Introduction

ETM

Experimental and Therapeutic Medicine

1792-0981 1792-1015

D.A. Spandidos

ETM-23-3-11145

10.3892/etm.2022.11145

Articles

Impact of metabolic disorders on endometrial receptivity in patients with polycystic ovary syndrome

Wang

Can

Wen

Yang-Xing

Mai

Qing-Yun

Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510080, P.R. China

Correspondence to: Professor Qing-Yun Mai, Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-Sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, P.R. China maiqy@mail.sysu.edu.cn

03 2022

14 01 2022

23 3

221

12 11 2021 22 12 2021

2020

The present study investigated the expression of endometrial receptivity-related molecules in patients with polycystic ovary syndrome (PCOS) and different androgen status, insulin resistance (IR) levels, and body mass indexes (BMI) to identify the mechanism underlying their effects on pregnancy outcomes. The present study recruited 43 participants from November 2020 to January 2021, which were classified into five groups: i) Hyperandrogenemia (HA) combined with impaired glucose tolerance group (n=8); ii) HA combined with diabetes mellitus group (n=8); iii) HA combined with non-IR (NIR) group (n=10); iv) non-HA (NHA) androgen combined with IR group (n=8); and v) NHA combined with NIR group (n=9). In addition, according to their BMIs, patients were sub-grouped into lean/normal (n=27), overweight (n=8) or obese (n=8) groups. The mRNA expression levels of endometrial receptivity-related molecules were detected using reverse transcription-quantitative PCR. In addition, flow cytometry was used to determine the phenotype and percentage of uterine natural killer cells (uNK). According to the results, patients with PCOS and IR status, HA and obesity (BMI ≥24 kg/m²) demonstrated significantly decreased mRNA expression levels of adiponectin, adiponectin receptor (AdipoR)1, AdipoR2, adapter protein containing PH domain, PTB domain and leucine zipper motif 1, estrogen receptor (ER) α, ERβ, progesterone receptor (PR), IL-15, integrin β3 avβ3, and insulin-like growth factor binding protein-1, but increased mRNA expression levels of IL-6 and IL-8 compared with NHA + NIR group or lean/normal group, respectively. In addition, obese patients with PCOS demonstrated increased mRNA expression levels of PR compared with overweight patients. This suggested that insulin resistant status, HA, and obesity could alter the endometrial receptivity of patients with PCOS, which may explain poorer embryo implantation and pregnancy outcomes in clinics.

polycystic ovary syndrome hyperandrogen status insulin resistance obesity endometrial receptivity

Funding: No funding was received.

Introduction

Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine and metabolic disease with a prevalence of 4-10% in reproductive-age women globally (1). The main manifestations of the disease are oligomenorrhea, chronic anovulation, polycystic ovarian ultrasonography changes and clinical or biochemical hyperandrogenism. Reportedly, 50-60% of patients with PCOS have different degrees of hyperandrogenemia (HA) (2), and 44-70% of patients have insulin resistance (IR) and hyperinsulinemia (3-5). In addition, 44-70% of patients with PCOS are obese (6). Although identification of the mechanism of PCOS remains elusive, the interaction of genetic, endocrine and environmental factors likely leads to PCOS (7).

In recent years, an increasing number of studies have confirmed that androgen status, IR and obesity play notable roles in the pathogenesis of PCOS (8-10). Previous studies have suggested that hyperandrogen status (11,12), IR (13-15) and obesity (16-19) increase the rate of miscarriage and decrease the pregnancy rate among patients with PCOS. Chang et al (13) indicated that IR affects endometrial function and the implantation process. Patel and Carr (20) revealed that hyperandrogen status and IR are associated with adverse fertility outcomes in patients with PCOS. Cohort studies of >9,500 cycles (21-23), indicate that obesity may induce embryo implantation failure by impairing endometrial receptivity.

Endometrial receptivity is an important factor for successful embryo implantation, which is accompanied by the fluctuations of steroid hormones and endometrial receptivity-related factors, the influx of uterine natural killer cells (uNK) cells and the inhibition of inflammatory factors. Endometrial receptivity is regulated by multiple factors, such as receptors of hormones (estrogen and progesterone), pro-inflammatory cytokines [IL-6, IL-8, IL-15 and monocyte chemoattractant protein-1 (MCP-1)] and endometrial decidualization-related factors [integrin β3 (avβ3) and insulin-like growth factor binding protein-1 (IGFBP-1)] (24).

In addition, adiponectin is also a biomarker of endometrial receptivity (25,26). Adiponectin, an insulin sensitizer secreted by adipocytes, is closely associated with the insulin signal transduction pathway (25,26). Adiponectin exerts its effects by binding to adapter protein containing PH domain, PTB domain and leucine zipper motif 1 (APPL1), together with AdipoR1 and AdipoR2, to activate different signaling pathways, such as MAPK, p38, ERK1/2, Akt and AMP-activated protein kinase (AMPK) (27). Adiponectin contains three main forms: Trimer, hexamer and high molecular weight (HMW). Some studies hypothesize that HMW adiponectin is the form most closely associated with insulin sensitivity (28). The binding of the APPL1 to adiponectin receptors is regulated by the APPL2; AdipoR1- and R2-dependent signaling is mediated through APPL 1 and APPL2. In the absence of adiponectin signal, APPL2 can bind to the adiponectin receptors or it can form an APPL1/APPL2 heterodimer which prevents the binding of APPL1/adiponectin receptors (29). The adiponectin signaling pathways play notable roles in anti-atherosclerosis, improving the insulin resistant state, reducing blood glucose and reducing anti-inflammatory effects.

Metabolic disorders (high androgen status, IR and obesity) may be the reason for embryo implantation failure, as androgen status and IR are two interacting factors. However, previous studies (13,21-23,30) did not assess different androgen status combining with different IR levels, nor consider the BMI cutoff value for Asian PCOS women. In addition, the majority of previous studies were performed in spontaneous ovulation. Therefore, based on previous findings (24,25) the present study investigated the endometrial receptivity in patients with PCOS with different metabolic abnormalities by assessing well-characterized endometrial receptivity markers, including adiponectin and its signaling protein, pro-inflammatory cytokines (MCP-1, IL-6 and IL-8) mediated through adiponectin, estrogen receptor (ERα), ERβ, progesterone receptor (PR), IL-15, avβ3 and IGFBP-1 in the endometrium during the window of implantation.

Materials and methods <sec> <title>Ethical approval

The present study was approved by the Clinical Scientific Research and Experimental Animal Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University [approval no. Ethics (2020) no. 422-1]. Written informed consent was obtained from all participants.

Participants

Infertile patients with PCOS attending the Department of Reproductive Endocrine of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) from November 2020 to January 2021 were recruited. The patients' PCOS diagnoses were based on the 2003 Rotterdam criteria (31). Each participant met at least two of the following criteria: i) Oligomenorrhea and/or chronic anovulation; ii) clinical and/or biochemical hyperandrogenism; and iii) polycystic ovarian ultrasonography changes. The exclusion criteria were patients with Cushing's syndrome, congenital adrenal hyperplasia, androgen-secreting tumor, tubal effusion, intrauterine adhesions, multiple endometrial polyps, chromosomal diseases, recurrent fertilization failures, organic diseases of the uterus or ovaries (such as endometriosis or adenomyosis), history of surgery, pelvic radiotherapy and chemotherapy in ovaries, thyroid diseases, hyperprolactinemia, adrenal gland tumors, cardiovascular and cerebrovascular diseases and mental illness and associated disorders. Patients who took hormones or drugs in the past 3 months (that would affect insulin) were also excluded. The diagnosis was confirmed by a pathologist, and the pathologist was independent from the study.

Levels of testosterone (T), sex hormone-binding globulin (SHBG), oral glucose tolerance test (OGTT) and fasting insulin were measured on the 2-5 days of menstruation. High androgen status was determined when the free androgen index (FAI) (%) [T (nmol/l)/SHBG (nmol/l)] was >4.5% (32). IR was considered when the homeostasis model assessment IR (HOMA-IR) [fasting insulin (µ international unit (IU)/ml) x fasting plasma glucose (FPG) (mmol/l)/22.5] was >2.4(33). According to the OGTT test, the present study defined FPG as ≥5.6 mmol/l and/or 2-h postprandial blood glucose (2hPBG) ≥7.7 mmol/l as impaired glucose tolerance (IGT); furthermore, FPG ≥7.0 mmol/l and/or 2hPBG ≥11.1 mmol/l was defined as diabetes mellitus (34).

The patients were classified into five different groups depending on their FAI, HOMA-IR, and OGTT results: i) Hyperandrogenemia combined with impaired glucose tolerance (HA + IGT) group (n=8), FAI ≥4.5% and HOMA-IR >2.4, 5.6 mmol/l ≤FPG ≤6.9 mmol/l, and/or 7.7 mmol/l ≤2 h PBG ≤11 mmol/l; ii) hyperandrogenemia combined with diabetes mellitus (HA + DM) group (n=8), FAI ≥4.5%, HOMA-IR >2.4, FPG ≥7.0 and/or 2 h PBG ≥11.1; iii) hyperandrogenemia combined with non-IR (HA + NIR) group (n=10), FAI ≥4.5%, HOMA-IR ≤2.4; iv) non-hyperandrogenemia androgen combined with IR (NHA + IR) group (n=8), FAI <4.5%, HOMA-IR >2.4; or v) non-hyperandrogenemia combined with non-IR (NHA + NIR) group (n=9), FAI <4.5%, HOMA ≤2.4. In addition, according to the BMI value, the subjects were divided into a lean/normal group (n=27; BMI<24 kg/m²), an overweight group (n=8; 24≤ BMI <28 kg/m²), and an obese group (n=8; BMI ≥28 kg/m²) (35).

Sample collection and treatments

The number of antral follicles was measured by vaginal B-ultrasound on the 2-5 days of menstruation for all participants, and ultrasound examinations were conducted every few days to monitor the number and size of the follicles as well as the endometrial thickness. In addition, the basic information of each patient was collected on the 2-5 days of menstruation, including age, BMI, years of infertility, and blood samples. In this sample, other possible interference in the diagnosis of PCOS [such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen, progesterone, testosterone, prolactin (PRL), anti-Mullerian hormone (AMH), thyroid-stimulating hormone (TSH), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG) and cholesterol (CHOL)] were included on Table I.

In order to guarantee the transformation of the endometrium to the secretory phase, hormone replacement therapy (HRT) was performed: 2 mg of estradiol was given daily (Progynova^®; Bayer) for endometrial proliferation support for ≥10 days. When the endometrium thickness reached 8 mm, 10 mg of dydrogesterone tablets were given twice a day (Duphaston^®; Abbott Biologicals B.V.) for 5 days for endometrial transformation. On the fifth day of endometrial transformation, intravenous blood and endometrium samples were collected. To minimize the biopsy difference, the endometrial biopsies were collected from the superficial layer of the endometrium instead of the basal layer. The receptive phase of endometrium was determined according to Noyes histological standard (36).

High molecular weight (HMW) adiponectin ELISA detection

The sensitivity of specific ELISA kit (cat. no. ml063723, MiBio) of HMW adiponectin was 10 ng/ml. The coefficient of variation within and between plates was <10 and 15%, respectively. The serum HMW adiponectin was determined using enzyme-linked immunosorbent assay (ELISA) with an enzyme-labeled instrument (SkanIt^™ software; Thermo Fisher Scientific, Inc.).

RNA extraction and PCR

The frozen endometrial samples were homogenized using TRIzol (cat. no.93289, Thermo Fisher Scientific, Inc.) reagent to obtain total ribonucleic acid (RNA). The RNA concentration was determined using spectrophotometry (A260:A280), and the integrity of RNA was determined using electrophoresis on a formaldehyde agarose gel. The tissue RNA extraction kit (EZBioscience) was used to extract 2 µg of total RNA from the endometrial sample; the RNA reverse transcription and SYBR Green qPCR Master Mix kit (EZBioscience) were used to perform the standard PCR according to the manufacturer's instructions. Human GAPDH was used as internal reference genes and as a biomarker of standardizing to the RNA load of each sample since the expression of this gene is relatively constant during the menstrual cycle (37). PCR amplification of target mRNA was assessed by using gene-specific primers in Table II. Primers were provided by Takara Biotechnology Co., Ltd. The PCR reaction was initiated with 95˚C for 5 min for amplification, melted at 95˚C for 10 sec, then annealing and extension were performed at 60˚C for 30 sec. The polymerase chain reaction products were analyzed using ethidium bromide agarose gel electrophoresis. A quantitative PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for real-time quantitative PCR and data analysis. Amplification efficiencies obtained from quantitative PCR were supported by using the method of 2^-ΔΔCq (38).

The relative expression levels of all target genes were calculated by normalizing to the reference gene (GAPDH) (2^-ΔCq), and the relative expression of target genes in different groups was presented as the fold-change relative to the control groups (2^-ΔΔCq). For the comparison of different metabolic disorders, the NHA + NIR group was used as the control group; for the comparison of different BMI values, the lean/normal group was selected as a control.

uNK cell cycle analysis

On the fifth day of endometrial transformation, endometrium samples were collected using the Endometrial Sampler (TAO Brush^™ IUMC). Endometrial tissue was weighed (weight, 300 µg) and immediately placed in PBS containing 0.25% collagenase from Clostridium histolyticum and 0.5% deoxyribonuclease bovine (all Sigma-Aldrich; Merck KGaA) All digestions were performed at 37˚C for 20 min with agitation. The tissue was filtered with a filter (100 um) and was centrifuged at a speed of 350 g at 4˚C for 5 min. The supernatant was removed and the endometrial cells were placed in PBS. The uNK cells are characterized by CD3^-/CD56⁺ granular lymphocytes, while the expression of CD16 is relatively low compared with peripheral blood natural killer cells (39). Cells were stained with the following monoclonal antibodies conjugated with FITC PE and APC at 20˚C for 20 min: CD3 (FITC anti-human; cat. no. 300402 UCHT1, BioLegend), CD16 (PE anti-human cat. no. 302003 3G8, BioLegend), CD56 (APC anti-human; cat. no. 362535 NCAM, BioLegend). Cells were acquired within 24 h of staining on a BD FACSAria flow cytometer (BD Biosciences). A gate set around CD3-lymphocytes was used to measure the proportion of NK-cell subsets within each sample. In the case of the NK-cell subset, a gate set around CD56+ lymphocytes was used to exclude NK-T-cell subset (Figs. S1 and S2). CELLQuest software (version 5.1 Becton Dickinson) was used for the data analysis.

Statistical analysis

In order to obtain statistical significance results, the number of subjects were calculated: n=2x [(tα + tβ)σ/δ]² (40), the test level α was set to 0.05 and the inspection power 1-β was set to 90%, checked the t boundary value table and only take one-sided, t (α=0.05)=1.645, one-sided t (β=0.1)=1.282. By consulting the literature (41), the discrimination (δ) and the standard deviation (σ) were substituted into the formula, samples required for each group was 8. SPSS software (version 20.0; IBM Corp.) was used for the statistical analysis. Normally distributed data were presented as mean ± standard deviation (SD), and the comparison between groups was performed using one-way analysis of variance. The data that was not normally distributed was presented as the median and interquartile range, and the comparison between groups was performed using the Kruskal-Wallis test. The Bonferroni test was used to analyze the post-hoc paired comparison. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Demographic data

As presented in Table I, there was no statistical difference in other clinical and endocrine characteristics among HA + IGT, HA + DM, HA + NIR, NHA + IR and NHA + NIR groups (P>0.05). However, only the comparison of BMI between the HA + IGT group (27.45 kg/m²) and the NHA + NIR group (19.11 kg/m²) demonstrated a significant difference (P<0.05).

HMW adiponectin

There was no significant difference in serum HMW adiponectin in patients across HA + IGT, HA + DM, HA + NIR, NHA + IR and NHA + NIR groups (P>0.05). In addition, no significant difference was revealed in serum HMW adiponectin in patients in lean/normal, overweight, and obese groups (P>0.05; Table III).

Endometrial receptivity-related factors

The relative mRNA expression levels of adiponectin, AdipoR1, AdipoR2, APPL1, ERα, ERβ, PR, IL-15, avβ3 and IGFBP-1 were significantly decreased, while the expression levels of IL-6 and IL-8 were significantly increased in HA + IGT, HA + DM, HA + NIR and NHA + IR groups when compared with the NHA + NIR group (all P<0.05). However, no significant difference was revealed amongst the HA + DM, HA + NIR, and NHA + IR groups (Fig. 1).

For the comparison of different bodyweight groups, the relative mRNA expression levels of adiponectin, AdipoR1, AdipoR2, APPL1, ERα, ERβ, PR, IL-15, avβ3, and IGFBP-1 were significantly lower, while the expression of MCP-1, IL-6 and IL-8 were significantly higher in the overweight and obese groups when compared with the lean/normal group (all P<0.05). Furthermore, the relative mRNA expression of PR in the obese group was significantly higher compared with that in the overweight group (P<0.01; Fig. 1).

Proportion of uNK cells

As presented in Fig. 2, the percentages of CD16^-/CD56⁺ and CD16⁺/CD56⁺ in endometrium were comparable across HA + IGT, HA + DM, HA + NIR, NHA + IR and NHA + NIR groups (P>0.05). In addition, no significant difference was revealed among patients in the lean/normal, overweight and obese groups (P>0.05).

Discussion

Wickham et al (42) hypothesized that IR can reduce serum HMW adiponectin, while O'Connor et al (43) claimed that serum HMW adiponectin is not dependent on IR. On the other hand, studies have indicated that high androgen status may indirectly reduce serum HMW adiponectin in patients with PCOS (44,45), but the evidence was still insufficient. Garcia et al (41) revealed that the serum adiponectin level in obese patients with PCOS was lower compared with that in the lean or obese patient groups. Nevertheless, other studies revealed no differences in the expression of adiponectin (46) or HMW adiponectin (47) among overweight/obese or normal-weight patients between PCOS and control groups. So far, there is no consensus on whether there are differences in serum adiponectin in different types of patients with PCOS (42-47). The present study revealed no significant difference in serum HMW adiponectin levels among different high androgen statuses, insulin-resistant levels or BMI levels in patients with PCOS. The various patient group settings and the detection method used for HMW adiponectin may explain the inconsistency in results compared with previous studies. Although the present study revealed no differences in HMW adiponectin, this does not mean that there was no difference in other forms of adiponectin. The alteration in other molecular forms of adiponectin should also be considered, and this needs further exploration.

The endometrium undergoes three different phases during the normal menstrual cycle, which is induced by the steroid hormones fluctuation (48). Estrogen (E2) promotes the expression of estrogen receptor α (Erα) and estrogen receptor β (Erβ); the expression levels of these receptors are highest in the late stage of proliferation. In addition, E2, together with ER, promotes progesterone receptor (PR) expression in the endometrium. After ovulation, progesterone inhibits the expression of ER in the endometrium, thus inducing decidualization. Decidualization is a necessary transformation of endometrium for successful embryo implantation, including endometrial stromal cells proliferation, the increase of glandular epithelial secretion and natural killer cells aggregation (24). Decidualization usually happens at 5-6 days after ovulation (49). The process of decidualization is accompanied by the alteration of ER, PR and inhibition of pro-inflammatory cytokines (interleukin and MCP-1) and endometrial pathological and physiological-related factors (avβ3, IGFBP-1) (24,50). Some researchers hypothesize that the decreased expression of ERα, ERβ, PR, IL-15, avβ3 and IGFBP-1 may indicate the decline of embryo implantation rate (51-54); however, the mechanism of how these factors affect endometrial receptivity remains unclear. Furthermore, previous studies have revealed increased expression of Erα (37,55,56) and PR (56), decreased expression of IL-15(39), avβ3(57) and IGFBP-1(58) and unchanged expression of Erβ (58) in secretory phase endometrium in patients with PCOS when compared with non-PCOS patients.

The present study demonstrated that the expression levels of ERα, ERβ, PR, IL-15, avβ3 and IGFBP-1 were significantly decreased in the presence of HA and/or IR and/or obesity (BMI ≥24 kg/m²), which were consistent with previous studies by Matteo et al (39), Cermik et al (57) and Piltonen et al (58). Nevertheless, the results were different from the findings by Margarit et al (56) and Quezada et al (37). The difference may be due to the diversity in studied population. Apart from the distinction of the hyperandrogenemia states, the IR levels and BMI, the control group used in previous studies were different. All previous studies were carried out with in untreated patients with PCOS (with spontaneous ovulation) and non-PCOS patients. By contrast, patients in the present study were treated with HRT to guarantee the endometrial transformation.

During the menstrual cycle, adiponectin and its receptors can be detected, the mRNA expression of adiponectin increased significantly in the early stage of proliferation; however, the mRNA expression levels of AdipoR1 and AdipoR2 increased significantly in the peri-implantation period (25). These changes have been confirmed in the artificial decidua model as well (59). Adiponectin has been indicated to be reduced in the endometrium of obese and PCOS patients (60). Garcia et al (41) demonstrated that the obese group (compared with the lean group) have an increased expression of endometrial AdipoR1 and a decreased expression of endometrial APPL1, while the AdipoR2 expression is similar (41). In addition, they demonstrated that when treating immortalized human endometrial stromal cell lines with testosterone and insulin, the mRNA expression levels of adiponectin, AdipoR1, AdipoR2 and APPL1 are significantly decreased (41).

Similarly, partly consistent with previous studies (37,41), the present study revealed that the mRNA expression levels of adiponectin, AdipoR1, AdipoR2 and APPL1 were significantly decreased in the presence of HA and/or IR and/or obesity (BMI ≥24 kg/m²) when compared with the NHA + NIR group and the lean/normal group, respectively. These findings were different from the results of Garcia et al (41), and this may be explained by the variance in sample collection. Garcia et al (41) collected samples in the proliferative phase and set untreated patients with PCOS (with spontaneous ovulation) as the control group. By contrast, all the patients with PCOS in the present study were treated with HRT, and endometrium samples were collected during the window of implantation in the HRT cycle.

MCP-1, IL-6 and IL-8 are pro-inflammatory factors which are involved in the morphological and pathological changes in the process of endometrial decidualization (61). It has been demonstrated that adiponectin exerts anti-inflammatory effects in the endometrium by inhibiting the production of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) (5). Compared with the NHA + NIR group and the lean/normal group, the expression levels of MCP-1, IL-6 and IL-8 were significantly increased in the endometrium in the presence of HA and/or IR and/or obesity (BMI ≥24 kg/m²) of patients with PCOS. It was hypothesized that the reduction of adiponectin may explain the increased levels of IL-6, IL-8 and MCP-1 in the endometrium.

Rosenbaum et al (30) revealed that obesity can affect endometrial decidualization in the mouse model and human embryonic stem cells. In addition, Comstock et al (62) claimed that obesity can change the expression of genes involved in implantation-related chemokine signaling pathways during implantation, especially in obese patients with metabolic syndrome (63). Notably, the present study revealed that the mRNA expression level of PR in the obese group was significantly higher compared with that in the overweight group. We hypothesized that obesity increased the expression of PR as the response to progesterone resistance (53). However, the underlying mechanism remains elusive and further studies are needed.

As a factor associated with endometrial receptivity (39,64), the percentage of uNK cells fluctuates along with the hormones changing during the menstrual cycle (65,66), which increases during the secretory phase (39). However, increases in the number of peripheral blood and endometrial NK cells (CD56⁺) have been used as an indicator to assess the risk of infertility or recurrent miscarriage (39). Piltonen et al (67) and Matteo et al (39) revealed that uNK cells decrease in the late menstrual secretion period, and the percentage of uNK cells CD16⁺/CD56⁺ is similar, while the percentage of CD16^-/CD56⁺ is lower in the secretion phase in patients with PCOS when compared with the control group. Nevertheless, no significant difference was revealed in the present study in uNK cells among patients with PCOS with different BMI, androgen status and IR levels. Considering the different phases of the menstrual cycle, the percentage of CD16^-/CD56⁺ was observed to decrease after implantation, while a previous study revealed the percentage of CD16^-/CD56⁺ was lower in the late menstrual secretion period (67). But this research needs to be explored further.

To avoid the impact of actual human embryo implantation on the endometrium, the present investigation was not performed in a conception cycle but in the HRT cycles. The HRT treatment was performed in all the patients with PCOS to promote endometrial transformation and cause implantation window-related changes. Although a previous study indicated that ER and PR expression is significantly decreased in the endometrium in the early luteal phase of HRT cycles (68), all the studied patients in the present study were treated with HRT; therefore avoiding the impact of internal hormone alteration caused by ovulation, and ensuring all patients were in the same endometrial phase. In the majority of previous studies (24,37,41) the expression of receptivity markers are measured from mRNA level. However, to clarify the differences in the expression level of theses markers, the detection from protein level is important; thus further exploration is needed to validate the present findings.

In conclusion, evidence regarding secretory endometrial receptivity factors in patients with PCOS is limited, so consistent conclusions cannot yet be made. The present study revealed that the IR status, hyperandrogenemia and obesity would impact the endometrial receptivity in patients with PCOS, which may explain the damaged embryo implantation and pregnancy outcomes. To develop the targeted therapies and improve pregnancy outcomes in patients with PCOS, further studies are needed to investigate the underlying mechanism of the impaired endometrium receptivity caused by metabolic disorders.

Supplementary Material

uNK cell subtype percentages of studied groups. Images represent flow cytometry plots of endometrial cells, including lymphocyte cells, single cells, and CD3<sup>-</sup> cells. (A) HA + IGT group; (B) HA + DM group; (C) HA + NIR group; (D) NHA + IR group; (E) NHA + NIR group. HA, hyperandrogenemia; IGT, impaired glucose tolerance; DM, diabetes mellitus; NIR, noninsulin resistance; NHA, non-hyperandrogenemia androgen; IR, insulin resistance; uNK cells, uterine natural killer cells.

uNK cell subtype percentages of studied groups. Images represent flow cytometry plots of endometrial cells, including lymphocytes cells, single cells, and CD3<sup>-</sup> cells. (A) lean/normal group; (B) overweight group; (C) obese group. HA, hyperandrogenemia; IGT, impaired glucose tolerance; DM, diabetes mellitus; NIR, non-insulin resistance; NHA, non-hyperandrogenemia androgen; IR, insulin resistance; uNK cells, uterine natural killer cells.

Acknowledgements

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

CW and QYM conceptualized the study and analyzed and interpreted the data. CW and YXW performed experiments and wrote the manuscript. CW and QYM confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Dabadghao

Roberts

Wang

Davies

Norman

Glucose tolerance abnormalities in Australian women with polycystic ovary syndrome

Med J Aust1873283312007

17874978

10.5694/j.1326-5377.2007.tb01273.x

Zhang

Yang

Wei

Song

Wang

Prevalence of polycystic ovary syndrome in women in China: A large community-based study

Hum Reprod28256292562013

23814096

10.1093/humrep/det262

Ovalle

Azziz

Insulin resistance, polycystic ovary syndrome, and type 2 diabetes mellitus

Fertil Steril77109511052002

12057712

10.1016/s0015-0282(02)03111-4

DeUgarte

Bartolucci

Azziz

Prevalence of insulin resistance in the polycystic ovary syndrome using the homeostasis model assessment

Fertil Steril83145414602005

15866584

10.1016/j.fertnstert.2004.11.070

Carmina

Lobo

Use of fasting blood to assess the prevalence of insulin resistance in women with polycystic ovary syndrome

Fertil Steril826616652004

15374711

10.1016/j.fertnstert.2004.01.041

Moran

Pasquali

Teede

Hoeger

Norman

Treatment of obesity in polycystic ovary syndrome: A position statement of the androgen excess and polycystic ovary syndrome society

Fertil Steril92196619822009

19062007

10.1016/j.fertnstert.2008.09.018

Greenwood

Huddleston

Insulin resistance in polycystic ovary syndrome: Concept versus cutoff

Fertil Steril1128278282019

31731944

10.1016/j.fertnstert.2019.08.100

Zeng

Xie

Liu

Long

Polycystic ovarian syndrome: Correlation between hyperandrogenism, insulin resistance and obesity

Clin Chim Acta5022142212020

31733195

10.1016/j.cca.2019.11.003

Xiao

Xue-Lian

The role of androgens in the pathogenesis and treatment of female reproductive endocrine diseases

J Int Obstet Gynecol462292322019

Patlolla

Vaikkakara

Sachan

Venkatanarasu

Bachimanchi

Bitla

Settipalli

Pathiputturu

Sugali

Chiri

Heterogenous origins of hyperandrogenism in the polycystic ovary syndrome in relation to body mass index and insulin resistance

Gynecol Endocrinol342382422018

29068242

10.1080/09513590.2017.1393062

Naver

Grinsted

Larsen

Hedley

Jørgensen

Christiansen

Nilas

Increased risk of preterm delivery and pre-eclampsia in women with polycystic ovary syndrome and hyperandrogenaemia

BJOG1215755812014

24418062

10.1111/1471-0528.12558

Elenis

Desroziers

Persson

Sundström Poromaa

Campbell

Early initiation of anti-androgen treatment is associated with increased probability of spontaneous conception leading to childbirth in women with polycystic ovary syndrome: A population-based multiregistry cohort study in Sweden

Hum Reprod36142714352021

33454768

10.1093/humrep/deaa357

Chang

Han

Seok

Lee

Yoon

Lee

Insulin resistance does not affect early embryo development but lowers implantation rate in in vitro maturation-in vitro fertilization-embryo transfer cycle

Clin Endocrinol7993992013

23176069

10.1111/cen.12099

Tian

Shen

Norman

Wang

Insulin resistance increases the risk of spontaneous abortion after assisted reproduction technology treatment

J Clin Endocrinol Metab92143014332007

17244790

10.1210/jc.2006-1123

Khattab

Mohsen

Foutouh

Ramadan

Moaz

Al-Inany

Metformin reduces abortion in pregnant women with polycystic ovary syndrome

Gynecol Endocrinol226806842006

17162710

10.1080/09513590601010508

Huang

Liao

Dong

Zhang

Effect of overweight/obesity on IVF-ET outcomes in Chinese patients with polycystic ovary syndrome

Int J Clin Exp Med7587258762014

25664123

Veleva

Tiitinen

Vilska

Hydén-Granskog

Tomás

Martikainen

Tapanainen

High and low BMI increase the risk of miscarriage after IVF/ICSI and FET

Hum Reprod238788842008

18281684

10.1093/humrep/den017

Fedorcsák

Storeng

Dale

Tanbo

Abyholm

Obesity is a risk factor for early pregnancy loss after IVF or ICSI

Acta Obstet Gynecol Scand7943482000

10646815

Dai

Guo

Zhai

Sun

Overweight and obesity adversely affect outcomes of assisted reproductive technologies in polycystic ovary syndrome patients

Int J Clin Exp Med69919952013

24260609

Patel

Carr

Oocyte quality in adult polycystic ovary syndrome

Semin Reprod Med261962032008

18302111

10.1055/s-2008-1042958

Jungheim

Schon

Schulte

DeUgarte

Fowler

Tuuli

IVF outcomes in obese donor oocyte recipients: A systematic review and meta-analysis

Hum Reprod28272027272013

23847110

10.1093/humrep/det292

Wattanakumtornkul

Damario

Stevens

Thornhill

Tummon

Body mass index and uterine receptivity in the oocyte donation model

Fertil Steril803363402003

12909496

10.1016/s0015-0282(03)00595-8

Bellver

Rossal

Bosch

Zúñiga

Corona

Meléndez

Gómez

Simón

Remohí

Pellicer

Obesity and the risk of spontaneous abortion after oocyte donation

Fertil Steril79113611402003

12738508

10.1016/s0015-0282(03)00176-6

Piltonen

Polycystic ovary syndrome: Endometrial markers

Best Pract Res Clin Obstet Gynaecol3766792016

27156350

10.1016/j.bpobgyn.2016.03.008

Takemura

Osuga

Yamauchi

Kobayashi

Harada

Hirata

Morimoto

Hirota

Yoshino

Koga

Expression of adiponectin receptors and its possible implication in the human endometrium

Endocrinology147320332102006

16601138

10.1210/en.2005-1510

Wang

Xie

Zhu

Serum adiponectin and resistin levels in patients with polycystic ovarian syndrome and their clinical implications

J Huazhong Univ Sci Technolog Med Sci306386422010

21063848

10.1007/s11596-010-0556-8

Barbe

Bongrani

Mellouk

Estienne

Kurowska

Grandhaye

Elfassy

Levy

Rak

Froment

Dupont

Mechanisms of adiponectin action in fertility: An overview from gametogenesis to gestation in humans and animal models in normal and pathological conditions

Int J Mol Sci2015262019

30934676

10.3390/ijms20071526

Fisher

Trujillo

Hanif

Barnett

McTernan

Scherer

Kumar

Serum high molecular weight complex of adiponectin correlates better with glucose tolerance than total serum adiponectin in Indo-Asian males

Diabetologia48108410872005

15902402

10.1007/s00125-005-1758-7

Yamauchi

Iwabu

Okada-Iwabu

Kadowaki

Adiponectin receptors: A review of their structure, function and how they work

Best Pract Res Clin Endocrinol Metab2815232014

24417942

10.1016/j.beem.2013.09.003

Rosenbaum

Haber

Dunaif

Insulin resistance in polycystic ovary syndrome: Decreased expression of GLUT-4 glucose transporters in adipocytes

Am J Physiol264E197E2021993

8447386

10.1152/ajpendo.1993.264.2.E197

Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group

Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome

Fertil Steril8119252004

14711538

10.1016/j.fertnstert.2003.10.004

Chang

Knochenhauer

Bartolucci

Azziz

Phenotypic spectrum of polycystic ovary syndrome: Clinical and biochemical characterization of the three major clinical subgroups

Fertil Steril83171717232005

15950641

10.1016/j.fertnstert.2005.01.096

Soonthornpun

Setasuban

Thamprasit

Chayanunnukul

Rattarasarn

Geater

Novel insulin sensitivity index derived from oral glucose tolerance test

J Clin Endocrinol Metab88101910232003

12629079

10.1210/jc.2002-021127

Salley

Wickham

Cheang

Essah

Karjane

Nestler

Glucose intolerance in polycystic ovary syndrome-a position statement of the androgen excess society

J Clin Endocrinol Metab92454645562007

18056778

10.1210/jc.2007-1549

Finucane

Stevens

Cowan

Danaei

Lin

Paciorek

Singh

Gutierrez

Bahalim

National, regional, and global trends in body-mass index since 1980: Systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants

Lancet3775575672011

21993114

10.1016/j.juro.2011.07.061

Noyes

Hertig

Rock

Reprint of: Dating the endometrial biopsy

Fertil Steril112 (4 Suppl 1)e93e1152019

31623748

10.1016/j.fertnstert.2019.08.079

Quezada

Avellaira

Johnson

Gabler

Fuentes

Vega

Evaluation of steroid receptors, coregulators, and molecules associated with uterine receptivity in secretory endometria from untreated women with polycystic ovary syndrome

Fertil Steril85101710262006

16580389

10.1016/j.fertnstert.2005.09.053

Livak

Schmittgen

TDL

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method

Methods254024082001

11846609

10.1006/meth.2001.1262

Matteo

Serviddio

Massenzio

Scillitani

Castellana

Picca

Sanguedolce

Cignarelli

Altomare

Bufo

Reduced percentage of natural killer cells associated with impaired cytokine network in the secretory endometrium of infertile women with polycystic ovary syndrome

Fertil Steril9422222227.e1-e32010

20236632

10.1016/j.fertnstert.2010.01.049

Sinclair

Haynes

Selecting participants that raise a clinical trial's population attributable fraction can increase the treatment effect within the trial and reduce the required sample size

J Clin Epidemiol648939022011

21420281

10.1016/j.jclinepi.2010.12.006

Garcia

Oróstica

Poblete

Rosas

Astorga

Romero

Vega

Endometria from obese PCOS women with hyperinsulinemia exhibit altered adiponectin signaling

Horm Metab Res479019092015

26197851

10.1055/s-0035-1555806

Wickham

ER III

Cheang

Clore

Baillargeon

Nestler

Total and high-molecular weight adiponectin in women with the polycystic ovary syndrome

Metabolism603663722011

20359725

10.1016/j.metabol.2010.02.019

O'Connor

Phelan

Tun

Boran

Gibney

Roche

High-molecular-weight adiponectin is selectively reduced in women with polycystic ovary syndrome independent of body mass index and severity of insulin resistance

J Clin Endocrinol Metab95137813852010

20080859

10.1210/jc.2009-1557

Panidis

Kourtis

Farmakiotis

Mouslech

Rousso

Koliakos

Serum adiponectin levels in women with polycystic ovary syndrome

Hum Reprod18179017962003

12923129

10.1093/humrep/deg353

Chan

Hoo

Wang

Tan

Zhang

Chen

Lam

Tse

Cooper

Lam

Testosterone selectively reduces the high molecular weight form of adiponectin by inhibiting its secretion from adipocytes

J Biol Chem28018073180802005

15760892

10.1074/jbc.M414231200

Lecke

Mattei

Morsch

Spritzer

Abdominal subcutaneous fat gene expression and circulating levels of leptin and adiponectin in polycystic ovary syndrome

Fertil Steril95204420492011

21419406

10.1016/j.fertnstert.2011.02.041

Barber

Hazell

Christodoulides

Golding

Alvey

Burling

Vidal-Puig

Groome

Wass

Franks

McCarthy

Serum levels of retinol-binding protein 4 and adiponectin in women with polycystic ovary syndrome: Associations with visceral fat but no evidence for fat mass-independent effects on pathogenesis in this condition

J Clin Endocrinol Metab93285928652008

18445670

10.1210/jc.2007-2759

Snijders

de Goeij

Debets-Te Baerts

Rousch

Koudstaal

Bosman

Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause

J Reprod Fertil943633711992

1593539

10.1530/jrf.0.0940363

Gellersen

Brosens

Cyclic decidualization of the human endometrium in reproductive health and failure

Endocr Rev358519052014

25141152

10.1210/er.2014-1045

Weyer

Tataranni

Bogardus

Pratley

Insulin resistance and insulin secretory dysfunction are independent predictors of worsening of glucose tolerance during each stage of type 2 diabetes development

Diabetes Care2489942001

11194248

10.2337/diacare.24.1.89

Benkhalifa

Madkour

Louanjli

Bouamoud

Saadani

Kaarouch

Chahine

Sefrioui

Merviel

Copin

From global proteome profiling to single targeted molecules of follicular fluid and oocyte: Contribution to embryo development and IVF outcome

Expert Rev Proteomics124074232015

26098221

10.1586/14789450.2015.1056782

Lessey

Palomino

Apparao

Young

Lininger

Estrogen receptor-alpha (ER-alpha) and defects in uterine receptivity in women

Reprod Biol Endocrinol4 (Suppl 1)S92006

17118173

10.1186/1477-7827-4-S1-S9

Young

Lessey

Progesterone function in human endometrium: Clinical perspectives

Semin Reprod Med285162010

20104424

10.1055/s-0029-1242988

Gnainsky

Granot

Aldo

Barash

Schechtman

Mor

Dekel

Local injury of the endometrium induces an inflammatory response that promotes successful implantation

Fertil Steril94203020362010

20338560

10.1016/j.fertnstert.2010.02.022

Gregory

Wilson

Apparao

Lininger

Meyer

Kowalik

Fritz

Lessey

Steroid receptor coactivator expression throughout the menstrual cycle in normal and abnormal endometrium

J Clin Endocrinol Metab87296029662002

12050280

10.1210/jcem.87.6.8572

Margarit

Taylor

Roberts

Hopkins

Davies

Brenton

Conlan

Bunkheila

Joels

White

Gonzalez

MUC1 as a discriminator between endometrium from fertile and infertile patients with PCOS and endometriosis

J Clin Endocrinol Metab95532053292010

20826587

10.1210/jc.2010-0603

Cermik

Selam

Taylor

Regulation of HOXA-10 expression by testosterone in vitro and in the endometrium of patients with polycystic ovary syndrome

J Clin Endocrinol Metab882382432003

12519859

10.1210/jc.2002-021072

Piltonen

Chen

Khatun

Kangasniemi

Liakka

Spitzer

Tran

Huddleston

Irwin

Giudice

Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro

Hum Reprod30120312152015

25750105

10.1093/humrep/dev055

Gamundi-Segura

Serna

Oehninger

Horcajadas

Arbones-Mainar

Effects of adipocyte-secreted factors on decidualized endometrial cells: Modulation of endometrial receptivity in vitro

J Physiol Biochem715375462015

25686566

10.1007/s13105-015-0393-0

Palin

Bordignon

Murphy

Adiponectin and the control of female reproductive functions

Vitam Horm902392872012

23017719

10.1016/B978-0-12-398313-8.00010-5

Yoshino

Osuga

Hirota

Koga

Hirata

Yano

Ayabe

Tsutsumi

Taketani

Endometrial stromal cells undergoing decidualization down-regulate their properties to produce proinflammatory cytokines in response to interleukin-1 beta via reduced p38 mitogen-activated protein kinase phosphorylation

J Clin Endocrinol Metab88223622412003

12727980

10.1210/jc.2002-021788

Comstock

Diaz-Gimeno

Cabanillas

Bellver

Sebastian-Leon

Shah

Schutt

Valdes

Ruiz-Alonso

Valbuena

Does an increased body mass index affect endometrial gene expression patterns in infertile patients? A functional genomics analysis

Fertil Steril107740748.e22017

27919438

10.1016/j.fertnstert.2016.11.009

Tierney

Tulac

Huang

Giudice

Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation

Physiol Genomics1647662003

14532334

10.1152/physiolgenomics.00066.2003

Gellersen

Brosens

Decidualization of the human endometrium: Mechanisms, functions, and clinical perspectives

Semin Reprod Med254454532007

17960529

10.1055/s-2007-991042

Giudice

Endometrium in PCOS: Implantation and predisposition to endocrine CA

Best Pract Res Clin Endocrinol Metab202352442006

16772154

10.1016/j.beem.2006.03.005

Laird

Tuckerman

Cork

Linjawi

Blakemore

A review of immune cells and molecules in women with recurrent miscarriage

Hum Reprod Update91631742003

12751778

10.1093/humupd/dmg013

Piltonen

Chen

Erikson

Spitzer

Barragan

Rabban

Huddleston

Irwin

Giudice

Mesenchymal stem/progenitors and other endometrial cell types from women with polycystic ovary syndrome (PCOS) display inflammatory and oncogenic potential

J Clin Endocrinol Metab98376537752013

23824412

10.1210/jc.2013-1923

Habiba

Bell

Al-Azzawi

The effect of hormone replacement therapy on the immunoreactive concentrations in the endometrium of oestrogen and progesterone receptor, heat shock protein 27, and human beta-lactoglobulin

Hum Reprod1536422000

10611185

10.1093/humrep/15.1.36

Figure 1

Relative mRNA levels of receptivity-related factors in the endometrium across studied groups. Relative mRNA levels of (A) adiponectin, AdipoR1, AdipoR2 and APPL1, (B) MCP-1, IL-6 and IL-8 and (C) ERα, ERβ, PR, IL-15, avβ3 and IGFBP-1 of receptivity-related factors in endometrium obtained from HA + IGT, HA + DM, HA + NIR and NHA + IR groups. For each gene, the mRNA expression levels were normalized to the mean value of NHA + NIR group (internal control). Relative mRNA levels of (D) adiponectin, AdipoR1, AdipoR2 and APPL1, (E) MCP-1, IL-6 and IL-8 and (F) ERα, ERβ, PR, IL-15, avβ3 and IGFBP-1 of receptivity-related factors in endometrium obtained from lean/normal, overweight and obese groups. For each gene, the mRNA expression levels were normalized to the mean value of lean/normal group (internal control). ^*P<0.05, ^**P<0.01. HA, hyperandrogenemia; IGT, impaired glucose tolerance; DM, diabetes mellitus; NIR, non-insulin resistance; NHA, non-hyperandrogenemia androgen; IR, insulin resistance; AdipoR, adiponectin receptor; APPL1, adapter protein containing PH domain, PTB domain and leucine zipper motif 1; MCP-1, monocyte chemoattractant protein-1; IL, interleukin; ER, estrogen receptor; PR, progesterone receptor; avβ3, integrin β3; IGFBP-1, insulin-like growth factor binding protein-1.

Figure 2

Percentage of subgroups of uNK cells in the endometrium across studied groups. Percentage of CD16-/CD56+ in endometrium obtained from (A) HA + IGT, HA + DM, HA + NIR and NHA + IR groups and (B) from lean/normal, overweight, and obese groups. (C) Percentage of CD16+/CD56+ in endometrium obtained from (C) HA + IGT, HA + DM, HA + NIR and NHA + IR groups and (D) from lean/normal, overweight and obese groups. HA, hyperandrogenemia; IGT, impaired glucose tolerance; DM, diabetes mellitus; NIR, non-insulin resistance; NHA, non-hyperandrogenemia androgen; IR, insulin resistance; uNK cells, uterine natural killer cells.

Table I

Chemical and endocrine characteristics of the studied groups.

Parameters	HA + IGT	HA + DM	HA + NIR	NHA + IR	NHA + NIR	P-value
Number	8.00	8.00	10.00	8.00	9.00
Age (years)	25.63±3.02	26.43±3.87	28.20±3.36	26.60±2.51	28.22±2.39	0.33
WHR	0.89±0.03	0.92±0.03	0.89±0.04	0.86±0.03	0.90±0.02	0.16
BMI (kg/m²)	25.05±3.2	27.45±5.05	21.25±1.93	23.37±3.69	19.11±2.37^a	<0.001
FSH (mIU/ml)	5.35±1.60	4.94±0.91	5.29±1.29	5.40±0.64	5.29 (4.68,5.97)	0.81
LH (mIU/ml)	7.26±4.39	7.53±2.51	8.68±4.83	40.94±11.03	7.58±3.66	0.76
E2 (pg/ml)	33.75±9.15	35.3±10.41	40.81±17.88	40.94±11.03	39.49±15.92	0.86
PRL (ng/ml)	13.01 (9.29,23.37)	17.67±8.06	15.22 (10.25,27.47)	12.41±5.29	14.39 (12.81,20.96)	0.78
AMH	11.44±5.17	7.57±4.56	11.05±5.57	10.46 (6.62,11.15)	9.04 (6.61,12.97)	0.38
TSH	1.80±1.11	1.55 (1.25,2.14)	2.16±1.09	1.52±0.63	1.97±0.58	0.62
CHOL	5.15±0.85	4.9(4.6,5.3)	5.11±0.99	4.6(4.6,5.95)	4.6±0.89	0.48
TG	1.01 (0.89,2.03)	1.3±0.65	1.05±0.39	1.11±0.43	0.85±0.13	0.42
HDL	1.28±0.36	1.24 (1.14,1.31)	1.54±0.30	1.35±0.20	1.40±0.25	0.34
LDL	3.29±0.62	3.22±0.22	1.54±0.30	1.35±0.20	1.40±0.25	0.22

^aP<0.05 compared with HA + IGT. Data are presented as means ± SEM (normal distribution) or as medium (interquartile ranges) (non-normal distribution). HA + IGT, hyperandrogenemia combined with impaired glucose tolerance; HA + DM, hyperandrogenemia combined with diabetes mellitus; HA + NIR, hyperandrogenemia combined with non-insulin resistance; NHA + IR, non-hyperandrogenemia androgen combined with insulin resistance; NHA + NIR, non-hyperandrogenemia combined with non-insulin resistance; WHR, waist to hip ratio; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estrogen; P, progesterone; PRL, prolactin; AMH, anti-mullerian hormone; TSH, thyroid-stimulating hormone; CHOL, cholesterol; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein; IU, international unit.

Table II

Gene-specific primer sequences.

Target genes	Primer sequences (5'-3')
GAPDH	Sense GAGTCAACGGATTTGGTCGT
	Antisense ATCCACAGTCTTCTGGGTG
Adiponectin	Sense TGCTGGGAGCTGTTCTACTG
	Antisense TACTCCGGTTTCACCGATGTC
AdipoR1	Sense AAACTGGCAACATCTGGACC
	Antisense GCTGTGGGGAGCAGTAGAAG
AdipoR2	Sense ACAGGCAACATTTGGACACA
	Antisense CCAAGGAACAAAACTTCCCA
APPL1	Sense TTAGCTGCCCGGGCCATCCATA
	Antisense ATCTTTTCCCCCTCATTGTTTG
MCP-1	Sense CAGCCAGATGCAATCAATGCC
	Antisense TGGAATCCTGAACCCACTTCT
IL-6	Sense CAGACAGCCACTCACCTCTTC
	Antisense TGCCAGTGCCTCTTTGCT
IL-8	Sense ATGACTTCCAAGCTGGCCGT
	Antisense TCCTTGGCAAAACTGCACCT
ERα	Sense GGTCAGTGCCTTGTTGGATG
	Antisense TGCCAGGTTGGTCAGTAAGC
ERβ	Sense ACTGGGATTGTGTGGTCAGC
	Antisense AGAGGATAGGCATCGGCATT
PR	Sense TTTAAGAGGGCAATGGAAGG
	Antisense CGGATTTTATCAACGATGCAG
IL-15	Sense GTCCGGAGATGCAAGTATTCA
	Antisense TCCTCACATTCTTTGCATCCA
avβ3	Sense AAGAGCCAGAGTGTCCCAAG
	Antisense AGTTTCCAGATGAGCAGGGC
IGFBP-1	Sense TTTTACCTGCCAAACTGCAACA
	Antisense CCCATTCCAAGGGTAGACGC

GAPDH, Human glyceraldehyde-3-phosphate dehydrogenase; AdipoR, Adiponectin receptor; APPL1, adapter protein containing PH domain, PTB domain and leucine zipper motif 1; AdipoR, adiponectin receptor; MCP-1, monocyte chemoattractant protein-1; IL, interleukin; ER, estrogen receptor; PR, progesterone receptor; avβ3, integrin β3; IGFBP-1, insulin-like growth factor binding protein-1.

Table III

Comparisons of HMW adiponectin levels among groups.

Groups	HMW adiponectin (ng/ml)	P-value
HA + IGT	4703.93 (4,148.54-4,768.53)
HA + DM	5,129.82±510.88	0.11
HA + NIR	4,625.06±1,084.25
NHA + IR	4,626.95±452.29
NHA + NIR	4,283.51±407.41
Lean/normal	4,520.65±779.47
Overweight	4,736.18±505.46	0.53
Obese	4,843.26±455.09

Data are presented as means ± SEM (normal distribution) or as medium (interquartile ranges) (non-normal distribution). HMW, High molecular weight; P, probability.