FMR1 expression in human granulosa cells increases with exon 1 CGG repeat length depending on ovarian reserve

A total of 229 women that underwent controlled ovarian hyperstimulation for either IVF or IVF with intracytoplasmic sperm injection (ICSI) treatment at the Department of Gynecological Endocrinology and Reproductive Medicine, University Women’s Hospital, Heidelberg, from February 2013 to August 2016 were prospectively recruited for our study. We collected GCs and blood samples from all patients. Additionally, their medical records and questionnaires were assessed in order to obtain demographic information (age at presentation and body mass index [BMI]), baseline hormone levels (serum follicle stimulating hormone [FSH], luteinizing hormone [LH], estradiol [E₂], and anti-Müllerian hormone [AMH]), and reproductive parameters (antral follicle count [AFC], total number of oocytes recovered, and mature [MII] oocytes retrieved). Patients were divided into three response groups. According to the Bologna Criteria [25], patients were included into the POR group (n = 70); in case of clinically documented PCOS, according to the Rotterdam Criteria [26], patients were included in the PCO group (n = 8). Those who did not fulfill criteria for POR or PCO groups were included in the NOR group, which served as a control (n = 151).

All patients signed an informed consent form and completed a clinical questionnaire. This study was approved by the local ethical committee of the University of Heidelberg, Germany (number S-602/2013), and conducted according to the principles expressed in the Declaration of Helsinki.

DNA samples were prepared as described previously [27] from 10 ml of blood samples with EDTA. To analyze CGG repeat length in the 5′-UTR of FMR1 exon 1, polymerase chain reaction (PCR) analysis and subsequent analysis of this region with the ALFexpress DNA sequencer (Amersham 1050; Pharmacia Biotech, Freiburg, Germany) or ABI 3100/3130xl sequencer (Life Technologies/Applied Biosystems, Foster City, CA, USA) were performed. PCR mixture (total volume, 30 ml) contained 0.25 μM of each primer (for forward and reverse primer sequences see Fu et al., 1991), 0.2 mM of dATP, dCTP, and dTTP each, 50 μM dGTP, 150 μM deaza-dGTP, 0.12 U KAPA Hot Start Taq polymerase, 1× PCR buffer, 1.5 mM MgCl₂, 1× Enhancer (Qiagen GmbH, Hilden, Germany), and 50 ng of genomic DNA. PCR conditions were as follows: 3 min at 94 °C for the first denaturation step; 35 cycles of amplification with a time-temperature profile of 15 s at 94 °C, 15 s at 66 °C, 15 s at 72 °C; and the additional incubation for 8 min at 72 °C in the last cycle. The forward primer was labeled with the fluorescent Cy5 or FAM dye (Eurofins Genomics, Ebersberg, Germany). For the analysis using ALFexpress sequencer, a 5 μl aliquot of PCR mix was mixed with 5 μl of 6× loading solution (5 mg/ml Blue Dextran (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) in formamide (Merck KGaA, Darmstadt, Germany)) and 1 μl of 250-bp internal marker. All samples following the denaturation at 95 °C for 5 min were analyzed on 6% denaturing polyacrylamide gel with 7 M urea. A 70–397 nucleotide size marker labeled with Cy5 dye was used for the determination of CGG repeat numbers. Allele sizes and peak areas of fluorescent products were analyzed with the Fragment Manager software (Pharmacia Biotech, Freiburg, Germany). To analyze the samples on the ABI 3100/3130xl sequencer, 1 μl of PCR product was mixed with 10.5 μl of Hi-Di-formamide and 0.5 μl of GeneScan ROX standard (Applied Biosystems, Foster City, CA, USA) and loaded. The obtained data were analyzed with the GeneMapper software (Applied Biosystems, Foster City, CA, USA). When the presence of PM was suspected, Southern blot was performed using a-³²P-dCTP radioactively-labeled p2 probe containing FMR1 exon 1 with CGG repeat as described previously [28].

A CGG repeat length of 26–34 was considered a normal repeat length range according to prior studies [4, 14‐16, 20]. We classified the patients according to the repeat lengths at both alleles (low < 26 repeats; normal 26–34 repeats; high 35–55 repeats) into six different genotypes: high/high, high/low, normal/high, normal/normal, normal/low, and low/low, as previously described [17]. PM carriers were not included in the study.

Ovarian stimulation was performed using either the long protocol of gonadotropin-releasing hormone (GnRH) agonist administration (long GnRH agonist protocol) or GnRH antagonist protocol. The appropriate protocol was selected by the physicians in charge. In the long GnRH agonist protocol an initial down-regulation using a GnRH-agonist at day 20+ 1 of the menstrual cycle was used. On day two of following cycle, gonadotropins (mainly recFSH or HMG) were injected daily to induce proper follicle maturation. When follicles reached 18 mm diameter, ovulation was induced by HCG injection and the oocytes were retrieved via ultrasound-guided follicular puncture after 36 h in 14 ml round bottom tubes containing PBS (1× Phosphate-buffered saline) and heparin (250 μl Heparin / 500 ml PBS). In the GnRH-antagonist protocol, gonadotropins (mainly recFSH or HMG) were injected daily to induce proper follicle maturation beginning at day 2 of menstrual cycle. When the leading follicle reached 14 mm average diameter, a GnRH-antagonist was used to prevent preterm spontaneous ovulation. At stage of 18 mm diameter follicle size, oocytes were retrieved after ovulation induction as described above. The cumulative dose of gonadotropins was determined based on the patients’ response and the decisions made by the physicians in charge.

GCs were retrieved from the follicular fluid after the transvaginal ultrasound-guided follicle puncture for IVF treatment that was performed with an ovum aspiration needle (Premium Fas Single Lumen, #4551 NS-AS1; Gynétics Medical Products N.V., Lommel, Belgium) connected to a vacuum pump (Cook Medical, K-MAR-5200, Bloomington, IN, USA). The aspirated follicular fluid was collected in 14 mL round-bottom tubes (Falcon, 352,001, NY, USA), and kept at 37 °C in a test-tube heater (Cook Medical, K-FTH-1012, Bloomington, IN, USA) or in a Thermo-Cell-Transporter (Labotect, Thermo-Cell-Transporter 3018, Rosdorf, Germany). Follicular fluid was transferred to a 100-mm cell culture dish (Thermo Fisher Scientific, Nunc, Waltham, MA, USA) on a table heated to 37 °C (Workstation L126 Dual, K-Systems, Birkerød, Denmark). GCs were identified morphologically as epithelial cell aggregates within the follicular fluid using a Nikon SMZ1500 zoom-stereomicroscope (Nikon Instruments Europe B.V., Amsterdam, Netherlands). In most cases granulosa cells (GCs) were picked up directly from the follicular fluid without additional washing. A brief washing step in either Multipurpose Handling Medium (MHM) or Sydney IVF Fertilization medium (Cook, K-SIFM-20, Bloomington, IN, USA) was considered necessary, if the follicular fluid was bloody. In some cases, i.e. bloody follicular fluid and a lot of Cumulus-Oocyte-Complexes (COCs), the MHM used for keeping COCs during the search also became bloody. In those cases GCs were briefly washed in Sydney IVF Fertilization medium that was also used to wash COCs before in vitro culture. We did not check for pH variations in Sydney IVF Fertilization medium after washing GCs and therefore cannot exclude a possible impact. However, only equilibrated Sydney IVF Fertilization medium was used for washing of COCs. The Sydney IVF Fertilization medium was then used immediately to wash GCs. Thus, the time during which Sydney IVF Fertilization medium was used outside the CO²-incubator was kept to a minimum.

Mural GCs were aspirated in a 2.5 μl volume with a sterile tip (ep Dualfilter T.I.P.S. 10 μl S, Eppendorf, Wesseling-Berzdorf, Germany), transferred to 1.5-ml tubes (Sarstedt, Nümbrecht, Germany) pre-filled with 12–13 μl of RNAlater stabilization solution (Ambion, AM7020, Life Technologies, Carlsbad, CA, USA), and stored at 4 °C.

GCs in the stabilizing solution were centrifuged at 5000×g for 5 min, and the supernatants were removed. mRNA was directly isolated from the GCs using TRIzol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions [29, 30] with PEQGOLD PHASETRAP A 1.5 ml tubes (VWR International GmbH, Darmstadt, Germany). mRNA was dissolved in RNAse-free water and the concentration and purity were detected using NanoDrop 2000c UV-spectrometer (NanoDrop Products, Wilmington, DE, USA). cDNA samples were synthesized after oligo-dT priming with SuperScript III First-Strand Synthesis System (Invitrogen by Life Technologies, Carlsbad, CA, USA) and the M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (Promega, Madison, WI, USA).

TaqMan predesigned gene expression assays for FMR1 (Hs00924544_m1) and two housekeeping genes, HPRT and TBP (Hs99999909_m1; Hs00427620_m1, respectively), as well as the TaqMan universal PCR master mix were purchased from Applied Biosystems (Life Technologies, Carlsbad, CA, USA) and the experiments were performed according to the manufacturer’s instructions. The samples were analyzed in triplicates, and standard PCR conditions were used, with Fast Forward 7500 real-time PCR-system (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Relative gene expression was analyzed using ΔΔCt method [31]. cDNA obtained from the COV 434 granulosa cells [32] was used as a calibrator in each run.

Data distribution was first determined by Shapiro-Wilk-Test. For simple comparison between ovarian reserve- (NOR, POR, or PCO) or CGG-subgroups, and the analysis of cohort demographic data, Kruskall-Wallis test or analysis of variance (ANOVA) were used. When statistically significant differences were obtained between groups, a post hoc test [Tukey’s honestly significant difference (HSD) or Dunn’s tests] was performed to identify which of the analyzed subgroups differed. In order to adjust the significance level, the Bonferroni correction was applied. Additionally, the χ2-test, supplemented by the adjusted residuals, was used for between- group comparisons (clinical ovarian reserve classification and genotype). Results are presented as mean ± standard deviation (SD), or median and interquartile range (percentile 25-percentile 75; respectively 1^rst-3rd quartile). For n < 3, the data are presented as median and minimum and maximum value (minimum–maximum). Statistical analyses were performed with SPSS (Statistical Package for the Social Sciences V. 22.0; IBM Corporation, NY, USA), and statistical significance was set at p ≤ 0.05.