Recruitment of patients and collection of blood, follicular fluid and ovarian granulosa cells

Blood, follicular fluid and ovarian granulosa cells were collected from patients who underwent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at Center for Reproductive Medicine between January 2018 and December 2020. Renji Hospital of Shanghai Jiao Tong University under a protocol approved by the Ethics Committee of Renji Hospital of Shanghai Jiao Tong University with informed consent (No. 2018072606). PCOS was diagnosed according to the revised Rotterdam consensus [26]. Women with tubal factor-related infertility were enrolled into the non-PCOS group. Enrolled PCOS and non-PCOS patients were sub-divided into groups with and without IR according to HOMA-IR (homeostasis model of assessment for insulin resistance index), which was calculated following the equation of fating insulin×fasting glucose/22.5). HOMA-IR ≥ 2.68 was diagnosed as IR [27]. Four groups were thus classified as following: non-PCOS without IR (n = 22), non-PCOS with IR (n = 22), PCOS without IR (n = 22) and PCOS with IR (n = 22). Insulin and glucose levels in the blood were measured with chemiluminescence assay (Beckman Access Health Co) and glucose oxidase method (Roche, Mannheim, Germany) respectively. Subjects with any of the following were excluded from this study: (1) history of unilateral oophorectomy; (2) endometriosis; (3) abnormal karyotype; (4) recurrent spontaneous miscarriage; and (5) other medical conditions such as acute inflammation screened by body temperature, blood routine test and high-sensitive C-reactive protein (CRP) test that contraindicated assisted reproductive technology and/or pregnancy. Patients diagnosed with IR were prescribed metformin treatment before IVF or ICSI treatment.

GnRH antagonist protocol was adopted for ovarian stimulation in both PCOS and non-PCOS patients under routine protocolperformed in our reproductive center. When two or more oocytes measured greater than 18 mm, urinary human chorionic gonadotropin was administered to trigger ovulation. Oocytes retrieval was performed 34-36 h later. Retrieved eggs were fertilized by IVF unless ICSI was indicated. Indications for ICSI included severe oligozoospermia and obstructive azoospermia. Fertilization was checked 16-18 h later. Embryos were cultured to cleavage stage. Cleavage embryos with ≥6 cells and < 20% fragmentation on day 3 were defined as good quality.

On the day of oocytes retrieval, peripheral venous blood and follicular fluid were collected. The first tube follicular fluid without blood contamination was collected. The remaining follicular fluid from the same patient was pooled. After centrifugation, the supernatant follicular fluid from first tube without blood contamination was collected. And the cell pellet from the pooled follicular fluid was resuspended in PBS for further dispersing in 0.1% hyaluronidase (Sigma Chemical Co, St-Louis, Mo). Dispersed cells were then centrifuged on Ficoll gradient to remove contaminating red blood cells. Purified granulosa cells were either frozen for later RNA extraction in − 80°C or cultured in DMEM/Hams F12 containing 10% FBS (Gibco, Grand Island, NY).

Examination of hormonal profiles

Hormonal profiles of recruited patients were determined by measuring follicular stimulating hormone (FSH, catalog number 07027346190), luteinizing hormone (LH, catalog number 07027575190), testosterone (T, catalog number 07027915190) and testerone (T, catalog number 07027915190) with chemiluminescence (Beckman Access Health Company, Chaska, MN). Anti-mullerian hormone (AMH) in the serum was determined by ELISA assay (Kangrun, Guangzhou, China, catalog number K-1401-100 N).

Determination of insulin sensitivity in granulosa cells from PCOS and non-PCOS patients

Six patients from each group (PCOS and non-PCOS patients with or without IR) were randomly chosen for this study. Granulosa cells from these patients were cultured overnight, and Akt phosphorylation and glucose uptake were then determined following insulin (100 nM, sigma) treatment for 15 min and 30 min. Akt phosphorylation was measured with Western blotting. For glucose uptake measurement, granulosa cells cultured in a 12-well plate were placed in glucose-free DMEM medium for treatment with insulin (100 nM) for 30 min. Then, the cells were incubated with 74 kBq [F18]-fluorodeoxyglucose ([F18]-FDG) at 37°C for 10 min. After washing with PBS, the cells were lysed with 5% NaOH. The lysate was countered along with a standard solution with a γ-counter (LS6500, Beckerman Coulter). The incorporation of [18F]-FDG was quantified by the percentage of the original concentration and normalized to the number of cells as an indicator of glucose uptake.

Measurement of SAA1 abundance in PCOS and non-PCOS patients with and without IR

SAA1 abundance in serum, follicular fluid and granulosa cells obtained from PCOS and non-PCOS patients with and without IR was measured with an immunoassay kit (R&D System, Minneapolis, MN, DY3019-05) following a protocol provided by the manufacturer. The detection limit was 1.6 ng/ml, and the cross reactivity to SAA2 was 2.7%. SAA1 mRNA abundance in granulosa cells was measured with quantitative real time PCR (qRT-PCR).

Study of SAA1 expression in granulosa cells

SAA1 expression was examined in cultured granulosa cells prepared from non-PCOS patients with immunofluorescent staining, qRT-PCR and immunoassay. For immunofluorescent staining, cultured granulosa cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% trixonX-100. After blocking the non-specific binding sites, the cells were incubated with primary antibody against SAA1 (1:100) (Abcam, Cambrige, UK) overnight at 4°C. Following washing with PBS, the cells were incubated with Alexa Fluor 488-labeled (green color) secondary antibody in darkness for 2 h. Nuclei were counterstained with DAPI (blue color). The staining was examined using a fluorescence microscope (Zeiss, DB, Germany).

For examination of SAA1 expression and production in the presence and absence of pro-inflammatory factor, granulosa cells were treated with interleukin-1β (IL-1β) (0 and 1 ng/ml, 24 h) (Sigma). After treatment, granulosa cells were collected for RNA extraction for measurement of SAA1 mRNA with qRT-PCR and conditioned culture medium was collected for determination of SAA1 production with an immunoassay kit (R&D System). Effect of SAA1 (0, 0.01, 0.1 and 1 μg/ml, 24 h) (endotoxin < 0.1 ng/μg, PeproTech Inc, Rocky Hill, NJ) on its own expression was also examined. SAA1 has been shown to bind TLR2/4 and activate NF-κB signaling pathway [11, 12, 28]. The role of TLR2/4 and NF-κB in the effect of SAA1 expression was investigated by treating the cells with SAA1 (1 μg/ml, 24 h) in the presence or absence of TLR2/4 antagonist OxpAPC (30 μg/ml, Invivogen, San Diego, CA) and NF-κB inhibitor JSH-23 (10 μM, Sigma) respectively.

Examination of effects of SAA1 on insulin sensitivity in granulosa cells

Dose-dependent effects of SAA1 (0, 0.01, 0.1 and 1 μg/ml, 24 h) on PTEN and GLUT4 abundance were examined in cultured granulosa cells. Then, effects of SAA1 on insulin (100 nM, 15 min or 30 min)-stimulated Akt phosphorylation, GLUT4 translocation (from cytoplasm to membrane) and glucose uptake were examined in granulosa cells pretreated with SAA1(1 μg/ml, 24 h). The involvement of TLR2/4 and NF-κB was studied by treating the cells with SAA1 (1 μg/ml, 24 h) in the presence or absence of OxpAPC (30 μg/ml) or JSH-23 (10 μM). Time-course of SAA1 (1 μg/ml, 0, 15, 30, 60 and 180 min) on p65 phosphorylation, a subunit of NF-κB, was also examined.

Extraction of RNA and analysis with qRT-PCR

Total RNA was extracted from frozen and cultured granulosa cells using a total RNA kit (OMEGA Bio-Tek, Norcross, GA). After determination of RNA quality, mRNA was reverse-transcribed to cDNA using PrimeScript® RT Master Mix (TaKaRa, Dalian, China). Amount of SAA1 mRNA was determined with qRT-PCR using the above reverse-transcribed cDNA and power SYBR Premix Ex Taq™ (TaKaRa) following a previously described protocol [29]. Abundance of mRNA in each sample was calculated using the 2-ΔΔCT method, and the ratio of SAA1 transcript over ACTB transcript, the house-keeping gene, was obtained to indicate relative SAA1 mRNA abundance. Primer sequences for PCR were as follows: SAA1, forward 5′-TTTCTGCTCCTTGGTCCTGG-3′ and reverse 5′-CTCTGGCATCGCTGATCACT-3′; ACTB, forward 5′-GGGAAATCGTGCGTGACATTAAG-3’and reverse 5′-TGTGTTGGCGTACAGGTCTTTG-3′.

Extraction of protein and analysis with Western blotting

Total cellular protein was extracted from treated granulosa cells in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Active Motif, Carlsbad, CA) containing protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Active Motif). For GLUT4 translocation assay, cytoplasmic and membrane protein fractions were separated using a membrane and cytoplasmic extraction kit (Sheng gong, Shanghai, China). Protein sample (30 μg) was electrophoresed in 10% SDS-polyacrylamide gel and transferred to a nitrocellulose blot. After blocking the non-specific binding sites with non-fat milk, the blot was incubated with antibodies to PTEN (1:800) (Abcam), p-Akt ser473 (1:2000) (Cell Signaling, Danvers, MA), total Akt (1:2000) (Cell Signaling), GLUT4 (1:1000) (Abcam), p-p65ser536 (1:1000) (Cell Signaling) and total p65 (1:1000) (Cell Signaling) at 4 °C overnight. After washing, the blot was further incubated with corresponding secondary antibodies conjugated with horseradish peroxidase (Sigma) for 1 h. Enhanced chemiluminescent detection system (Millipore, Billerica, MA) was then applied for band detection. To control sampling error, the same blot was probed with an antibody against the house keeping protein GAPDH (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). For GLUT4 translocation study, the blot was incubated with antibodies against GAPDH and Na+-K+-ATPase (1:10000) (Cell Signaling) as cytoplasm and cell membrane protein markers respectively. The ratio of target protein over GAPDH or Na+-K+-ATPase was obtained to indicate relative abundance of the target protein.

Statistical analysis

All the data were presented as mean ± SEM. The Kolmogorov-Smirnov test was used to determine whether the continuous variables were of normal distribution. Paired or unpaired Student T-test or One-way ANOVA test followed by Student-Newman-Keuls test were used to assess the differences in normally distributed variables. All data analysis was performed with the Statistical Package for Social Science (SPSS, version 18.0 for windows). P < 0.05 was considered as statistically significant.

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