hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells: promising bio-markers of live birth after IVF/ICSI

Background MicroRNAs (miRNAs) are considered potential biomarkers for various diseases. This study investigated whether hsa-miR-320a-3p and hsa-miR-483-5p levels in human ovarian granulosa cells derived from follicular fluids are associated with embryo developmental competence. Methods We collected 195 granulosa cells samples and analyzed the treatment outcomes in patients undergoing in vitro fertilization (n = 147) or intracytoplasmic sperm injection (n = 48) cycles. The hsa-miR-320a-3p and hsa-miR-483-5p levels in granulosa cells were measured using quantitative reverse transcription-polymerase chain reaction. Results Patients were subdivided into four groups according to the granulosa cells hsa-miR-320a-3p and hsa-miR-483-5p levels quartiles (Q1–Q4). Embryo developmental competence was compared using the chi-square test. Patients in Q3 were less likely to achieve a normal fertilization rate for in vitro fertilization and blastocyst formation than those in Q1 as they expressed high levels of hsa-miR-320a-3p and hsa-miR-483-5p (P < 0.05). Patients in Q3 and Q4 were less likely to achieve a good-quality embryo as they expressed high levels of hsa-miR-483-5p and hsa-miR-320a-3p (P < 0.05). The hsa-miR-320a-3p and hsa-miR-483-5p levels were not associated with clinical pregnancy. However, multiple regression analysis indicated that in Q3 and Q4 intervals had experienced a decreased chance of live birth due to high expression levels of hsa-miR-320a-3p and hsa-miR-483-5p levels. The relative hsa-miR-320a-3p expression levels in granulosa cells were weakly and positively correlated with the patient age (P = 0.0033). Moreover, both the basal follicle stimulating hormone (P = 0.0003) and ovarian stimulation protocols (P = 0.006 and P = 0.004) significantly and positively affected hsa-miR-320a-3p levels. The days of stimulation was negatively correlated with the relative hsa-miR-320a-3p expression level (P = 0.047). Conclusions The hsa-miR-320a-3p and hsa-miR-483-5p levels in human granulosa cells negatively correlated with the good-quality embryo rate and live birth, indicating that hsa-miR-320a-3p and hsa-miR-483-5p can be used as potential negative indicators to predict good-quality embryos and live births.

Page 2 of 7 Liu et al. Reproductive Biology and Endocrinology (2022) 20:160 Background MicroRNAs (miRNAs) are highly conserved, singlestranded, small, non-coding, and functional RNAs of 19-25 nucleotides, which regulate post-transcriptional RNA levels by binding to the 3′-untranslated region of messenger RNAs (mRNAs) and causing destabilization or translation repression [1]. They are widely expressed in various biological systems. Although many miRNAs are commonly expressed, the selective specific expression of miRNAs is common in tissues, suggesting that the requirement for specific miRNAs in different tissues and specific roles of miRNAs in tissues. Owing to their tissue-specific expression, miRNAs are considered potential biomarkers [2]. Several studies have identified miRNAs that are expressed in ovarian follicle cells [3]. MiRNAs are involved in the regulation of various biological processes, including granulosa cell proliferation, apoptosis [4,5], and oocyte maturation [5,6]. Recent studies have reported that the miRNAs expression led to downstream events that will affected fertilization and day 3 embryo morphology [7]. Moreover, miRNAs could be promising biomarkers for ovarian responses during in vitro fertilization (IVF) [8]. Some miRNAs are also differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome, making them potential biomarkers for predicting IVF success [9]. These findings suggest that miRNAs play important roles in the oocyte development.
The aim of this study was to investigate the relationship between miRNAs (hsa-miR-320a-3p and hsa-miR-483-5p) in human granulosa cells expression levels and oocyte developmental competence and explored the effect of patient clinical characteristics on miRNAs (hsa-miR-320a-3p and hsa-miR-483-5p) expression levels in human granulosa cells.

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/ m 2 (mean ± SD: 22.60 ± 3.23 kg/m 2 ). 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 × 10 5 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 transcriptionpolymerase 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.

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

Identification of granulosa cells from human follicular fluids
As shown in Fig. 1, the 96% cells in the dishes were granulosa cells, which were characterized by a positive FSHR staining. Non-specific staining was not detected. This proves that all the cells isolated were granulosa cells, and directly extracted granulosa cells can be used in subsequent studies.

Discussion
In this study, our results indicated that hsa-miR-320a-3p and hsa-miR-483-5p expression levels in the human granulosa cells were negatively associated with good-quality embryos and live births. Moreover, hsa-miR-483-5p levels were negatively associated with blastulation. Further studies revealed that hsa-miR-320a-3p levels positively correlated with patient age and basal follicle stimulating hormone (FSH) levels.
Follicular fluid content can be used as a noninvasive marker to predict oocyte quality. In our study, we found that a significantly difference in normal fertilization rate those with high hsa-miR-320a-3p expression levels during IVF cycles. Further, multi-variable logistic regression analysis indicated that the high expression levels of hsa-miR-320a-3p and hsa-miR-483-5p in granulosa cells seemed to reduce the number of good-quality embryos and live births (P < 0.0001). Notably, patients with higher levels of hsa-miR-483-5p exhibited a decreasing trend in blastulation. These results suggested a negative effect of hsa-miR-320a-3p and hsa-miR-483-5p on oocyte development and pregnancy outcomes. Additionally, hsa-miR-320a-3p and hsa-miR-483-5p have been reported to plays important roles in inhibiting cell proliferation and migration [14][15][16]. These processes have been proven to affect oocyte development [17]. hsa-miR-320a-3p and hsa-miR-483-5p in granulosa cells may also acts as apoptosis factors and decrease oocyte development via a paracrine mechanism. Consequently, higher levels of hsa-miR-320a-3p and hsa-miR-483-5p  20:160 in granulosa cells may reduce the developmental competency of oocytes. Furthermore, our results suggested that hsa-miR-320a-3p expression is weakly and positively correlated with patient age (r 2 = 0.209, P = 0.0033). Ansere et al. revealed that cellular senescence may contribute to ovarian aging, and the subsequent decline in ovarian follicular reserve [18]. In our study, the hsa-miR-320a-3p levels were positively associated with basal FSH levels (r 2 = 0.257, P = 0.0003). It is a useful predictor of ovarian reserve [19,20], indicating an association between hsa-miR-320a-3p and ovarian reserve function. However, no obviously relationship was observed between hsa-miR-320a-3p and AMH, AFC or BMI, which have also been reported to affect ovarian functions [21,22]. These conflicting results may be due to several factors, such as the cause of infertility and ovarian stimulation protocols. The potential associations between hsa-miR-320a-3p and FSH may provide a new direction to predict ovarian reserve function.
Many positive regulatory indicators predict the ART outcomes in human granulosa cells, such as circRNA [23], AQP7 [24] and telomerase activity [25]. As negative regulatory indicators, hsa-miR-320a-3p and hsa-miR-483-5p, can be combined with positive regulatory indicators to make the prediction results more reliable.

Conclusion
The current study indicated that the expression levels of hsa-miR-320a-3p and hsa-miR-483-5p in granulosa cells are negatively associated with good-quality embryos and live births in women undergoing IVF/ICSI. Notably, patients with higher levels of hsa-miR-483-5p exhibited a decreasing trend in blastulation. These results suggest that hsa-miR-320a-3p and hsa-miR-483-5p could be used as potential indicators to predict the quality of embryos and live births.