Diego Duarte Alcoba

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus-oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbeccos Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 μM BCB for 60 min; and 60, 90 or 120 min to 13 μM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 μM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 μM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 μM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

Selection of developmentally competent human oocytes aspirated during cesarean section

Abstract Purpose: To evaluate the efficiency/safety of Brilliant Cresyl Blue (BCB) staining as a selection method of developmentally competent immature human oocytes. Materials and methods: Immature oocytes of 32 pregnant women were recovered during cesarean section (CS). After retrieval, 92 oocytes were randomly divided into two groups: control (directly disposed to in vitro maturation – IVM) and treated – exposed to BCB 26 μM during 60 min. After staining, the treated group was classified as cytoplasm coloration, BCB positive (blue) or negative (colorless), and then disposed to IVM. Nuclear status was checked after 24 and 48 h of IVM. Nuclear maturation (polar body extrusion), meiosis resumption (absence of germinal vesicle) and degeneration rates were evaluated among the three groups (control, BCB positive and BCB negative) using Generalized Estimating Equations, followed by Bonferroni’s correction for multiple comparisons. Results: Nuclear maturation was higher in BCB positive compared to BCB negative, after 24 and 48 h of IVM (p = .004 and p = .032). The control group was equal to BCB positive. There was no difference among groups analyzing meiosis resumption and degeneration rates. Conclusion: The BCB test can be a good marker in pre-selection procedures of developmentally competent human oocytes aspirated during CS.

FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells

Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26-34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system.

Brilliant cresyl blue staining does not present cytotoxic effects on human luteinized follicular cells, according to gene/protein expression, as well as to cytotoxicity tests

In vitro oocyte maturation presents many advantages and its success is related to the selection of fully grown oocytes. In animal models, staining of cumulus-oocyte complexes (COCs) with Brilliant Cresyl Blue (BCB) is widely used for this selection. However, a lack of information about the safety of BCB makes its applicability in humans questionable. Because granulosa and cumulus cells have a close relationship with the oocyte and taking into account that follicular cells are also exposed to BCB when this pre-selection method is applied, we aimed to evaluate the effects of BCB on human follicular cells exposed to BCB. Cytotoxicity tests (Sulforhodamine B and Neutral Red Uptake) and gene and protein expression of elements related to the cell cycle (BAX, BCL2, TP53 and CDKN1A), as well as to cell death and metabolism (GAPDH), glucose consumption, and estradiol and progesterone secretion, were examined in granulosa and cumulus cells with and without exposure to BCB. Regardless estradiol secretion and glucose consumption, all other evaluations presented similar results between control and treated (BCB) groups, independently of cell type. Therefore, our results demonstrate convincingly that BCB seems to be safe for use in humans and it should encourage future studies to evaluate the development of embryos derived from human oocytes selected by BCB staining.

Developmental potential of in vitro or in vivo matured oocytes

This study compared the embryological features of mature and immature oocytes (different stages) collected from stimulated cycles of in vitro fertilization (IVF). Immature oocytes were identified, classified as PI (prophase I - germinal vesicle, GV) or MI (metaphase I), were matured in vitro and fertilized using the intra-cytoplasmic sperm injection (ICSI) technique. Fertilization potential, cleavage, and subsequent transfer/cryopreservation of the embryos derived from these in vitro matured oocytes were compared with those of in vivo matured oocytes (collected at the MII stage). The characteristics of embryos derived from gametes recovered in the MI and MII stages were similar. The fertilization rate of immature oocytes recovered in PI was significantly lower than that of MII oocytes (P = 0.031), and the cleavage rate of the PI group was also lower than that of the MI (P = 0.004) and MII (P < 0.001) groups. In vitro maturation of MI oocytes is a suitable alternative when immature oocytes are recovered, as their characteristics and development are similar to those of in vivo matured oocytes. Optimization of outcomes for PI oocytes will require development of techniques that can distinguish which of these gametes will mature and fertilize.

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

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.