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

Abstract

BackgroundFragile-X-Mental-Retardation-1- (FMR1)-gene is supposed to be a key gene for ovarian reserve and folliculogenesis. It contains in its 5’-UTR a triplet-base-repeat (CGG), that varies between 26 and 34 in general population. CGG-repeat-lengths with 55–200 repeats (pre-mutation = PM) show instable heredity with a tendency to increase and are associated with premature-ovarian-insufficiency or failure (POI/POF) in about 20%. FMR1-mRNA-expression in leucocytes and granulosa cells (GCs) increases with CGG-repeat-length in PM-carriers, but variable FMR1-expression profiles were also described in women with POI without PM-FMR1 repeat-length. Additionally, associations between low numbers of retrieved oocytes and elevated FMR1-expression levels have been shown in GCs of females with mid-range PM-CGG-repeats without POI. Effects of FMR1-repeat-lengths-deviations (n < 26 or n > 34) below the PM range (n < 55) on ovarian reserve and response to ovarian stimulation remain controversial.MethodsWe enrolled 229 women undergoing controlled ovarian hyperstimulation for IVF/ICSI-treatment and devided them in three ovarian-response-subgroups: Poor responder (POR) after Bologna Criteria, polycystic ovary syndrome (PCO) after Rotterdam Criteria, or normal responder (NOR, control group). Subjects were subdivided into six genotypes according to their be-allelic CGG-repeat length. FMR1-CGG-repeat-length was determined using ALF-express-DNA-sequencer or ABI 3100/3130 × 1-sequencer. mRNA was extracted from GCs after follicular aspiration and quantitative FMR1-expression was determined using specific TaqMan-Assay and applying the ΔΔCT method. Kruskall-Wallis-Test or ANOVA were used for simple comparison between ovarian reserve (NOR, POR or PCO) and CGG-subgroups or cohort demographic data. All statistical analysis were performed with SPSS and statistical significance was set at p ≤ 0.05.ResultsA statistically significant increase in FMR1-mRNA-expression-levels was detected in GCs of PORs with heterozygous normal/low-CGG-repeat-length compared with other genotypes (p = 0.044).ConclusionFemale ovarian response may be negatively affected by low CGG-alleles during stimulation. In addition, due to a low-allele-effect, folliculogenesis may be impaired already prior to stimulation leading to diminished ovarian reserve and poor ovarian response. A better understanding of FMR1 expression-regulation in GCs may help to elucidate pathomechanisms of folliculogenesis disorders and to develop risk-adjusted treatments for IVF/ICSI-therapy. Herewith FMR1-genotyping potentially provides a better estimatation of treatment outcome and allows the optimal adaptation of stimulation protocols in future.

FMR1 and AKT/mTOR Signaling in Human Granulosa Cells: Functional Interaction and Impact on Ovarian Response

“…GCs were recovered from the follicular fluid after transvaginal ultrasound-guided follicle puncture for in vitro fertilization (IVF), as described previously [17]. The follicles were aspirated with a specific needle (Premium Fas Single Lumen, #4551 NS-AS1; Gynétics Medical Products N.V., Lommel, Belgium) that was connected to a vacuum pump (Cook Medical, K-MAR-5200, Bloomington, IN, USA).…”

“…GCs in RNAlater were centrifuged at 5000× g for 5 min, and the supernatants were removed. Total RNA was isolated from these GCs using TRIzol (Life Technologies by Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer's instructions [38,39] with PEQGOLD PHA-SETRAP A 1.5 mL tubes (VWR International GmbH, Darmstadt, Germany) or MaXtract TM High Density 1.5 mL tubes (Qiage Germantown, MD, USA), as described previously [17]. RNA was dissolved in RNase-free water, and concentration and purity were determined using a NanoDrop 2000c UV-spectrometer (NanoDrop Products, Wilmington, DE, USA).…”

“…Increased FMR1 expression thus appears to be an important ovarian pathomechanism [15,16]. Accordingly, elevated FMR1 expression analyzed in native GCs of women with distinct ovarian responses demonstrated an association with poor ovarian reserve in women with CGG repeats below the norm of 26-34 repeats [17]. Additionally, variable FMR1 genotypes with CGG repeats below or above 26-34 impact ovarian reserve [18][19][20].…”

Genetic determination of the ovarian reserve: a literature review

“…The FMR1 gene contains a 5’-UTR triplet repeat (GGG) region, the length of which varies individually. It has been shown that FMR1 gene expression depends on the length and number of these triplet repeats [ 13 ]. Four allelic forms of the FMR1 gene are identified according to the number of GGG repeats (Fig.…”

Development and Validation of a Mathematical Model to Predict the Complexity of FMR1 Allele Combinations

“…The incidence of normal pure alleles (without interspersions) is low and their origin as well as the phenotypic impact in females, are still debatable. It has been proposed that "low zone" alleles, variably determined to be CGG ≤ 26 or CGG ≤ 23, are associated with different phenotypic outcomes (Mailick et al, 2014;Gleicher et al, 2015;Rehnitz et al, 2018). Some studies show that they are associated with decreased ovarian reserve and fertility issues, due to a mechanism not yet elucidated, possibly different from that involved in premutated alleles (Gleicher et al, 2015;Wang et al, 2017), although such negative effects were not corroborated by others (Spitzer et al, 2012;Ruth et al, 2016).…”