Key message

hsa-miR-320a-3p in human granulosa cells can be used as a potential indicator to predict good-quality embryos and live births.

Background

MicroRNAs (miRNAs) are highly conserved, single-stranded, 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.

Materials and methods

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.

Results

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.

Fig. 1
figure 1

A representative image of immunofluorescence staining in human ovarian granulosa cells (n = 5). The Red (× 400) expressed FSHR, the blue (× 400) expressed nuclear staining using 4 ‘, 6-diamino-2-phenylindole (DAPI). Non-specific staining can be observed with PBS instead of primary or secondary antibodies

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Relationship of the hsa-miR-320a-3p and hsa-miR-483-5p levels in the human granulosa cells and embryo developmental competence

The patients were subdivided into four groups according to the relative expression of hsa-miR-320a-3p levels quartile in the granulosa cells: Q1: 0.46-6.17 × 103, n = 49; Q2: 6.41 × 103-2.35 × 105, n = 49; Q3: 2.63 × 105-2.34 × 106, n = 49; and Q4: 2.51 × 106-9.38 × 107, n = 48. In the Q3 intervals, the normal fertilization rate for IVF was lower compared to Q1 and Q2 (Table 1, P < 0.05). In the Q3 and Q4 intervals, the good-quality embryo rate was lower than Q2 (Table 1, P < 0.0001). However, the normal fertilization rate for ICSI and blastulation rate did not differ (Table 1, P > 0.05). Multiple regression analysis showed that in Q3 and Q4 intervals experienced a decreased chance of live birth when Q1 group was used as reference (Table 2, P < 0.0001). And there was no difference in clinical pregnancy (Table 2, P > 0.05).

The patients were subdivided into four groups according to the relative expression of hsa-miR-483-5p levels quartile: Q1: 0.002-0.18, n = 49; Q2: 0.18-1.13, n = 49; Q3:1.21-5.80, n = 49; and Q4: 5.81-3.52 × 103, n = 48, respectively. In the Q3 intervals, the good-quality embryo rate and blastulation rate were lower than Q1 group (Table 1, P < 0.05). The normal fertilization rate for IVF or ICSI was no significant differences among groups (P > 0.05), as shown in Table 1. Multiple regression analysis showed that in Q3 and Q4 intervals had a decreased chance of live birth (Table 2, P < 0.0001). Clinical pregnancy had no differ among four group (Table 2, P > 0.05).

Table 1 Association between the levels of hsa-miR-320-3p and hsa-miR-483-5p in granulosa cells from human follicular fluids and reproductive outcomes of assisted reproductive technology (ART)
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Table 2 Multi-variable logistic regression analysis of the levels of hsa-miR-320a-3p and hsa-miR-483-5p in human granulosa cells and clinical outcomes (OR 95% CI)
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Effect of patients’ clinical characteristics on the hsa-miR-320a-3p and hsa-miR-483-5p levels in the human granulosa cells

The relative expression of hsa-miR-320a-3p in the human granulosa cells were weak positively correlated with age (β ± SE: 4.79 × 105 ± 1.61 × 105, P = 0.0033) (Table 3). Moreover, both the basal FSH (β ± SE: 7.90 × 105 ± 2.14 × 105, P = 0.0003) (Table 3) and ovarian stimulation protocol, including mild stimulation protocol and luteal phase stimulation (β ± SE: 8.27 × 10− 9 ± 2.92 × 10− 9, 6.29 × 10− 9 ± 2.09 × 10− 9, respectively; P = 0.006, P = 0.004, respectively) (Table 3) significantly and positively affected hsa-miR-320a-3p levels in the human granulosa cells. The days of stimulation were negatively correlated with the relative expression of hsa-miR-320a-3p in the human granulosa cells (β ± SE: − 6.85 × 105 ± 3.42 × 105, P = 0.0466) (Table 3). The relative expression of hsa-miR-320a-3p in the human granulosa cells were not associated with BMI, basal LH, basal E2, AMH, AFC and total dose of gonadotropins (Table 3, P > 0.05).

The relative expression of hsa-miR-483-5p in the human granulosa cells were not associated with age, BMI, female baseline levels, AFC, days of stimulation, total dose of gonadotropins and ovarian stimulation protocol (Table 3, P > 0.05).

Table 3 Patients’ characteristics association with the hsa-miR-320a-3p and the hsa-miR-483-5p levels in granulosa cells from human follicular fluids
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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 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 (r2 = 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 (r2 = 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.