Melatonin Dependent Modulation in microRNAs Relative Expression Pattern and Levels of cfDNA in Mature Oocytes of Unexplained Infertile Patients.

Intrafollicular melatonin maintains the DNA integrity of granulosa cells and protects them against apoptosis. This ubiquitous indoleamine compound serves as a potent free radical scavenger. It reduces oxidative stress and modulates DNA damage response, which improves oocyte's quality with a higher fertilization rate. Methods: This prospective study was designed to investigate the antioxidant property of intrafollicular melatonin and its impact on IVF outcome parameters by exploring the relative expression of ve microRNAs (miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p) and levels of cfDNA by real-time PCR in unexplained infertile patients. We collected 425 follicular uid (ff) samples containing mature oocytes from 295 patients undergoing IVF.

receptors. However, it was initially uncovered as a pleiotropic regulatory molecule that has shown considerable antioxidant/antinitrosant activity in various cell types and biological uids, including ff [1]. Intrafollicular melatonin concentration is almost 3-fold higher than that in blood plasma, which directly protects oocyte from oxidative stress within the ovarian follicle [2]. Human follicular uid (ff) may be considered a "biological window" that re ects allosteric metabolic regulation. It comprises various hormones, polysaccharides, reactive oxygen species (ROS), and antioxidant defense systems [3].
Paradoxically, ROS act as powerful signaling molecules that initiate the process of various growth-related responses and are primarily involved in cellular metabolism. However, the acquisition of ROS has harmful effects on the follicular microenvironment. Surprisingly, it favors the process of ovulation, thus might function as a double-edged-sword in the cellular processes [4]. Under moderate concentrations, ROS are responsible for mediating inter-and intracellular signaling cascades provided protection against apoptosis while massive production of ROS result in oxidative stress. Apoptosis is mainly due to the upregulation of oxidant species and the antioxidant defense system's limited e cacy. However, within the follicles, a delicate equilibrium exists between the pro-oxidant and antioxidant defense system, maintained by antioxidants' prominence. Non-enzymatic antioxidants, such as vitamins, minerals, superoxide dismutase (SOD), Glutathione (GSH), ascorbic acid, and melatonin, are required necessarily during ovulation [5].
Oxidative stress is an incessant cause of DNA damage and epigenetic modi cations in developing oocytes. The molecular mechanism of biological processes underlying cellular response to broadspectrum actions of melatonin is not restricted to its interaction with intracellular proteins but also has functional effects on non-protein-coding RNA species [6]. Owing to their wide plethora of functional activities and dynamic stability, miRNAs have a promising function as diagnostic and prognostic biomarkers [7]. Notably, miRNAs expression pro le has been recognized as a novel mechanism of intercellular communication, thereby acting similarly to hormones and a potential source of non-invasive biomarkers in the oocyte microenvironment to predict embryo quality [8].
Oxidative or nitro-oxidative stress leads to DNA damage when the antioxidant capability is insu cient, thereby playing a crucial role in intrinsic/extrinsic pathways of apoptosis by the integrated release of various pro-apoptotic factors. The amount of cfDNA in ff signi cantly related to the oocyte's quality and re ects the extent of cell damage by apoptosis [9]. In contrast, intrafollicular melatonin maintains the DNA integrity of granulosa cells, protects them against apoptosis, and stimulates estradiol and progesterone production that improves oocyte's quality with a higher fertilization rate [10]. Considering the possible effect of melatonin on different miRNA expression levels, which affects oocyte maturation quality, it should be of primary importance to focus on this relationship. Currently, limited studies are focused on the interactions of melatonin concentration and miRNAs relative expression pro le in the ff samples, which might serve as a potent non-invasive tool for predicting oocyte development capability in infertile patients. Therefore, this study was designed to investigate the melatonin-dependent modulation in ve miRNAs (miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p) relative expression patterns constitutively in mature oocytes' ff microenvironment to predict embryo quality. Previous studies demonstrating their involvement in oocyte maturation and embryo quality [11,12]. Additionally, we sought to explore whether melatonin's antioxidative property affects the concentration of apoptotic cfDNA in ff samples from which mature oocytes were obtained.

Methods
Participant's Selection: Subjects This prospective study included 425 individual ff samples related to mature oocytes from 295 women (mean age; 33.87 ± 1.98 years) with unexplained infertility. The subjects were registered in the tertiarycare hospital's assisted reproductive center between January 2017 and December 2018. The participants were sub-grouped based on intrafollicular melatonin concentration (Group A; ≤ 30 pg/mL, Group B; >70 to ≤110 pg/mL), Group C; >111 to ≤ 385 pg/mL). The study was approved by the Institutional Review Board (IRB). All patients provided written consent to participate.

Inclusion Criteria
Unexplained infertile women with less than four previous attempts and have normal physical and mental health, basal FSH level ≤8.85 IU/mL, and ovaries appeared in normal shape and sizes, were the part of this study. Moreover, male partners have normal semen parameters.

Exclusion Criteria
We excluded patients with metabolic disorders, communicable diseases, body mass index (BMI) ≥35 kg/m 2 , hyperandrogenemia, polycystic ovary syndrome, hyperprolactinemia, and reported any pelvic surgery. Moreover, we excluded females with known chromosomal translocation, endometriosis, diminished ovarian reserve, and autoimmune diseases.

Assessment of Clinical Parameters
BMI was calculated based on height and weight. While baseline hormones such as follicular stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E 2 ), thyroid-stimulating hormone (TSH) and antimullerian duct hormone (AMH) were assessed on 2 nd day of the menstrual cycle through electrochemiluminescence immunoassay, according to the manufacturer's instructions (Elecsys® Roche Diagnostics, Indianapolis, USA). The antral follicle count (AFC) was assessed using transvaginal ultrasonography (TVS) on the 2 nd or 3 rd day of the menstrual cycle.

Therapeutic Regimen
To minimize the possible confounding bias by varied controlled ovarian stimulation (COS) procedures, we only include patients in which ovarian stimulation was done through long GnRH agonist (decapeptyl ® : Ferring, USA, ATCO pharma) administered in the middle of the luteal phase of the previous cycle. Ovarian stimulation with rFSH (Follitropin β, Purigon ® : Organon Schering-plough, Oss, France) was evaluated by TVS and by quantifying serum 17β-estradiol level. The dosage of rFSH was adjusted according to ovarian response, BMI, bFSH levels, and AFC. A single dose of human chorionic gonadotrophin (6500-10,000 IU: Merk Serono, Lyon, Spain) was injected when more than two follicles reached a mean diameter of 18 mm or more by TVS inspection.

Follicular uid collection and estimation of melatonin and E 2 concentration
Oocytes were retrieved by TVS guided puncture after 36 hours of hCG treatment. Clear follicular uid without blood contents was aspirated independently from the two or three individual follicles. FF samples of mature MII-oocytes were centrifuged separately at 1500 × g for 20 minutes at 4 °C by preventing highintensity bright light exposure. The supernatant was ltered through a 0.85 µL lter and stored immediately as aliquots of 500 µL × 2 at -80 °C. Each mature oocyte, its related embryo, and ff sample were handled separately in the IVF laboratory. Subsequently, to avoid potential confounding bias by dissimilar follicles of different maturation statuses, as they might contain a varied miRNA and cfDNA pro le, we only include those follicles with greater than 18mm diameter. Finally, mature oocytes were subject to intracytoplasmic sperm injection (ICSI) procedure. Melatonin and E 2 concentrations were evaluated by diluting ff samples 1:100 through radioimmunoassay kits (MP® diagnostics, Santa Ana, California, USA). Intra-assay variations for melatonin and E 2 was <10%.

Assessment of embryo quality
Fertilization check was done 18-24 h after ICSI, and the embryo quality was determined through manual grading, using standard criteria based on cytoplasmic appearance, the extent of fragmentation, number, and regularity in the symmetry of blastomeres [13].
RNA extraction from follicular uid and relative expression analysis by RT-qPCR 500 µL frozen aliquots were thawed on ice, and cell debris was removed by centrifugation for 20 min at 3000 ×g at 4 °C. RNA was extracted from individual follicles using the Silica-based membrane puri cation technique (miRNAeasy kit, Qiagen, USA) following the given instructions except that we diluted the sample 3:1 ratio with XBP buffer to optimize its use with the ff. Total RNA was dissolved in 30 µL of RNAs free water, and its concentration was measured through Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Expression-based digital gel electrophoresis (Bio-Rad, Hercules, CA, USA) was also used to con rm the total RNA concentration. MicroRNA pro ling and data normalization were achieved as narrated by (Mestdagh et al 2009). Complementary DNA (cDNA) was generated using the TaqMan MicroRNA reverse transcription kit (Life Technologies, USA) in combination with RNA speci c stem-loop Megaplex primers (Applied biosystems).
A total of 15 µL reaction mixture contains 5 µL of a sample (10 ng miRNA), 0.5 µL of dNTP (100mM), 1.5 µL RT buffer (10X), 1 µL /50 IU of multiScribe RT enzyme, 0.19 µL of RNase inhibitor, 3 µL of stem-loop RT primers and 4.16 µL of nuclease-free water. Reverse transcription was performed in pulsating RT reaction: 40 cycles of 16 °C for 2 min, 42 °C for 60 seconds, and 50 °C for 60 seconds. Inactivation of reverse transcriptase was done at 85 °C for 5 min and hold step at 4 °C. Ampli cation was done with the following conditions: enzyme inactivation at 95 °C for 10 min and 40 cycles of two thermal ampli cation steps of 95 °C for 15 seconds 60 seconds for 1 min and a hold step at 4 °C. Q-PCR was duplicated for each sample using a CFX-96 ® touch RT-PCR detection system (Bio-Rad, Life Sciences, USA). We used Allele ID software ® to design primers and probs. For quanti cation of follicular uid miRNAs expression levels, PCR reaction was performed in a total volume of 20 µL, having 3 µL of cDNA, 10 µL of TaqMan Universal PCR MasterMix (Applied Biosystems), 0.8 µL of each primer and 5.4 µL double distilled water.
Ampli cation was carried out in a 96-well plate, and thermocycling conditions were10 minutes at 95 °C for enzyme activation, followed by 45 cycles of 95 °C for 20 seconds, 60 °C for 60 seconds. miRNA expression levels were normalized against the expression of MiR-16, which was used as an internal control because of its constant expression in ff samples. The relative expression of the ve miRNAs such as miR-320a, miR766-3p, miR-132-3p, miR-16-5p, and miR-663b was calculated using equation 2 -∆Ct, while ∆Ct = Ct target miRNA -Ct miR-16. To calculate the fold change (FC), we estimated the relative expression levels between high quality and impaired quality embryo on day 3 using 2 -∆∆Crt formula [11].
Extraction and assessment of follicular uid cell-free DNA (cfDNA) CfDNA was quanti ed, as previously described [14]. For cfDNA extraction, each ff sample was diluted with an equal volume of buffer solution (Tween-20, Tris-50 mmol/l, EDTA-1mmol) and incubated with proteinase K (Qiagen) at 55°C for at least 30 min, followed by inactivation at 98 °C for 10 min. After denaturation, each ff sample was centrifuged at 3000 rpm for 15 min and then immediately stored at -80°C until quanti cation. The cfDNA concentration in each follicle with mature oocyte was estimated relative to the corresponding ampli cation of β-globin and GAPDH measured by the real-time PCR-SYBR green detection method previously described [15].

Antioxidant status and oxidative stress markers measurements in follicular uid samples
The frozen (-80 °C) ff samples were thawed and evaluated for oxidative status. Average values of triplicate measurements were carried out from each ff sample to avoid inter-assay variations. ROS levels were measured by chemiluminescence assay using luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as a prob [16]. The total antioxidant capacity (TAC) was assessed using the colorimetric assay based on the manufacturer's instructions (BioVision, Inc, CA, USA). Lipid peroxidation was evaluated by calculating the concentration of Thiobarbituric acid reactive substances (TBARS) [17] while 8-hydroxy-2'deoxyguanosine (8-OHdG) was measured using a kit based on the manufacturer's instructions (BioVision, Inc, CA, USA). Both TBARS and 8-OHdG values were expressed as µM/L and ng/mL, respectively.

Pathway analysis:
We performed in-silico analysis to predict targets of miRNAs using web-based bioinformatics tool DIANA miRPath-v3 available on http://snf-515788.vm.okeanos.grnet.gr. Pathways were identi ed in both the regression analysis and fold-change. The results were demonstrated as a heat map. The more intense red color directed an increased probability that a speci c miRNA targets a unique pathway supplemented with target genes.

Statistical Analysis:
Baseline characteristics are presented as means ± SD, number percentage [n%], and median with 95% population limits as applicable. We used Kruskal-Wallis/two-tailed test to explore the outcome differences in parameter levels between patients with low (≤30 pg/ml), intermediate (>70 to ≤110 pg/ml)), and high intrafollicular melatonin concentration (>111 to ≤385 pg/ml). Based on the evaluation of the normality of the distribution by Kolmogorov-Smirnov test and Shapiro-Wilk test, we used the Mann-WhitneyU test to determine the Pairwise comparison between different groups. χ 2 -test was used to address the categorical variables. Spearman rank test was used to determine the correlation between intrafollicular melatonin levels and other parameters. Receiving operating characteristics (ROC) curves were used to calculate AUC with a 95% con dence interval (Cl). The sensitivity and speci city for optimal cut-off were calculated using XLSTAT 2020 software. SPSS (version 27; SPSS Inc., Chicago, IL. USA) was used for further statistical analysis. P <0.05 was considered statistically signi cant.

Baseline clinical characteristics of participants
The demographic parameters of participants are listed in Supplementary Table 1. Patients were divided into three groups based on melatonin concentration in their follicular uid samples: Group A; ≤ 30 pg/mL, Group B; >70 to ≤110 pg/mL), and Group C; >111 to ≤ 385 pg/mL. Of the 425 oocytes with associated ff samples, 55 oocytes did not fertilize nor reach the 1PN stage. From the results of our study, it was evident that increased concentration of cfDNA is found in ff samples of group A (median, 95% Cl: 2.01 (1.66; 3.42) compared to groups B (median, 95% Cl: 1.03 (0.41; 1.98) and C (median, 95% Cl: 0.86 (0.11; 1.03; p<0.001). Subsequently, ROS, TBARS, and 8-OHdG were signi cantly higher in group A than that found in group B and C patients (p <0.001). Similarly, we observed that patients exhibiting decreased intrafollicular melatonin concentration (≤ 30 pg/mL) have a signi cantly lower total antioxidant capacity [median, 95% Cl: 238 (198; 305; Kruskal-Wallis test, p<0.001] (Table 1). However, we could not nd any signi cant association among subgroups of patients regarding age, BMI, and baseline endocrine parameters such as FSH, AMH, LH, and TSH levels. Additionally, we also screened for ve selected microRNAs and detected miR-663b in 266/295 samples, miR-320a (284/295 samples), miR-766-3p (279/295 samples), miR-132-3p (268/295) and miR-16-5p in 273 out of 295 samples.  Intrafollicular cfDNA content and IVF outcome parameters among idiopathic patients The signi cant impact of intrafollicular melatonin concentration on cfDNA content of ff is given in Table  3, which shows that the higher level of intrafollicular melatonin signi cantly reduces the cfDNA content between groups. Among the analyzed idiopathic patients, no statistically signi cant differences have existed for the association of cfDNA concentration, and the number of oocytes retrieved, normally fertilized oocytes and early cleaved zygotes. However, when we compared the IVF outcome parameters such as number of MII-oocytes, blastomeres with a regular symmetry, high-quality day 3 embryos and fragmentation rate, we observed that cfDNA content was signi cantly higher in group A than group B and C (p<0.001) ( Table 3). Studying the correlation between cfDNA levels and IVF outcome parameters shows that the highest cfDNA levels are related to the lowest IVF outcome parameters and decreased melatonin concentration (Supplementary Table 3 and Fig.1S). Melatonin Concentration >111 to ≤385 pg/ml). Intra-follicular data for patients are also subjected to statistical differences, P* = Kruskal-Wallis test, P** = pairwise comparisons between sub-groups, and p<0.05 considered statistically signi cant.
Effect of melatonin concentration on intrafollicular oxidative balance and day 3 embryo quality The association of intrafollicular melatonin levels and oxidative stress markers is shown in Supplementary developed from oocytes containing higher levels of intrafollicular melatonin (>111 pg/ml). Likewise, they distinctively exhibit a positive correlation (R 2 = 0.870; p<0.001) (Fig. 2S). Melatonin mediated miRNAs expression pro le in follicular uid samples is associated with IVF outcome parameters Under the in uence of melatonin, miRNAs have shown different expression patterns that vary from group A to C with the same trend as the intrafollicular melatonin levels differ among the groups (see Fig. 1). As shown in Table 4, the relative expression levels of miR-663b, miR-320a, miR-766-3p, miR-132-3p, and miR-16-5p were signi cantly upregulated in group B and C (Raw Ct < 30) compared to group A (Raw Ct > 30). In group A the relative expression levels of miR-766-3p and miR-132-3p were signi cantly reduced (p<0.002) in comparison to group B and C. While the relative expression of miR-766-3p and miR-132-3p in ff of groups B and C were slightly upregulated but did not reach the signi cant threshold (see Fig. 1).
To examine the possible correlation between miRNAs in ff and day 3 embryo quality, we compared cases of high-quality embryos to impaired-quality embryos. The correlation analysis results were summed up in Supplementary  Table S5).

Predictive model for high-quality embryo selection
A predictive model for high-quality embryos is depicted in Fig. 2 Fig. 2A). These results showed that miR-320a predicts IVF outcome parameters better than miR-132-3p (see Table 5). Likewise, combination of all miRNAs did not improve the AUC of miR-320a (0.89) but enhance the senstivity to 87% and slightly decrease the speci city to 83% which may offer a potent non-invasive diagnostic tool in the selection of high-quality day 3 embryos (see Table 5 . 2B).
Furthermore, the AUC value for the combination of all evaluated stress markes was 0.78 with sensitivity of 83.2 % and speci city of 78.3% (p = 0.001) as given in (see Table 5 . 2C). Note: *The null hypothesis was true area = 0.5, after the adjustment of a number of attempts and the number of embryos. P-values in bold letters considered statistically signi cant p<0.05.
** Estimated cut points that maximize sensitivity and speci city for observed range predictors.

Multitargeting activity of melatonin upregulated miRNAs
MicroRNAs are the genetic switches that ne-tune essential cellular responses and are required to streamline the signal transductions in several cell types. They are depicted as multivalent with single miRNA able to target numerous genes, thus regulating structural and functional molecules' expression within a pathway. In the present study, we identi ed several pathways that involve at least one of the studied miRNAs, including cytoskeletal organization, post-translational protein modi cation, cell cycle, oocyte meiosis, p53 signaling pathway, TGF-beta signaling pathway, and estrogen signaling pathway (see Fig. 3 and 3S).

Discussion
The intrafollicular microenvironment is a highly complex and critical indicator of an individual oocyte's developmental capability to be fertilized and mature into a good quality embryo. Primarily melatonin's positive impact on the intrafollicular microenvironment has been reported through melatonin-mediated gene expression [18]. This is the rst attempt in unexplained infertile patients to establish a direct association between melatonin's antioxidant property and its impact on oocyte quality, fertilization rates, and embryo quality by exploring the relative expression patterns of ve miRNAs and levels of cfDNA.
Our results established that patients with higher melatonin concentration were associated with elevated 17β-estradiol (E 2 ) levels in their ff samples. E 2 is a key player in the nal steps of oocyte's nuclear and cytoplasmic maturation [19]. The non-human primate study demonstrated that it can improve oocytes' developmental competence during in vitro maturation (IVM) [20]. Our study showed that patients with lower intrafollicular melatonin concentration exhibited a considerable oxidative imbalance in their ff (e.g., lower TAC levels and higher ROS, 8-OHdG, and TBARS levels), which jeopardizes the quality of oocytes and thus, hampers the oocyte's maturation along with limiting IVF outcome parameters. These ndings follow much earlier research reporting that an imbalance between lipid peroxidation marker (TBARS) and antioxidant system plays a signi cant role in the pathogenesis of unexplained infertility [21]. Our study's outcome agrees with Jana et al, which revealed a direct relationship between low TAC levels and poor embryo quality and a sharp decline in fertilization rate [22]. Additionally, recent studies validated that decreased melatonin concentration is responsible for reduced TAC levels in the ff samples [23,24]. These studies suggest that elevated follicular lipid peroxidation and lower TAC levels have a negative impact on IVF outcomes.
Over the past decade, advances in scienti c knowledge have established that depreciation in intrafollicular melatonin assets resulted in excessive ROS production, which is responsible for single or double-strand DNA breaks, thus introducing mutations in nuclear DNA and reduces in mitochondrial function [25]. Together with this reference, our study tends to con rm that the concentration of ROS raised high in those patients who had lower levels of melatonin (≤30 pg/mL) than those who had moderate to higher concentrations in their ff samples. ROS production might be a result of suboptimal intrafollicular microenvironment or impaired metabolism of the developing oocyte. Increasing evidence highlights that melatonin is responsible for the upregulation of speci c miRNAs, which control antioxidative enzymes' expression, more likely by acting as a potent free radical scavenger [26]. Similarly, another group reported that melatonin signi cantly upregulates genes' expression and the formation of proteins responsible for synthesizing antioxidant enzymes. Therefore, compromised antioxidant capacity leads to oxidative stress and are-up the process of apoptosis [27].
One of the signi cant obstacles for IVF is oxidative damage to nuclear DNA that can be estimated by assessing intrafollicular cfDNA concentration and levels of 8-OHdG. The increased concentrations of cfDNA and 8-OHdG levels also indicate the embryo's inappropriate quality and often have a low success rate of pregnancy [9,28]. In the same context, our results showed that intrafollicular cfDNA concentration and 8-OHdG levels were signi cantly raised in good quality embryos of those patients who have decreased intrafollicular melatonin concentration (≤30 pg/mL). Alternatively, those patients who exhibited a higher concentration of melatonin (>111 pg/mL) in their ff samples have decreased intrafollicular cfDNA concentration and levels of 8-OHdG, which is in agreement with previous observations [29,30]. Furthermore, in the present study, higher intrafollicular melatonin resulted in an enrichment of high-quality day 3 embryos leading to an increase in the availability of embryos suitable for transfer, ultimately resulting in an augmented future pregnancy rate per embryo transfer [31].
Our exploratory analysis exhibited that miRNAs such as miR-320a, miR766-3p, miR-132-3p, miR-16-5p, and miR-663b were signi cantly decreasing their relative expression in ff samples that have a lower concentration of intrafollicular melatonin (≤30 pg/mL) and yielded a smaller number of high-quality embryos on day 3. In agreement with our study, Feng et al compared miRNAs from the ff that generate poor-quality and top-quality embryos. They found that miR-132-3p and miR-16-5p were downregulated in the ff containing mature oocytes that produce a higher number of poor-quality embryos than top quality embryos and vice-versa [12,32]. However, the ndings were statistically insigni cant, and the effect of intrafollicular melatonin to regulate these miRNAs is mostly unknown. Bioinformatic analysis of studied miRNAs reveals a fundamental role in mediating genes that regulate vital aspects of multiple biological functions. They mediate cell-to-cell communication and target genes associated with follicular development, growth, and oocyte maturation, further indicating that these endogenous messengers have a critical role in oogenesis [33]. Subsequently, the molecular signature based on these miRNAs' differential levels enabled them to participate in the cell junction assembly, TGF-beta signaling, MAPK signaling, Wnt signaling PI3K-Akt pathway, Notch signaling, Estrogen signaling, and Hippo signaling pathways [34,35].
Our study showed that miR-320 was among the highest expressed miRNA in ff samples, which have intrafollicular melatonin concentration >111 pg/mL. Similarly, Diez-frail et al observed that miR-320 was over the top-ten highest expressed miRNA in ff samples of good quality embryos [36]. Further, they reported that knockdown of miR-320a expression in mouse metaphase-II oocytes resulting in embryos arrested at rst cleavage. While very few can develop into top-quality embryos, indicating that the miR-320a has a potential role in modulating gene expression and regulating embryonic development [37].
Intriguingly, another investigation did not nd miR-320 in ff [38]. This inconsistency in the results may be explained by genetic heterogeneity due to the population's different ethnic origins, resulting in varied gene expression in body uids. Moreover, different stimulation protocols may be responsible for a varied expression of genes or miRNAs. Unsurprisingly, studies focused on the relationship between miR-320a relative expression levels in ff and embryonic development provides additional support to physiological and molecular mechanisms underlying in-vivo and in-vitro fertilization [32,39,40]. Evidence from another investigation has indicated that melatonin exerts its antioxidant activities by coordinating crosstalk between miRNAs and interrelated pathways [12].
Additionally, a transcriptome analysis reveals that high quality human preimplantation embryos secrete miR-320a that regulates the decidualized human endometrial stromal cells (hESCs) migration by targeting cell adhesion and cytoskeleton organization [32]. The outcome of their study speci es the promising effect of miR-320a to boost success rates in assisted reproduction. Previously, another piece of evidence indicates that miR766-3p, miR-132-3p, miR-16-5p, and miR-663b have decreased relative expression in the ff samples, yielding impaired quality embryos when compared with the top-quality embryos on day 3 [11]; this is in line with our ndings. In the same frame of reference, a recent study conducted by Ragusa et al. con rmed the hypothesis that miR766-3p ne-tunes cellular responses, especially in the control of the cell cycle, play a vital role in the rst phase of embryogenesis [41].
Conversely, Fu et al con rmed that miR-663b has a substantial negative correlation in ff of oocytes that produce viable blastocyst than those yielding poor-quality blastocyst [42]. However, the authors of the study did not mention the exact cause of infertility in the subjects. We speculated that it is potentially resulting from vast differences in patiens' selection, detection methodology and biological variability.
This study still holds some signi cant limitations, as other confounding factors were not addressed. First, the oocyte and embryo quality are associated with morphological scores and the number and composition of chromosomes; therefore, it is imperative to nd the numerical chromosome euploidy by Preimplantation Genetic Diagnosis (PGD). Second, we were not able to knockdown miRNAs to assess their actual effects on embryonic development. Third, the primary goal is embryo quality, not live birth.
Further studies should be planned to nd a relationship between these miRNAs' expression in ff and chromosomal anomalies.

Conclusion
Conclusively, our study showed that melatonin antioxidant capability signi cantly impacts cfDNA concentration and speci c miRNAs relative expression pro le in the follicular ambient microenvironment on oocyte development and embryo quality. Therefore, it may be a potent non-invasive diagnostic tool to select high-quality day 3 embryos in the future with such promise. Furthermore, information compiled herein will improve our understanding that physiology of reproduction may categorize these miRNAs as non-invasive biomarkers. These unique miRNAs signature will enhance the quality of embryo selection and minimize the chance of multiple gestations, thus, eventually, improve the probability of successful IVF pregnancies. More comprehensive studies should be conducted on a genome-wide scale that must con rm melatonin's effect in the follicular microenvironment to regulate miRNA expression pro les related to oocyte maturation and embryo quality. Ethics approval and consent to participate: The study was approved by our Institutional Ethical Committee (IEC). Informed consent was obtained from all subjects before the research and publishing of the results of the investigation.

Consent for Publication:
Not applicable

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.