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 tertiary-care 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/m2, 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 (E2), thyroid-stimulating hormone (TSH) and anti-mullerian duct hormone (AMH) were assessed on 2nd 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 2nd or 3rd 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 fluid collection and estimation of melatonin and E2 concentration
Oocytes were retrieved by TVS guided puncture after 36 hours of hCG treatment. Clear follicular fluid 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 high-intensity bright light exposure. The supernatant was filtered through a 0.85 µL filter 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 profile, we only include those follicles with greater than 18mm diameter. Finally, mature oocytes were subject to intracytoplasmic sperm injection (ICSI) procedure. Melatonin and E2 concentrations were evaluated by diluting ff samples 1:100 through radioimmunoassay kits (MP® diagnostics, Santa Ana, California, USA). Intra-assay variations for melatonin and E2 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 fluid 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 purification 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 confirm the total RNA concentration. MicroRNA profiling 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 specific 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. Amplification was done with the following conditions: enzyme inactivation at 95 °C for 10 min and 40 cycles of two thermal amplification 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 quantification of follicular fluid 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. Amplification 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 five 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 fluid cell-free DNA (cfDNA)
CfDNA was quantified, 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 quantification. The cfDNA concentration in each follicle with mature oocyte was estimated relative to the corresponding amplification 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 fluid 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 identified 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 specific 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. χ2-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% confidence interval (Cl). The sensitivity and specificity 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 significant.