Patients’ characteristics

This study recruited 195 women enrolled in IVF (n = 147) or ICSI (n = 48) cycles at the Center for Reproductive Medicine of Tongji Medical College in the Huazhong University of Science and Technology from December 2019 to January 2021. Participants were required to meet the following eligibility requirements: conventional controlled stimulation protocols were used. Patients were excluded if they were diagnosed with infectious disease, malignant tumors, premature ovarian failure, polycystic ovary syndrome, systemic diseases and hereditary diseases. The women’s ages ranged from 21 to 46 years (mean ± SD: 34.39 ± 5.19 years) and their body mass index (BMI) ranged from 15.80 to 32.40 kg/m2 (mean ± SD: 22.60 ± 3.23 kg/m2). Baseline hormonal levels including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and 17β-estradiol (E2) and anti-Mülerian hormone (AMH) were measured on the third day of menstruation. The number of days of stimulation ranged from 5 to 22 days (mean ± SD: 9.97 ± 2.48 days), and the total dose of gonadotropins received ranged from 900 to 6450 IU (mean ± SD: 2344.27 ± 842.52 IU).

The controlled ovarian stimulation protocols were used included ultra-long protocol, long protocol, antagonist protocol, progestin-primed ovarian stimulation (PPOS), mild stimulation protocol, and luteal phase stimulation. FSH stimulation was monitored by measuring serum E2 levels and follicular size. Human chorionic gonadotrophin (hCG) (Livzon, Zhuhai, China) was injected when at least three follicles are 18 mm or larger in diameter by ultrasound. After hCG injection 36 h, oocytes were extracted by transvaginal ultrasound-guided puncture.

Human granulosa cells collection and identification

Granulosa cells were collected from the follicular fluid of 195 patients as described [10]. Briefly, after the isolation of the cumulus-oocyte complexes (COCs) for conventional IVF or ICSI procedures, the follicular fluids were centrifuged and granulosa cells were collected and resuspended in 1× phosphate-buffered saline (PBS). Then, it was added to a 50% Percoll gradient (GE Healthcare Life Sciences, Piscataway, NJ, USA) and centrifuged at 400 g for 30 min at 4 °C. The cells in the middle layer were collected, resuspended in PBS.

To confirm the purity of granulosa cells, it was seeded and cultured on coverslips at a density of 1 × 105 cells/ coverslips for 48 h. Then the granulosa cells were fixed in 4% (v/v) paraformaldehyde for 20 min for immunofluorescence as before [11]. The FSH receptor (FSHR) was used to detect the purity of granulosa cells. To exclude the non-specific staining from antibodies, the primary and secondary antibodies were omitted as negative control groups, respectively.

RNA isolation, cDNA synthesis, and real-time quantitative PCR (qPCR)

Total RNA was extracted from granulosa cells using the RNA-easy Isolation Reagent (Vazyme Biotech Co., Ltd., Nanjing), and transcribed into cDNA using the All-in-One™ miRNA quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Detection Kit 2.0 (GeneCopoeia, Inc., United States) according to the manufacturer’s protocol. The cDNA synthesis reaction conditions were the following: 37 °C for 60 min and 85 °C for 5 s.

The hsa-miR-320a-3p and hsa-miR-483-5p primers were purchased by the GeneCopoeia Company. hsa-miR-320a-3p primer forward:5′-TTGAGAGGGCGAAAAAAA-3′. hsa-miR-483-5p primer forward: 5′-CGGGAGGAAAGAAGGGAGAA-3′. Reverse primers are universal reverse primers in the All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Inc. USA). U6 was used as a housekeeping gene. The reaction was performed in a total volume of 20 μL contained 10 μL 2× All-in-One™ qPCR Mix, 2 μL All-in-One™ miRNA qPCR Primer (2 μM), 2 μL Universal Adaptor PCR Primer (2 μM) and 2 μL First-strand cDNA. The cycling conditions used were the following: 95 °C for 600 s, 40 cycles at 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s. The relative quantity of miRNA expression was calculated using the 2−△△CT method.

Morphological assessment of oocytes, good-quality embryos, and blastocysts

The appearance of prokaryotic zygote 18 to 20 hours after microinjection or artificial insemination is a representative of fertilization. IVF normal fertilization rate = number of 2PN/total number of oocytes × 100%. ICSI normal fertilization rate = number of 2PN/total number of MII oocytes × 100%. Morphological scores of embryos at day 3 were consistent with the current consensus system [12]. Good-quality embryos and blastocysts were defined as previous [13]. Good-quality embryo rate = number of day 3 good-quality embryos/normal fertilization number of cleavage embryos × 100%. Blastulation rate = number of blastocysts at stage 2 and above/total number of cleavage embryos in blastocyst culture × 100%.

Statistical analysis

The hsa-miR-320a-3p and hsa-miR-483-5p levels, expressed as means ± standard deviation (SD), median values and the interquartile range (IQR), or as OR (95% CI), if appropriate. Linear regression was carried out for the effect of patients’ characteristics information on the hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells. To evaluate the correlation between hsa-miR-320a-3p and hsa-miR-483-5p levels and embryo developmental competence, we first subdivided all 195 samples according to their granulosa cells hsa-miR-320a-3p and hsa-miR-483-5p levels quartile, then the normal fertilization rate, good-quality embryo rate and blastulation rate were compared by Chi-square test. Multi-variable logistic regression analysis was used to analyze clinical pregnancy and live birth. Statistical analyses were performed using the Statistical Package for Social Sciences program, Version 12.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.