Background
Polycystic ovary syndrome (PCOS) is one of the most common reproductive, endocrine, and metabolic disorders in women. It affects about 5 to 10% of women of reproductive age and is usually a lifelong disease. PCOS is characterized by chronic anovulation, hyperandrogenism, and, consequently, infertility [
1]. Metabolic disorders, including obesity, insulin resistance, and diabetes, are cofactors as well as predisposing factors of PCOS [
2]. Therefore, understanding the molecular mechanisms of the metabolic diseases underlying the pathophysiology of PCOS will help to identify novel diagnostic and therapeutic strategies.
Previously, it was shown that microRNAs (miRNAs) play an important role in follicular development and fertility [
3]. The miRNAs are highly conserved, 19 to 25 nucleotide-long, single-stranded RNA molecules that post-transcriptionally regulate gene expression, and they perform their functions by mediating translational repression or by directing the cleavage of target mRNAs. Recent evidence suggests that miRNAs play fundamental roles in all cellular and tissue activities under both normal and pathological conditions, but the currently available information on the expression and function of miRNAs in the ovary, especially in oocyte development, is still very limited. It was discovered, however, that miRNAs might be involved in the turnover of many maternal transcripts whose degradation might be essential for the successful completion of meiotic maturation by oocytes [
3].
The analysis of expression of miR-23a and miR-23b in follicular cells from women undergoing assisted reproductive technology (ART) including the patients with tubal factor and endometriosis showed that significant increase in the levels of miR-23b directly correlated with CYP19A1 (aromatase gene) expression, miR-23a, compared to normal women [
4]. Aromatase which convert androgens to estrogen has role in pathogenesis of PCOS [
5]. Therefore, in the present study, serum samples and other information were collected from women with PCOS and healthy controls to study the correlation between serum miR-23a/b expression, obesity, and sex hormones in women with PCOS.
Discussion
We found a negative influence of decrease of miR-23a on occurrence of PCOS and increase of testosterone. On the other hand, although a positive effect of BMI on the expression levels of miR-23b (without considering presence or absence of PCOS) was observed in logistic regression analysis, but decrease in miR-23b fold-changes as well as miR-23a decrease was observed in PCOS women. Considering this fact that miR-23a alterations was not affected by BMI in contrast with miR-23b, suggests it as a better indicator for evaluation of PCOS than the miR-23b.
Furthermore, the BMI among the women with PCOS was higher on average compared with the healthy controls. The mean BMI of healthy women of the same ethnicity and in the same age range (20.99 ± 3.31 kg/m
2) [
6] is similar to the BMI among healthy controls in the current study. Therefore, all of the PCOS patients in this study could be considered to be obese (the mean BMI of the PCOS patients was 23.96 ± 4.44 kg/m
2). Obesity in the PCOS patients increased the expression of both miR-23a and miR23b, but BMI was correlated with decreased expression of these miRNAs in the healthy controls. The pattern of correlations between obesity and the expression of miR-23a and miR23b that was observed in the present study resembles the pattern of correlations of serum testosterone, LH, and FSH concentrations with the expression of the miR-23a and miR23b, but the effects of obesity on the relationship with miR-23a/b expression in the women with PCOS was greater than the association of hormone changes with miR-23a/b expression. In the healthy controls and the endometrial-phase subgroups, obesity and testosterone concentrations had negative correlation coefficients, whereas in the women with PCOS a positive but non-significant correlation in increased BMI and testosterone concentrations was observed. Consistent with our findings, Murri et al. [
7] reported opposite patterns of association between obesity and testosterone concentrations in PCOS patients and healthy controls. According to the normal range of testosterone levels among healthy women of the same ethnicity and in the same age range of the current study (0.32 ± 0.16 ng/mL) [
6], all of the PCOS patients who were selected in this study based on the Rotterdam consensus criteria were defined as having hyperandrogenemia (0.59 ± 0.19 ng/mL).
The patterns of changes in the expression of miR-23a and miR-23b were the same in the PCOS patients and in the healthy controls and in the endometrial-phase subgroups, and in the present study the expressions of both miR-23a and miR-23b in serum were significantly lower in the women with PCOS compared to the healthy controls. The one exception was that the mean expression of miR-23b in the women with PCOS was higher than that in the healthy controls in the late secretory phase, and it can be speculated that this difference might be due to the involvement of miR-23b in ovulation. In support of this, it has been shown that miR-23b targets the X-linked inhibitor of apoptosis and can induce apoptosis in human granulosa cells in vitro [
8]. In addition, a comparison of seasonally ovulatory and anovulatory follicles in horses revealed increased expression of miR-23b in the anovulatory follicles [
9]. Furthermore, in our current work we found that the pattern of expression of miR-23a and miR-23b changed from a positive correlation in the proliferative phase to a negative correlation in the late secretory phase; whereas the women with PCOS showed a positive correlation in the expressions of miR-23a and miR-23b.
In the present study, the increase in serum E
2 concentrations in the proliferative phase was negatively correlated with the expression of miR-23a and miR-23b in the healthy controls. Furthermore, in the evaluated population, a negative influence of increase of testosterone concentrations on miR-23a expression was observed. In addition, the same non-significant effect of testosterone was observed on miR-23 expression in whole blood. A previous study on the expression of miR-23a and miR-23b in follicular fluid showed that expression of these miRNAs along with the expression of their target gene could regulate the expression of aromatase, CYP19A1, in ovarian cells and, therefore, might have a role in E
2 biosynthesis [
4]. Therefore, it can be speculated that alterations in the expression of these miRNAs in serum might affect follicular growth and ovulation via other target genes than those that play a role in E
2 hormone synthesis, including target genes that are functionally related to cell growth and apoptosis. In the present study, a decrease in miR-23a expression in women with PCOS was observed, and overexpression of pre-miR-23 has previously been shown to play a role in apoptosis in cultured human luteinized granulosa cells [
8]. Therefore, altered miR-23a expression in PCOS patients might induce down-regulation of apoptotic processes in ovarian cells.
With the help of bioinformatics tools, we have shown that miR-23a and miR-23b target large numbers of genes and that many of these genes are targeted by several other miRNAs. Target genes of miR-23a and miR-23b are involved in many biological functions, including metabolic, cellular, and reproductive processes that are important in PCOS pathogenesis [
2,
10]. One of the metabolic disorders that has a definite role in PCOS is obesity [
11], and several studies have shown that cellular communication can be altered in PCOS and obesity, including communication between inflammatory cells and metabolic cells [
12‐
14]. Additionally, inflammatory and immune gene targets that regulate cellular processes such as apoptosis can influence follicular function and steroid production [
10,
15].
The major limitation of the present study was possibly the evaluation of serum miRNA expression, which represents miRNAs from several unknown origin cell types. However, several studies have highlighted the potential of serum samples to act as developmental markers of diseases, including PCOS [
7,
16‐
18]. Therefore, sampling of serum might represent the overall state of the entire body instead of specific cells at the time of collection.
Methods
Subjects and selection criteria
In the current study, 18 Han Chinese women (with a mean ± SD age of 25.8 ± 4.5 years) were recruited from the Affiliated Obstetrical and Gynecological Hospital of Fudan University between September 2011 and January 2012. The women were all diagnosed with PCOS based on the revised diagnostic criteria announced in the Rotterdam consensus [
1], and patients with Cushing syndrome, late-onset congenital adrenal hyperplasia, thyroid dysfunction, hyperprolactinemia, or androgen-secreting tumors were excluded. Other exclusion criteria included diabetes, hypertension, chronic renal disease, smoking, and the use of alcohol or medications. Thirty healthy age-matched Han Chinese women (25.5 ± 2.3 years old) with no previous history of reproductive system diseases or appendicitis served as controls. The control women had normal and regularly cycling menstrual periods, and their ovaries appeared normal on ultrasound. The exclusion criteria of the healthy women in the study were taking drugs, including oral contraceptives or other hormone drugs, intrauterine device placement, smoking, and/or pregnancy in the past 3 months. Control subjects were divided into four groups according to their endometrial cycle phase – proliferative phase (days 4–14,
n = 8), early secretory phase (days 15–18,
n = 6), mid-secretory phase (days 19–24,
n = 8), and late secretory phase (days 25–30,
n = 8).
Assessment of BMI and sex hormones
The BMI in both normal women and PCOS patients was calculated as weight (kg) divided by the square of the height (m2). Measurements and blood samples were conducted within the first 10 days from the onset of menstruation in PCOS cases with mild oligomenorrhea, and they were conducted at random times for PCOS cases with severe oligomenorrhea or amenorrhea. Measurements and blood samples were conducted at different phases of the endometrial cycle in controls as described above. Total testosterone, LH, and FSH were measured by radioimmunoassay (RigorBio Scientific and Technology Co., Beijing) according to the manufacturer’s instructions.
Quantification of miR 23a/b in peripheral blood
Venous blood samples (5 ml) were drawn from every subject. Serum was separated by centrifuging at 3000 × g for 10 min at 4 °C and was stored at −20 °C. Whole RNA was extracted from 200 μL of serum with the miRcute miRNA Isolation kit (DP501, Tiangen Biotech, Beijing) according to the manufacturer’s instructions. The RNA was then reverse transcribed using the miRcute miRNA first-strand cDNA synthesis kit (KR201, Tiangen Biotech, Beijing), and quantitative real-time polymerase chain reaction (qPCR) was performed using the miRcute miRNA qPCR detection kit (Tiangen Biotech, Beijing). The qPCR was performed under the following conditions: initial PCR denaturation at 94 °C for 120 s followed by 42 combined cycles of denaturation of 20 s at 94 °C and annealing and extension of 34 s at 60 °C. Fluorescence was measured at 55 °C in 81 cycles of 10 s. Results were calculated using the 2−ΔΔCt method, and U6 was used as the controls for miR-23a and miR-23b. The sequences of primers were as follows (Invitrogen, Shanghai):
Primer sequence of miR-23a: 5′-ATCACATTGCCAGGGATTTCCA-3′
Primer sequence of miR-23b: 5′-GCACATTGCCAGGGATTACCA-3′
U6 as the internal control: 5′-CTCGCTTGGGCAGCACA-3
Statistical analysis
Data are described as the mean ± SD. An independent sample t-test or one-way ANOVA with correction of p-values with the Bonferroni post-hoc test was used to test for differences in demographic variables and laboratory measurements between PCOS patients and healthy controls. Spearman correlation coefficients were calculated to evaluate the relationship between miRNA levels and other measurements in both the PCOS and control groups. All data were analyzed using SPSS version 22.0 (SPSS, Inc., Chicago, IL), and p < 0.05 was considered statistically significant.
To evaluate the power of study regarding to the selected sample size and the five groups of normal and PCOS women, the observed power of the dependent variables (miR-23a/b expression, BMI and testosterone concentrations) were estimated using univariate analysis of variance in general linear model of SPSS [
19].
Possible effects of the miR-23a and miR-23b on occurrence of PCOS were explored using logistic regression analysis. The data of the normal women was used as reference. The data were compared by logistic regression analysis using the presence of PCOS as the dependent variable (0 denotes normal and 1 denotes PCOS) and the expression of miR-23a and the expression of miR-23b as the independent factors were entered into equation. Furthermore, possible effects of the BMI and serum testosterone concentration on the fold change of miR-23a and miR-23b were explored using logistic regression analysis. The data of the fold change of miR-23a/b less than 1 was used as reference. The data were compared by logistic regression analysis using the fold change of miR-23a or miR-23b as the dependent variable (0 denotes less than 1 fold change and 1 denotes more than or equal 1 fold change) and the BMI and serum testosterone, concentration as the independent factors were entered into equation.
Regression analyses were conducted according to the method of Hosmer and Lemeshow [
20]. The
p-values for data inclusion and exclusion were set at 0.05 and 0.10, respectively. The variable that had been selected or retained entered the final likelihood ratio (LR), in which the final odds ratio estimates with 95% confidence intervals were derived. The constants were not included in the model.
Acknowledgements
Not applicable.