miR-320a-3p Levels in Human Granulosa Cells: A Promising Biomarker of Good Quality Embryo and Clinical Pregnancy after IVF/ICSI.

Background miRNAs in body uids are considered potential biomarkers of diseases. This study investigated whether miR-320a-3p and miR-483-5p levels in human granulosa cells from follicular uids were associated with embryo developmental competence. Methods We collected 195 patients’ granulosa cells samples undergoing in vitro fertilization (n =147) or intracytoplasmic sperm injection (n =48) cycles, and gathered information about the outcomes of the treatment. miR-320a-3p and miR-483-5p levels were measured using qRT-PCR. Results The miR-320a-3p levels in human granulosa cells across different patient groups were signicantly different in the good quality embryo rates, with lowest levels in the Q4 intervals (P<0.05). The relative expression levels of miR-320a-3p were negatively associated with clinical pregnancy rate (P<0.05) and positively correlated with the patient age (P=0.0033). Moreover, both the basal FSH (P=0.0003) and ovarian stimulation protocol (P=0.006 and P=0.004) signicantly and positively affected miR-320a-3p levels. The days of stimulation was negatively correlated with the relative expression of miR-320a-3p (P=0.0466). The relative expression levels of miR-483-5p were signicantly positively correlated with AMH (P=0.0047). Neither miR-320a-3p nor miR-483-5p levels in granulosa cells were associated with normal fertilized rate, blastulation rate and abortion rate. the quality embryo between miRNAs (miR-320a-3p and miR-483-5p) in human granulosa cells expression prole and oocyte developmental competence and explored the effect of patient clinical characteristics on miRNAs (miR-320a-3p and miR-483-5p) expression prole in human granulosa cells. Then the granulosa cells were xed in 4% (v/v) paraformaldehyde for 20 min for immunouorescence as before (20). The FSH receptor (FSHR) was used to detect the purity of granulosa cells. To exclude the non-specic staining from antibodies, the primary and secondary antibodies were omitted as negative control groups, respectively. the interquartile range (IQR), if appropriate. Linear regression was carried out for the effect of patients’ characteristics information on the miR-320a-3p and miR-483-5p levels in follicular uid. To evaluate the correlation between miR-320a-3p and miR-483-5p levels and embryo developmental competence, we rst subdivided all 195 samples according to their follicular uid miR-320a-3p and miR-483-5p levels quartile, then the normal fertilized rate, good quality embryo rate and blastocysts rate were compared by ANOVA or Kruskal–Wallis test. Reproductive outcomes of assisted reproductive technology of the miR-320a-3p and miR-483-5p levels were compared with an Furthermore, our results suggested that the expression of miR-320a-3p levels were positively correlated with patient’s age (r 2 =0.209, P=0.0033). Findings by Victor A Ansere et al revealed that cellular senescence may contribute to ovarian aging, and subsequently declines in ovarian follicular reserve (27). Indeed, in our study, the miR-320a-3p levels were positively associated with basal FSH (r 2 =0.257, P=0.0003). Studies showed that miRNA is involved in hormone regulation during follicular formation. FSH plays a crucial role in folliculogenesis by a novel pathway of miRNAs (28). With the occurrence of aging, miR-320a-3p could regulate the level of basal FSH. Mianmian Yin et al demonstrate that miR-320 regulates steroid production by targeting E2F1 and SF-1 in the follicular development (29). FSH highly capable of forecasting marker of ovarian reserve function (30, 31). Potential associations between miR-320a-3p and FSH provide new direction for predicting ovarian response. In conclusion, the current study showed that the expression levels of miR-320a-3p are related to age and embryo development ability for women undergoing IVF/ICSI, suggesting that miR-320a-3p can be used as a potential indicator to predict treatment outcomes. A deeper understanding of the specic role of miRNA in the development and maturation of follicles remains to be further studied. The current research showed that changes in the miR-320a-3p levels of human follicular uid were correlated with the good quality embryo rate and clinical pregnancy rate and the patient age, suggesting that miR-320a-3p levels have potential use in evaluating embryo development ability and clinical pregnancy. A deeper understanding of the mechanism of miR-320a-3p affecting oocyte development ability could promote the future clinical application of miR-320-3p.


Background
MicroRNAs (miRNAs) are highly conserved, single stranded, small non-coding functional RNAs of 19-25 nucleotides, which contribute to post-transcriptional levels by binding the 3'-untranslated region of messenger RNAs (mRNAs), with destabilization or translation repression (1). Moreover, miRNAs are widely expressed in biological systems. Although many miRNAs are commonly expressed, speci c expression of miRNAs are common in tissues, suggesting that different tissues have unique requirements for miRNAs and that these miRNAs have speci c functional roles in different tissues. Owing to tissue-speci c miRNAs expression, miRNAs are considered potential biomarkers (2). MiRNAs are very stable in biological uids (3) and resistant to a wide range of storage conditions making them biomarkers in some states (4), such as retinoblastoma (5), Parkinson's disease (6), and pregnancy (7).
In reproduction, several studies have identi ed miRNAs are not only expressed in ovarian follicles cells, also found in the biological uids, such as follicular uid (8-10). The miRNAs in follicular uid are involved in regulating various biological processes, include ovarian cell proliferation and apoptosis (11,12), and oocyte quality and maturation (12,13). Recent studies have reported that the miRNAs expression in the follicular uid can lead to downstream events that will affect fertilization and day 3 embryo morphology (14) and are signi cantly negatively related to viable blastocyst formation (15). Moreover, miRNAs might be represented as promising biomarkers during in vitro fertilization (IVF) (16), and polycystic ovarian syndrome (PCOS) (17). In human embryos culture media, some of miRNAs are differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome, which makes them potential biomarkers for predicting IVF success (18). These studies suggest that miRNAs play an important role in the oocyte development and fertilization. Our previous study found that the expression of miR-320a-3p and miR-483-5p levels were decreased in the follicular uid exosomes of elderly women. However, to date, no studies have reported the relationship of the two miRNAs expression pro le in the human mural granulosa cells and ART outcomes during IVF/ICSI. Therefore, the aim of this study was to investigated the relationship between miRNAs (miR-320a-3p and miR-483-5p) in human granulosa cells expression pro le and oocyte developmental competence and explored the effect of patient clinical characteristics on miRNAs (miR-320a-3p and miR-483-5p) expression pro le in human granulosa cells. 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 ovulation induction schemes 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 ultrasoundguided puncture.

Human granulosa cell collection and identi cation
Granulosa cells were collected from the follicular uid of 195 patients as described (19). After collection, granulosa cells were seeded and cultured on coverslips for 48 h. Then the granulosa cells were xed in 4% (v/v) paraformaldehyde for 20 min for immuno uorescence as before (20). The FSH receptor (FSHR) was used to detect the purity of granulosa cells. To exclude the nonspeci c 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 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 ℃ for 60 min and 85 ℃ for 5 s.
The miR-320a-3p and miR-483-5p primers were purchased by the GeneCopoeia Company. 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 TM qPCR Mix, 2 μl All-in-One TM 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 ℃ for 600 s, 40 cycles at 95 ℃ for 10 s, 60 ℃ for 20 s and 72℃ for 10 s. The relative quantity of miRNA expression was calculated using the 2 −△CT method.

Morphological assessment of oocytes, good qualityembryos, and blastocysts
The appearance of prokaryotic zygote 18 to 20 hours after microinjection or arti cial insemination is a representative of fertilization. Morphological scores of embryos at day 3 were consistent with the current consensus system (21). High-quality embryos and blastocysts were de ned as previous (22).

Statistical analysis
The miR-320a-3p and miR-483-5p levels, expressed as means ± standard deviation (SD), or as median values and the interquartile range (IQR), if appropriate. Linear regression was carried out for the effect of patients' characteristics information on the miR-320a-3p and miR-483-5p levels in follicular uid. To evaluate the correlation between miR-320a-3p and miR-483-5p levels and embryo developmental competence, we rst subdivided all 195 samples according to their follicular uid miR-320a-3p and miR-483-5p levels quartile, then the normal fertilized rate, good quality embryo rate and blastocysts rate were compared by ANOVA or Kruskal-Wallis test.
Reproductive outcomes of assisted reproductive technology of the miR-320a-3p and miR-483-5p levels were compared with an unpaired t test or Mann-Whitney test. 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 signi cant.

Results
Human granulosa cells in follicular uids identi cation As shown in Figure 1, most of the cells in the dishes were granulosa cells, which were characterized by a positive FSHR staining. Nonspeci c staining was not detected.
Relationship of the miR-320a-3p and miR-483-5p levels in the granulosa cells and embryo developmental competence The patients were subdivided into four groups according to the relative expression of miR-320a-3p levels quartile in the granulosa cells: The relative expression of miR-320a-3p levels across different patient groups were signi cantly different in the good quality embryo rates, with lowest levels in the Q4 intervals ( Table 2, P < 0.01). However, the normal fertilized rate for IVF or ICSI and blastocyst rate did not differ (Table 1 and Table 2, P > 0.05).
The relative expression of miR-483-5p levels across different patient groups were no difference (P > 0.05) in the normal fertilization rates for IVF or ICSI, good quality embryo rate, and blastulation rate, as shown in Table 1 and Table 2.

Discussion
In this study, our results showed that miR-320a-3p expression levels in the granulosa cells were associated with the oocyte potential that had developed to the good quality embryo stage, and with clinical pregnancy for women undergoing IVF or ICSI. Moreover, it was found to be positively correlated with patient age and basal FSH.
In our study, the expression levels of miR-320a-3p in mural granulosa cells was associated with the good quality embryo rate, and the highest expression group of miR-320a-3p had a lowest rate of good quality embryo rate (P<0.0001). This could be because of an adverse effect of miRNAs on the quality of the embryo. Moreover, studies showed that follicular uid miRNAs signi cantly in uence oocyte mature (23), developmental (24) and the quality of the embryo (15). In addition, increasing evidences implicated that a good day-3 embryo is apt to form a high-quality blastocyst (25). Meanwhile, a good day-3 embryo also have an optimistic pregnancy rate (25). Our results demonstrated that there were lower expressed miR-320a-3p in pregnant group, compared to unpregnant group (P=0.0477). Older women generally face reduced ovarian function and reduced pregnancy rates (26). Thus miR-320a-3p can be used as a non-invasive marker to predict embryonic development quality and clinical pregnancy outcome (14)(15)(16)18).
Furthermore, our results suggested that the expression of miR-320a-3p levels were positively correlated with patient's age (r 2 =0.209, P=0.0033). Findings by Victor A Ansere et al revealed that cellular senescence may contribute to ovarian aging, and subsequently declines in ovarian follicular reserve (27). Indeed, in our study, the miR-320a-3p levels were positively associated with basal FSH (r 2 =0.257, P=0.0003). Studies showed that miRNA is involved in hormone regulation during follicular formation. FSH plays a crucial role in folliculogenesis by a novel pathway of miRNAs (28). With the occurrence of aging, miR-320a-3p could regulate the level of basal FSH. Mianmian Yin et al demonstrate that miR-320 regulates steroid production by targeting E2F1 and SF-1 in the follicular development (29). FSH highly capable of forecasting marker of ovarian reserve function (30,31). Potential associations between miR-320a-3p and FSH provide new direction for predicting ovarian response.
In conclusion, the current study showed that the expression levels of miR-320a-3p are related to age and embryo development ability for women undergoing IVF/ICSI, suggesting that miR-320a-3p can be used as a potential indicator to predict treatment outcomes. A deeper understanding of the speci c role of miRNA in the development and maturation of follicles remains to be further studied.

Conclusion
The current research showed that changes in the miR-320a-3p levels of human follicular uid were correlated with the good quality embryo rate and clinical pregnancy rate and the patient age, suggesting that miR-320a-3p levels have potential use in evaluating embryo development ability and clinical pregnancy. A deeper understanding of the mechanism of miR-320a-3p affecting oocyte development ability could promote the future clinical application of miR-320-3p.

Consent for publication
Consent for publication have be obtained from that person.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.