on hCG day, and in granulosa cells, miR-329-3p exhibited high expression levels in the serum of POR patients.
Conclusion
miR-329-3p exhibited increased expression in granulosa cells and in the sera of POR patients. Consequently, we propose that miR-329-3p may be a potential biomarker for the diagnosis of POR.
Introduction
Over the past four decades, assisted reproductive technology has seen exponential growth, with more than 8 million babies born through in vitro fertilization (IVF) [1]. Despite these advances, numerous challenges remain. Controlled ovarian hyperstimulation (COH) is a method employed in assisted reproduction that uses gonadotropins to induce ovulation and promote the growth of multiple ovarian follicles, facilitating the optimal development of early embryos [2]. However, many patients undergoing IVF experience poor follicular development and low 17β-estradiol (E2) levels despite hyperstimulation with gonadotropins, leading to an insufficient number of oocytes. This condition is known as poor ovarian response (POR) [3]. The ovarian response to gonadotropins can vary widely among individuals, from poor to high. This variability is significant because a poor response to hyperstimulation typically results in the retrieval of very few oocytes [4].
There are many different opinions regarding the precise definition of POR. To facilitate a consensus, the European Society of Human Reproduction and Embryology (ESHRE) paper in 2011 cited 24 previous definitions [3]. According to the Bologna criteria, patients must exhibit at least two of the following three characteristics: (1) advanced maternal age or any other risk factor for POR; (2) a history of POR; or (3) an abnormal ovarian reserve test. A diagnosis of POR can be made after two episodes of POR following maximal stimulation, even in the absence of advanced maternal age or an abnormal ovarian reserve test [3]. However, the molecular mechanisms underlying this poor response to hyperstimulation remain largely unknown [5].
Our initial study was predicated on the hypothesis that abnormal function of the follicle-stimulating hormone receptor (FSHR) is a primary cause of POR [6]. Previous research demonstrated that female FSHR+/− mice exhibited characteristics similar to those observed in POR patients, including reduced fertility, significant oocyte loss, and low E2 levels [7]. Furthermore, a clinical study involving POR patients revealed that FSHR protein levels were markedly lower in granulosa cells. Additionally, there was a positive correlation between serum E2 levels and the number of mature oocytes retrieved [8].
MicroRNAs (miRNAs) are small noncoding RNAs, approximately 19 to 25 base pairs in length, that function as translational repressors and play a role in regulating a wide array of biological processes [9]. In various fields, miRNAs are being explored as potential biomarkers for numerous diseases, including cancers [10]. They can be detected in tissues, blood, body fluids, urine, and saliva [11]. miRNAs related to ovarian steroidogenesis have been reported, such as miR-133b [12] and miR-378 [13], which are associated with E2 synthesis and target forkhead box L2 (Foxl2) and aromatase, respectively. It has been reported that miR-15a and miR-188 regulate ovarian cell proliferation and apoptosis [14], while miR-224 regulates granulosa cell proliferation and function by targeting Smad4 [15]. However, the role of miR-329-3p in ovarian physiology has not yet been studied.
As miRNAs have been identified as crucial mediators of steroidogenesis, proliferation, and apoptosis in granulosa cells [12,13,15], it was hypothesized that miRNAs could play a significant role in patients with POR. In a previous study, we developed an in vitro POR model using KGN cells, prompted by findings that FSHR expression was reduced in POR patients. We discovered that four miRNAs—miR-130a-3p, miR-185-5p, miR-329-3p, and miR-4463—were differentially expressed between normal KGN cells and those in the POR model [6]. Notably, miR-4463 directly targeted aromatase, the critical enzyme for ovarian steroidogenesis, thereby reducing E2 production in KGN cells. This suggests that FSHR dysfunction in POR may be linked to elevated miR-4463 levels in granulosa cells, indicating that miR-4463 could be a significant regulator of POR and a potential biomarker for this condition [6].
The objectives of the present study were: (1) to identify target genes of miR-329-3p and their regulatory mechanisms in granulosa cells associated with the follicle-stimulating hormone (FSH) signaling pathway; (2) to determine and evaluate the regulatory molecular pathways of miR-329-3p using both dry and wet laboratory methods; and (3) to assess the expression levels of miR-329-3p in control and POR patients to explore its potential as a biomarker for diagnosing POR.
Methods
1. Bioinformatics analysis
Our previous studies on microarray and bioinformatics analysis have been adapted to predict the target genes of miR-329-3p. We conducted a functional pathway analysis of 485 miRNA target genes using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis via the Database for Annotation, Visualization and Integrated Discovery (DAVID) annotation tool. The TargetScan Human database was utilized to predict the binding sites between the miRNA and the target genes.
2. KGN cell culture
The immortalized human granulosa cell line, KGN, was sourced from the Riken BioResource Center (Riken Cell Bank). These cells were cultured in Dulbecco's Modified Eagle’s Medium:Nutrient Mixture F-12 (Gibco), enriched with 10% fetal bovine serum. Cultivation took place in a humidified environment with 5% CO2 at a constant temperature of 37 °C.
3. Transfection of KGN cells with a miR-329-3p mimic and inhibitor
KGN cells were cultured in 6-well dishes at a density of 2×105 cells per well under a humidified atmosphere with 5% CO2 at 37 °C for 24 hours. The miR-329-3p mimic and inhibitor were sourced from Bioneer. Transfection of KGN cells was performed using 10 nM of the miR-329-3p mimic or 50 nM of the miR-329-3p inhibitor for 48 hours, employing Lipofectamine 3000 (Invitrogen).
4. Total RNA extraction and quantitative real-time polymerase chain reaction of KGN cells
Total RNA was extracted from KGN cells using a miRNeasy Micro Kit (Qiagen), following the manufacturer’s instructions. For gene expression analysis, 1 μg of RNA was reverse-transcribed into first-strand cDNA using oligo (dT) and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). The quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using iQ SYBR Green Supermix PCR reagents (Bio-Rad) to monitor the amplification. The results were analyzed using CFX Maestro Software. The amplification mixture included cDNA, 5 pmol of both forward and reverse primers, and SYBR Green Supermix. The amplification protocol consisted of 40 cycles, each including denaturation at 95 °C for 40 seconds, annealing at 60 °C for 40 seconds, and extension at 72 °C for 40 seconds. Following PCR, fluorescence was monitored as the samples were gradually heated from 55 to 95 °C in 0.5 °C increments. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the endogenous reference for mRNA normalization.
For the miRNA expression analysis, we utilized the HB miR Multi Assay Kit System I (HeimBiotek) following a two-step process. Initially, 500 ng of RNA was reverse-transcribed using the HB_I RT Reaction Kit and its reagents at 37 °C for 60 minutes, followed by a 5-minute incubation at 95 °C for one cycle. Subsequently, the synthesized cDNAs were amplified using the HB_I Real-Time PCR Master Mix Kit in a C1000 Thermal Cycler (Bio-Rad). The qRT-PCR protocol included an initial activation step at 95 °C for 15 minutes, followed by 45 cycles of 10 seconds at 95 °C and 40 seconds at 60 °C, and standard melting conditions. The melting conditions involved a temperature ramp from 55 to 95 °C, increasing by 0.5 °C at each step, using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Primers for miRNA amplification were sourced from HeimBiotek, with the small non-coding RNAs RNU6B serving as the endogenous reference for miRNA normalization. Following mRNA or miRNA normalization, fold changes were calculated using the 2-ΔΔCt method. All experiments were performed in triplicate and replicated at least three times.
5. Western blot analysis
Cellular proteins were extracted from treated cells and homogenized in radioimmunoprecipitation assay lysis buffer containing 1% protease inhibitor cocktail (Thermo Scientific). We separated 20 μg of protein extracts using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred them onto polyvinylidene difluoride membranes. These membranes were blocked for 1 hour in Tris-buffered saline/Tween (TBST; 0.2 M NaCl, 0.1% Tween-20, and 10 mM Tris [pH 7.4]) with 5% skim milk. They were then incubated overnight at 4 °C with primary antibodies diluted against cytochrome P450 family 19 subfamily A member 1 (CYP19A1; 3599-100; BioVision) and GAPDH (SC-47724; Santa Cruz Biotechnology). Following this, the membranes were washed several times with TBST and incubated with diluted secondary antibodies for 1 hour at room temperature. After additional washes, signals were developed using an enhanced chemiluminescence system (Amersham Biosciences), and the relative expression of the protein bands was quantified using a ChemiDoc XRS+ imaging system with Image Lab software (Bio-Rad).
6. Measurement of E2 levels
The concentrations of E2 secreted by KGN cells were measured using a competitive enzyme-linked immunosorbent assay (ELISA) with a human estrogen ELISA kit (MyBioSource). KGN cells were plated in 6-well plates and transfected with either small interfering RNAs (siRNAs) or a miRNA mimic. Following a 48-hour incubation, the culture media were collected to assess the levels of E2 secreted by the KGN cells, as per the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm using an automated microplate reader (E-Max; Molecular Devices).
7. Patients
A total of 32 patients were recruited from the CHA Fertility Center at CHA University between November 2019 and April 2020. Patients with POR were classified according to the Bologna criteria, which require meeting at least two of the following three conditions: (1) advanced maternal age (≥40 years) or other risk factors for POR; (2) a history of POR (≤3 oocytes with a conventional stimulation protocol); or (3) an abnormal ovarian reserve test (antral follicle count [AFC] <5–7 follicles or anti-Müllerian hormone [AMH] <0.5–1.1 ng/mL). Patients who were normal responders were classified as the control group. The exclusion criteria included: (1) age >45 years; (2) body mass index (BMI) >30 kg/m2; (3) FSH >40 mIU/mL; (4) hyperresponse (≥20 retrieved oocytes); and (5) medical conditions such as diabetes mellitus and other endocrine disorders. All participants provided informed consent. The study received approval from the Institutional Research Ethics Committee (1044308-201806-BR-024-02). Four types of samples were collected from each patient: (1) serum on menstrual days 2–3 (day 2 serum); (2) serum on the day of human chorionic gonadotropin (hCG) injection (hCG day serum); (3) granulosa cells after ovum pick-up (OPU); and (4) cumulus cells from 32 patients undergoing intracytoplasmic sperm injection. The diagnosis of POR was established based on the Bologna criteria [3].
Sixteen patients were assigned to the control group, which consisted of individuals with normal ovarian reserve tests. Their infertility was attributed to either male infertility factors or tubal factors, and they had no history of menstrual abnormalities. We evaluated the clinical characteristics of both control and POR patients, including the number of previous IVF cycles, duration of infertility, BMI, AFC, E2 levels on day 2 and on the day of hCG administration, AMH, FSH levels on day 2, and the number of retrieved metaphase II (MII) oocytes. Control and POR patients underwent stimulation using a flexible gonadotropin-releasing hormone (GnRH) antagonist protocol. The gonadotropin dosages were adjusted based on the patient's age, estradiol levels, BMI, and AFC. When a patient's follicle reached a size greater than 18 mm, hCG was administered. Approximately 36 hours after the hCG injection, cumulus-oocyte complexes (COCs) were collected via transvaginal ultrasound-guided aspiration.
8. Collection of serum samples, cumulus cells, and granulosa cells
Blood samples from patients were collected on two occasions: on day 2 of the menstrual cycle and on the day of hCG administration. Serum samples were placed in separate tubes and processed by centrifugation at 3,000 rpm for 10 minutes. The serum supernatant was then snap-frozen using liquid nitrogen and stored at −80 °C until RNA isolation. Approximately 36 hours post-hCG injection, COCs were immersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) medium containing hyaluronidase. The cumulus cells were then mechanically separated from the oocytes and similarly snap-frozen in liquid nitrogen, followed by storage at −80 °C until RNA isolation. Following OPU, granulosa cells were extracted from the follicular fluid using a previously established protocol [16]. The granulosa cells were also stored at −80 °C after snap freezing with liquid nitrogen until RNA isolation.
9. Human serum miRNA extraction and qRT-PCR analysis
Total RNA, including small RNA, was extracted from 200 µL of serum samples using the miRNeasy Serum/Plasma Extraction Kit (QIAGEN). To normalize gene expression, Caenorhabditis elegans miR-39_1 was added as an RNA spike-in (miRNeasy Serum/Plasma Spike-in Control; QIAGEN). The miRNA expression analysis was conducted using a two-step process with the HB miR Multi Assay Kit System I (HeimBiotek). For the qRT-PCR analysis, a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) was utilized.
10. Statistical analysis
The data are presented as the mean±standard error of the mean from at least three separate and independent experiments. Statistical analyses of cell data were conducted using one-way analysis of variance with the Newman-Keuls multiple comparison test to identify differences between the treatment groups and the control. Differences between the two groups were analyzed using the Student t test for statistical evaluation. Significant differences (p<0.05) between the mean values of triplicate samples were determined for all experiments.
Results
1. miR-329-3p expression changes in an in vitro model of POR
In our previous study, we developed an in vitro POR cell line model using KGN cells transfected with FSHR siRNA, which resulted in increased miR-329-3p levels and decreased FSHR expression [6]. To confirm these results, in the current study, KGN cells were transfected with FSHR siRNA for 48 and 72 hours to reduce FSHR expression. We then employed qRT-PCR to assess the expression levels of miR-329-3p in both nontargeting siRNA-treated KGN cells and siRNA targeting FSHR (siFSHR)-treated KGN cells. In both the 48- and 72-hour siFSHR-treated KGN cells, miR-329-3p expression was significantly elevated (p<0.05) (Figure 1).
The expression of miR-329-3p was upregulated in follicle-stimulating hormone receptor (FSHR)-knockdown KGN cells, as demonstrated by quantitative real-time polymerase chain reaction. KGN cells were transfected with FSHR small interfering RNA (siRNA) for 48 and 72 hours. The expression of miR-329-3p was normalized to that of small non-coding RNAs RNU6B and then evaluated using the 2-ΔΔCt method, and the relative expression was calculated. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis was repeated three times. The error bars represent the mean±standard error of the mean. NC, negative control group; siFSHR, siRNA targeting FSHR. a)Statistical significance at p<0.05.
2. Bioinformatics analysis to identify miRNA target genes
To identify the biological functions of miR-329-3p, we utilized bioinformatics tools to search for target genes. We selected approximately 1,000 to 7,000 genes using eight distinct in silico miRNA prediction tools: miRWalk, miRanda, miRDB, PicTar 2, PITA, RNA22, RNAhybrid, and TargetScan [6]. Next, we identified genes targeted by more than three of the four miRNAs previously selected in miRNA microarray data: miR-130a-3p, miR-185-50, miR-4463, and miR-329-3p. The workflow is depicted in Figure 2. In total, 485 genes were chosen for further analysis. We conducted a KEGG enrichment pathway analysis using the DAVID online database (ver. 6.8). This analysis revealed that the ovarian steroidogenesis pathway was among the targeted pathways, as indicated in Table 1. Nine genes within this pathway—adenylyl cyclase 1 (ADCY1), ADCY2, ADCY9, insulin like growth factor 1 (IGFR1), IGF1, insulin receptor (INSR), low density lipoprotein receptor (LDLR), phospholipase A2 group IVF (PLA2G4F), and protein kinase A subunit beta (PRKACB)—were predominantly associated with the FSH signaling pathway.
Workflow of the bioinformatics analysis of putative miR-329-3p target genes. FSH, follicle-stimulating hormone; FSHR, follicle-stimulating hormone receptor; siRNA, small interfering RNA; miRNA, microRNA.
KEGG pathway analysis of miR-329-3p target genes
3. miR-329-3p regulates the FSH signaling pathway by targeting ADCY9 and PRKACB in KGN cells
The relationship between miR-329-3p and the 3’ untranslated regions (3’-UTRs) of ADCY9 and PRKACB in granulosa cells is depicted in Figure 3A. Adenylyl cyclase and protein kinase A (PKA) are key components of the second messenger system in cellular signaling pathways, playing crucial roles in the proliferation, differentiation, and steroid production of granulosa cells [17]. We found that miR-329-3p suppressed the gene expression of ADCY9 and PRKACB, as predicted by the TargetScan Human database web tool, and the putative binding sites of miR-329-3p in the 3’-UTRs were also identified (Figure 3B).
The ovarian steroidogenesis pathway in granulosa cells and TargetScan Human analysis. (A) Follicle-stimulating hormone (FSH) is a major hormone that induces signaling when it binds to follicle-stimulating hormone receptor (FSHR). Adenylyl cyclase 9 (ADCY9) and protein kinase A subunit beta (PRKACB) were predicted as target genes of miR-329-3p and were analyzed through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (B) The predicted target site in the 3’ untranslated region (3’-UTR) of ADCY9 and PRKACB mRNA contains the sequence for hsa-miR-329-3p binding. The nucleotides in the red box represent the seed region of miR-329-3p. GDP, guanosine diphosphate; GTP, guanosine triphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CREB, cAMP-response element binding protein.
To verify the data obtained through bioinformatics tools, we conducted qRT-PCR to assess the expression levels of the target genes. We transfected KGN cells with the miR-329-3p mimic and miR-329-3p inhibitor for 48 hours to induce overexpression and suppression of miR-329-3p, respectively. Following the transfection of miR-329-3p into KGN cells, we observed a downregulation of five types of ADCY isoforms (ADCY1, ADCY5, ADCY6, ADCY7, and ADCY9) and PRKACB in the miR-329-3p mimic group compared to the control group. However, only the changes in ADCY9 and PRKACB were statistically significant (p<0.05). Conversely, the expression of ADCY9 and PRKACB significantly increased after transfection with the miR-329-3p inhibitor (p<0.05). These findings indicate that miR-329-3p regulates ADCY9 and PRKACB (Figure 4A). Consequently, we further investigated the role of miR-329-3p in the steroidogenic activity of granulosa cells by targeting ADCY9 and PRKACB. Additionally, a dual luciferase reporter assay demonstrated that miR-329-3p could target the 3’-UTR of PRKACB [18].
After KGN cells were transfected with a miR-329-3p mimic or miR-329-3p inhibitor, the target genes and steroidogenesis were confirmed to be regulated by miR-329-3p. (A) The mRNA expression levels of adenylyl cyclase 1 (ADCY1), ADCY5, ADCY6, ADCY7, ADCY9, and protein kinase A subunit beta (PRKACB) were calculated from the cycle threshold (CT) values obtained from quantitative real-time polymerase chain reaction. The mRNA levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) The protein levels were measured by Western blot analysis. GAPDH was used as an internal control gene. The relative protein levels were calculated by measuring the density and area of the bands. (C) 17β-Estradiol (E2) levels were measured using competitive enzyme-linked immunosorbent assay with cell culture supernatant from miR-329-3p mimic or miR-329-3p inhibitor-transfected KGN cells. Experiments were repeated at least three times, and data are expressed as the mean±standard error of the mean. NS, not significant. a)p<0.05; b)p<0.01 indicate statistical significance.
4. miR-329-3p regulates steroidogenesis in KGN cells
The overexpression of miR-329-3p inhibited FSH signaling by suppressing ADCY9 and PRKACB in KGN cells, indicating repression of FSH transcription. CYP19A1, which encodes the key enzyme aromatase involved in estrogen synthesis, is regulated by the FSH signaling pathway. To assess whether miR-329-3p affected aromatase expression, Western blotting was performed. The findings revealed that aromatase expression was significantly reduced in KGN cells transfected with the miR-329-3p mimic (p<0.05), whereas the miR-329-3p inhibitor did not significantly alter expression levels (Figure 4B). ELISA analysis was used to measure E2 levels. Transfection with the miR-329-3p mimic resulted in a decrease in E2 levels by approximately 16% compared to control KGN cells, though this change was not statistically significant (p<0.16). Conversely, transfection with the miR-329-3p inhibitor led to an approximate 36% increase in E2 levels compared to controls (Figure 4C). These findings strongly suggest that miR-329-3p plays a critical role in modulating both aromatase expression and E2 synthesis indirectly by targeting ADCY9 and PRKACB.
5. The clinical characteristics of control and POR patients
To verify our data from cellular and bioinformatics analyses, we measured the levels of miR-329-3p and related factors, FSHR and aromatase, in patients from both the control and POR groups. The clinical characteristics of these patients are detailed in Table 2. There were significant differences between the control and POR groups in several categories: age (36.6 years vs. 39.6 years, p<0.05), number of previous IVF cycles (2.4 cycles vs. 6.7 cycles, p<0.001), AFC (11.8 vs. 3.4, p<0.001), hCG day E2 levels (2,596.1 pg/mL vs. 348.8 pg/mL, p<0.001), day 2 AMH levels (3.4 ng/mL vs. 0.8 ng/mL, p<0.001), day 2 FSH levels (8.7 mIU/mL vs. 14.2 mIU/mL, p<0.001), and the number of retrieved MII oocytes (8.2 vs. 1.5, p<0.001). However, no significant differences were observed in the duration of infertility, BMI, or day 2 E2 levels between the two groups (Table 2). Patients in the POR group underwent approximately four more IVF cycles than those in the control group, likely due to their lower response to COH during IVF and a higher rate of cycle cancellation. Additionally, AMH levels were significantly lower in POR patients compared to control patients, as was the AFC. Both AMH and AFC are crucial in assessing ovarian reserve and influencing the response to COH [3]. The significantly lower number of retrieved MII oocytes in POR patients can be attributed to reduced follicular development, despite the administration of maximal gonadotropin doses for ovarian stimulation.
Clinical characteristics of control and POR patients
6. FSHR and aromatase expression in granulosa cells of control and POR patients
Our in vitro model of POR (siFSHR+FSH KGN cells) was initially validated by confirming the well-documented finding that many POR patients exhibit low FSHR expression. Consequently, we assessed the expression levels of FSHR and aromatase in granulosa cells from 16 control subjects and 16 POR patients. We observed that FSHR expression was significantly reduced by approximately 66% in POR patients compared to controls (p<0.05) (Figure 5A), aligning with the results from our in vitro model of POR. However, the alteration in aromatase expression did not reach statistical significance, as depicted in Figure 5B.
Quantification of the mRNA levels of follicle-stimulating hormone receptor (FSHR) and aromatase by quantitative polymerase chain reaction by comparing granulosa cells from control patients and poor ovarian response (POR) patients. In the 16 control patients and 16 POR patients, (A) the expression of FSHR was significantly lower in the POR patients, and (B) the expression of aromatase was not significantly different. The data are presented as the mean±standard error of the mean. NS, not significant. a)p<0.05 indicates statistical significance.
7. The expression of miR-329-3p is elevated in POR patients
Day 2 serum, hCG day serum, granulosa cells, and cumulus cells were collected from 16 control and 16 POR patients. The E2 levels in the day 2 serum and hCG day serum of patients were markedly different. However, miR-329-3p expression in both day 2 serum and hCG day serum was significantly increased, showing a 4.6-fold increase (p<0.01) and a 3.5-fold increase (p<0.05), respectively, in the POR group compared to the control group (Figure 6A, 6B). These findings indicate that miR-329-3p levels in the serum are not influenced by E2 levels. Therefore, miR-329-3p could serve as a reliable diagnostic marker for POR, as it is not related to E2 and does not exhibit fluctuating expression patterns. Additionally, miR-329-3p expression was significantly upregulated by 70% in the granulosa cells of POR patients (p<0.05) (Figure 6C). However, there was no significant difference in miR-329-3p expression between control and POR patients in cumulus cells (Figure 6D).
The expression level of miR-329-3p is elevated in in vitro fertilization patient serum and granulosa cells, but the difference was not significant in cumulus cells. (A, B) The expression levels of miR-329-3p were examined by quantitative real-time polymerase chain reaction (qRT-PCR) in sera from control patients and poor ovarian response (POR) patients. Caenorhabditis elegans miR-39 was used as an RNA spike-in to normalize gene expression. (C, D) The expression levels of miR-329-3p were examined by qRT-PCR in granulosa cells and cumulus cells in control patients and POR patients. RNU6B was used for miRNA normalization. The data are presented as the mean±standard error of the mean. hCG, human chorionic gonadotropin; NS, not significant. a)p<0.05; b)p<0.01 indicate statistical significance.
Discussion
In this study, we used bioinformatics tools to determine that miR-329-3p levels were elevated in FSHR-knockdown KGN cells and were associated with the ovarian steroidogenesis molecular pathway in granulosa cells. ADCY9 and PRKACB were identified as targets of miR-329-3p, influencing aromatase expression and E2 production in miR-329-3p-transfected KGN cells. We observed a decrease in FSHR expression in granulosa cells from POR patients, with a concurrent increase in miR-329-3p levels in day 2 serum, hCG day serum, and granulosa cells from these patients. Our findings suggest that the upregulation of miR-329-3p expression may be a phenomenon associated with POR, characterized by low E2 production, and that miR-329-3p could serve as a potential biomarker for POR.
It is well known that the FSH signaling pathway in granulosa cells is initiated through the activation of a G-protein coupled receptor, which stimulates adenylyl cyclase to convert adenosine triphosphate into cyclic adenosine monophosphate (cAMP). cAMP serves as a second messenger, activating cAMP-dependent PKA, which subsequently phosphorylates cAMP-response element binding protein (CREB) [19,20]. Additionally, the FSH signaling pathway in granulosa cells interacts with several other cell signaling pathways, including the p38 mitogen-activated protein kinase (MAPK) pathway, p42/44 MAPK pathway, and phosphoinositide 3-kinase (PI3K)/PKB (AKT) pathway, all of which are activated during granulosa cell differentiation [21-25]. Among these pathways, PKA is considered a master kinase that initiates the intracellular signaling and target gene expression profiles associated with granulosa cell differentiation [17]. FSH stimulation induces the expression of transcripts related to granulosa cell differentiation and steroidogenesis, including aromatase, the key enzyme for estrogen synthesis. In this study, reducing FSHR expression with siFSHR in KGN cells led to increased levels of miR-329-3p, which in turn influenced its target genes. The downregulation of ADCY9 and PRKACB by miR-329-3p significantly reduced aromatase expression in the miR-329-3p mimic group, resulting in decreased E2 synthesis. Consequently, we concluded that miR-329-3p plays a crucial role in regulating ovarian steroidogenesis.
FSHR expression is a critical marker in patients with POR, as dysfunctional FSHR is linked to impaired proliferation and differentiation of granulosa cells, along with a marked decrease in E2 production [26]. The ovarian response to FSH hyperstimulation is significantly influenced by polymorphisms in the FSHR gene [27,28]. Stimulation by FSH typically leads to increased expression of transcripts associated with steroidogenesis in granulosa cells, as well as the rate-limiting enzymes necessary for the synthesis of estrogen and progesterone [17]. This indicates that FSH is the principal hormone regulating granulosa cell functions. In the presence of FSHR abnormalities, however, granulosa cells may not adequately respond to FSH stimulation, potentially resulting in their dysfunction.
Recent studies have shown that miR-329-3p plays a regulatory role in various molecular pathways across multiple clinical disciplines. Specifically, miR-329-3p influences GnRH synthesis in the hypothalamus by targeting PKA subunits [18], regulates SMAD2 in cervical cancer tissues [29], inhibits neural stem cell proliferation by targeting E2F transcription factor 1 (E2F1) [30], is highly expressed in mouse ischemia by targeting hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) [31], and is expressed in rat prostate cancer [32]. These findings are summarized in Table 3. Despite these advances, research on the role of miR-329-3p in ovarian physiology has been lacking. Our study is the first to explore this area, revealing that the overexpression of miR-329-3p in granulosa cells targets adenylyl cyclase and PKA, subsequently regulating ovarian E2 production and inducing POR.
List of articles and proposed functions related to miR-329-3p
Several reports have described the use of miRNAs as biomarkers for disease diagnoses [33]. Studies have shown a correlation between miRNAs and POR; specifically, miR-21-5p expression is elevated in cumulus cells, while E2 levels in POR patients are decreased [5]. Another study found that miR-15a-5p levels were higher in follicular fluid, influenced the PI3K-AKT-mammalian target of rapamycin (mTOR) pathway, and triggered apoptosis by targeting B-cell lymphoma 2 (BCL2) and Bcl-2-associated death (BAD) [34]. However, these studies are limited by the need for granulosa cells, cumulus cells, and follicular fluids to measure miRNA levels, which restricts the clinical utility of these biomarkers in setting the gonadotropin dose for COH or diagnosing the patient’s condition. The treatment of suspected POR patients requires a tailored approach at every stage of assisted reproduction, including the selection of GnRH analogs, gonadotropin dose and type, ovulation trigger, and the potential use of adjuvant therapies [35]. Additionally, in cases of polycystic ovarian syndrome (PCOS), it is crucial to assess the expression of serum miRNA biomarkers, as PCOS patients undergoing COH have a high risk of developing ovarian hyperstimulation syndrome [36]. For these reasons, detecting miRNA expression in serum or other body fluids may offer a more practical alternative for diagnosing ovarian conditions compared to using ovarian cells and follicular fluids. We confirmed that the serum level of miR-329-3p was elevated in POR patients on day 2 and on the hCG day, despite varying E2 levels on these days. A significant amount of research is currently focused on the potential of miRNAs as biomarkers and therapeutic candidates for a wide range of diseases. The application of miRNAs as biomarkers and therapeutic targets holds great promise. However, their use as precise targets remains challenging because a single miRNA can function differently at various stages of a disease [37]. Consequently, miRNA-based drugs, including miRNA mimics and miRNA inhibitors, are still in development as innovative therapeutic targets [38]. To address these challenges, it is crucial to consider factors such as ensuring a sufficient sample size to mitigate the impact of potential mutations and minor effects, conducting prospective studies to verify the results of observational studies, and undertaking the validation process only after comprehensive epidemiological and functional studies have been completed [10]. Despite these obstacles, if measurable, elevated miR-329-3p expression in the serum of POR patients could serve not only as a valuable biomarker for diagnosing POR but also as a potential therapeutic target in the future.
In conclusion, we found that miR-329-3p regulates ovarian steroidogenesis in granulosa cells by targeting ADCY9 and PRKACB. Additionally, we observed that miR-329-3p was highly expressed in serum at the initial basal E2 point (day 2 of the cycle) and at maximal E2 conditions (hCG day of the cycle) (Figure 7). Given the demonstrated potential of miRNA as a biomarker for predicting diseases, our findings may herald a new era in the use of miRNA as a biomarker for POR. Overcoming existing limitations could make miR-329-3p expression in IVF patients a crucial clinical biomarker for determining gonadotropin dosage and might also position it as a future therapeutic target for POR.
The predicted molecular mechanism underlying the effect of miR-329-3p on adenylyl cyclase 9 (ADCY9) and protein kinase A subunit beta (PRKACB) in granulosa cells for follicle-stimulating hormone (FSH)-responsive 17β-estradiol (E2) synthesis. In this study, we determined that abnormal follicle-stimulating hormone receptor (FSHR) in granulosa cells increased miR-329-3p expression levels in these cells, followed by the inhibition of adenylyl cyclase and protein kinase A (PKA). Furthermore, aromatase expression and E2 levels were reduced. AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; RISC, RNA-induced silencing complex.
Notes
Conflict of interest
Sooyeon Lee was affiliated with CHA University, Seongnam, Republic of Korea, but is currently affiliated with CHAYON Laboratories Inc., Seoul, Republic of Korea.
Eun Jeong Yu is an associated managing editor and Hye-Ok Kim is an editorial board member of the journal, but they were not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts.
Author contributions
Conceptualization: JHK, HOK, SYL, EAP, KHC, KK, EJY, MKK, KAL. Methodology: JHK, SYL, KAL. Formal analysis: JHK, SYL. Data curation: JHK, HOK, EAP, KHC, KK, EJY. Funding acquisition: HOK, MKK. Project administration: MKK, KAL. Visualization: JHK, EAP, KHC, KK. Validation: JHK, KAL. Investigation: JHK, SYL. Writing-original draft: JHK, HOK. Writing-review & editing: SYL, KAL. Approval of final manuscript: MKK, KAL.