Significance of serum AMH and antral follicle count discrepancy for the prediction of ovarian stimulation response in Poseidon criteria patients.

PURPOSE: To determine the risk of not being a poor responder in ovarian stimulation (OS) for in vitro fertilization (IVF) when ovarian reserve markers are discordant-one falling within Poseidon's criteria normal range (e.g., anti-Müllerian hormone (AMH) ≥ 1.2 ng/mL or antral follicle count (AFC) ≥ 5), and the other in the poor ovarian reserve range. METHODS: A tri-center retrospective cohort study (2015-2017) involving women with discordant AMH and AFC values undergoing their first IVF/ICSI cycle using conventional OS (cOS, ≥ 150 IU/day of follicle-stimulating hormone). Discordant serum AMH and AFC values were defined according to Poseidon's criteria (AMH < 1.2 ng/mL and AFC ≥ 5 or AMH ≥ 1.2 ng/mL and AFC < 5). Poor ovarian response (POR) was < 4 retrieved oocytes. Receiver operating characteristic (ROC) curves were used to determine AMH and AFC cut-offs for non-POR. Logistic regression analysis evaluated factors associated with non-POR. RESULTS: Out of 8797 patients who underwent assessment with both AMH and AFC, 1172 (13.3%) exhibited discordant values. Of these, 854 (72.9%) had ≥ 4 oocytes retrieved. Within this group, 726 (85.0%) had "low" AMH values, whereas 128 (15.0%) had "low" AFCs. An AFC of 6 had 77% sensitivity and 52% specificity (AUC = 0.700), while AMH of 1.19 ng/mL had 31% sensitivity and 85% specificity (AUC = 0.492) for non-POR. AFC and the use of recombinant gonadotropins were positive predictors of non-POR. CONCLUSIONS: When serum AMH is < 1.19 ng/mL, but AFC is ≥ 6, there is a moderate likelihood of a non-POR during stimulation. Conversely, if AFC is < 5 but serum AMH is ≥ 1.19 ng/mL, the chances of non-POR are low. Among patients with discordant markers, AFC emerges as the primary predictor of oocyte yield.

Personalized prediction of the secondary oocytes number after ovarian stimulation: A machine learning model based on clinical and genetic data.

Controlled ovarian stimulation is tailored to the patient based on clinical parameters but estimating the number of retrieved metaphase II (MII) oocytes is a challenge. Here, we have developed a model that takes advantage of the patient's genetic and clinical characteristics simultaneously for predicting the stimulation outcome. Sequence variants in reproduction-related genes identified by next-generation sequencing were matched to groups of various MII oocyte counts using ranking, correspondence analysis, and self-organizing map methods. The gradient boosting machine technique was used to train models on a clinical dataset of 8,574 or a clinical-genetic dataset of 516 ovarian stimulations. The clinical-genetic model predicted the number of MII oocytes better than that based on clinical data. Anti-Müllerian hormone level and antral follicle count were the two most important predictors while a genetic feature consisting of sequence variants in the GDF9, LHCGR, FSHB, ESR1, and ESR2 genes was the third. The combined contribution of genetic features important for the prediction was over one-third of that revealed for anti-Müllerian hormone. Predictions of our clinical-genetic model accurately matched individuals' actual outcomes preventing over- or underestimation. The genetic data upgrades the personalized prediction of ovarian stimulation outcomes, thus improving the in vitro fertilization procedure.

Quantitative analyses of insulin-like peptide 3 and sex steroid hormones in dominant follicles and corpora lutea during the estrus cycle and in follicular cysts in beef heifers.

Insulin-like peptide 3 (INSL3) and sex steroids were measured in bovine dominant follicles and corpora lutea during the estrus cycle and in follicular cysts. Paired ovaries from beef heifers (n = 47) were classified, by their morphological features, either into four stages of the estrus cycle (Day 1 = day of ovulation, Day 20 = day of estrus) as Stage I (Days 1-4; n = 8), Stage II (Days 5-10; n = 10), Stage III (Days 11-17; n = 8), and Stage IV (Days 18-20; n = 11) or follicular cystic (n = 10). Cysts (n = 15) were subdivided into estrogen-active (n = 7) and estrogen-inactive (n = 8) cysts. INSL3, testosterone, and estradiol-17ß concentrations in the dominant follicular fluid of Stage IV were higher than those in Stages II and III (P < 0.05). INSL3 concentrations in the cystic fluid were similar to those in dominant follicles at Stage IV, whereas testosterone and estradiol-17ß concentrations were lower in cysts (P < 0.05). INSL3 content per estrogen-inactive cyst was higher than that of Stage IV (P < 0.05). INSL3 and progesterone concentrations in luteal tissue and contents per corpus luteum were higher in Stages II and III (P < 0.05). In conclusion, INSL3 secretion in bovine dominant follicles increased with maturation. Follicular cysts may retain the production of INSL3 during their formation but tend to lose the capacity for testosterone secretion. Estrogen-inactive cysts subjected to advanced atresia may accumulate more INSL3. INSL3 production in bovine corpora lutea is enhanced during maturation.

Protein Palmitoylation in Bovine Ovarian Follicle.

Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

The endocrine milieu in naturally matured follicles is different in women with high serum anti-Müllerian hormone concentrations.

RESEARCH QUESTION: Is the endocrine milieu different in women with low serum anti-Müllerian hormone (AMH) concentration compared with women with high concentration? DESIGN: Cohort study of 84 women (four groups) classified according to AMH concentration and age undergoing natural cycle IVF treatment. Concentrations of LH, oestradiol, testosterone, androstenedione and AMH were determined in follicular fluid (FF), associations analysed and clinical outcome parameters evaluated. RESULTS: A positive correlation between serum and FF AMH concentrations was confirmed. Follicular fluid androstenedione concentration was positively correlated with serum AMH concentration (P < 0.0001, r2 = 0.197). The correlation between FF LH and FF testosterone concentration in all women was not significant (P = 0.050, r2 = 0.046); however, the correlation between FF androstenedione in women with high serum AMH concentration was significant (P = 0.032, r2 = 0.220). Follicular fluid testosterone and androstenedione were positively correlated with FF oestradiol overall and in some individual groups. The high serum AMH concentration group showed the highest FF AMH and androstenedione concentrations and lowest oestradiol-testosterone and oestradiol-androstenedione ratios. High FF AMH concentration was associated with a higher clinical pregnancy rate and high FF oestradiol concentration with a slightly better embryo quality. CONCLUSIONS: Differences in the endocrine milieu in women with high serum AMH concentration seem to be caused by increased follicular LH concentration. In women with high serum AMH concentration, FF androstenedione is increased and ratios of oestradiol-testosterone and oestradiol-androstenedione are decreased, suggesting a disturbed endocrine milieu caused by reduced metabolization of FF androgens into oestrogens. In natural cycles, FF AMH concentrations are positively associated with higher clinical pregnancy rates and oestradiol concentrations with a higher embryo score.

Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor ß.

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erßnull ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERß is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erßnull mutant model that fails to show follicle maturation was also included in order to identify the ERß-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erßnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erßnull GCs. These latter ERß-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERß-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erßnull GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERß.

Identification of circular RNAs in the ovarian follicles of Meishan and Duroc sows during the follicular phase.

Circular RNAs (circRNAs) are a newly discovered class of endogenous non-coding RNAs that play an important role in growth and development by regulating gene expression and participating in a variety of biological processes. However, the role of circRNAs in porcine follicles remains unclear. Therefore, this study examined middle-sized ovarian follicles obtained from Meishan and Duroc sows at day 4 of the follicular phase. High-throughput RNA sequencing (RNA-seq) was utilized to construct circRNAs, and differential expression was identified. The findings were validated using reverse transcription PCR (RT-PCR) and DNA sequencing, GO and KEGG analyses were performed, and potential miRNA targets were identified. The RNA-seq identified a total of 15,866 circRNAs, with 244 differentially expressed in the Meishan relative to the Duroc (111 up-regulated and 133 down-regulated). The RT-PCR finding confirmed the RNA-seq results, and quantitative real-time PCR (qPCR) analysis examining a subset of the circRNAs showed that they are resistant to RNase R digestion. Bioinformatics analysis (GO and KEGG) showed that the host genes associated with the differentially expressed circRNAs are involved in reproduction and follicular development signaling pathways. Furthermore, many of the circRNAs were found to interact with miRNAs that are associated with follicular development. This study presents a new perspective for studying circRNAs and provides a valuable resource for further examination into the potential roles of circRNAs in porcine follicular development.

An Ovarian Reserve Assessment Model Based on Anti-Müllerian Hormone Levels, Follicle-Stimulating Hormone Levels, and Age: Retrospective Cohort Study.

BACKGROUND: Previously, we reported a model for assessing ovarian reserves using 4 predictors: anti-Müllerian hormone (AMH) level, antral follicle count (AFC), follicle-stimulating hormone (FSH) level, and female age. This model is referred as the AAFA (anti-Müllerian hormone level-antral follicle count-follicle-stimulating hormone level-age) model. OBJECTIVE: This study aims to explore the possibility of establishing a model for predicting ovarian reserves using only 3 factors: AMH level, FSH level, and age. The proposed model is referred to as the AFA (anti-Müllerian hormone level-follicle-stimulating hormone level-age) model. METHODS: Oocytes from ovarian cycles stimulated by gonadotropin-releasing hormone antagonist were collected retrospectively at our reproductive center. Poor ovarian response (<5 oocytes retrieved) was defined as an outcome variable. The AFA model was built using a multivariable logistic regression analysis on data from 2017; data from 2018 were used to validate the performance of AFA model. Measurements of the area under the curve (AUC), sensitivity, specificity, positive predictive value, and negative predicative value were used to evaluate the performance of the model. To rank the ovarian reserves of the whole population, we ranked the subgroups according to the predicted probability of poor ovarian response and further divided the 60 subgroups into 4 clusters, A-D, according to cut-off values consistent with the AAFA model. RESULTS: The AUCs of the AFA and AAFA models were similar for the same validation set, with values of 0.853 (95% CI 0.841-0.865) and 0.850 (95% CI 0.838-0.862), respectively. We further ranked the ovarian reserves according to their predicted probability of poor ovarian response, which was calculated using our AFA model. The actual incidences of poor ovarian response in groups from A-D in the AFA model were 0.037 (95% CI 0.029-0.046), 0.128 (95% CI 0.099-0.165), 0.294 (95% CI 0.250-0.341), and 0.624 (95% CI 0.577-0.669), respectively. The order of ovarian reserve from adequate to poor followed the order from A to D. The clinical pregnancy rate, live-birth rate, and specific differences in groups A-D were similar when predicted using the AFA and AAFA models. CONCLUSIONS: This AFA model for assessing the true ovarian reserve was more convenient, cost-effective, and objective than our original AAFA model.

Confirmation of ovarian follicles in an enantiornithine (Aves) from the Jehol biota using soft tissue analyses.

The remains of ovarian follicles reported in nine specimens of basal birds represents one of the most remarkable examples of soft-tissue preservation in the Early Cretaceous Jehol Biota. This discovery was immediately contested and the structures alternatively interpreted as ingested seeds. Fragments of the purported follicles preserved in an enantiornithine (STM10-12) were extracted and subjected to multiple high-resolution analyses. The structures in STM10-12 possess the histological and histochemical characteristics of smooth muscles fibers intertwined together with collagen fibers, resembling the contractile structure in the perifollicular membrane (PFM) of living birds. Fossilized blood vessels, very abundant in extant PFMs, are also preserved. Energy Dispersive Spectroscopy shows the preserved tissues primarily underwent alumino-silicification, with minor mineralization via iron oxides. No evidence of plant tissue was found. These results confirm the original interpretation as follicles within the left ovary, supporting the interpretation that the right ovary was functionally lost early in avian evolution.

Follicular fat-soluble vitamins as markers of oocyte competency.

The main aim of this prospective study was to investigate the effect of the concentration of fat-soluble vitamins A, D, E and K in individual follicles on oocyte quality and developmental competence. The analysis was performed on 313 follicular fluid (FF) samples from 50 patients undergoing ovarian stimulation with intracytoplasmic sperm injection. We demonstrated that the mean concentration of individual vitamins in FF correlated with their level in serum (p < 0.0001). The levels of vitamin D in FF were higher than in serum, while the opposite was observed for other analyzed vitamins. We did not observe a correlation between FF vitamin D concentration with fertilization success. However, we observed its association with embryo development status on day 3. Moreover, we showed a statistically significant negative correlation between the mean day 5 embryo score and the concentration of vitamin D in serum (rS = -0.68 p = 0.01) and follicular fluid (rS = -0.71 p = 0.01). Our study showed that FF concentration of vitamin A and E was helpful in the prediction of fertilization success of each individual oocyte. Moreover, vitamin A and E concentrations in FF were associated with status of embryo development on the third day of culture. Vitamin A was also associated with the embryo quality on day 2 and the embryo development status on day 5 after fertilization. In conclusion, a combination of FF vitamin analysis and routine morphological assessment could allow for a more accurate and sensitive method of determining embryonic developmental competence and enable the selection of a better embryo to transfer and perhaps translating into an increased chance of pregnancy.Abbreviations: in vitro fertilization: IVF; anti-Mullerian hormone: AMH; follicular fluid: FF; intracytoplasmic sperm injection: ICSI; top quality: TQ; vitamin D binding globulin level: VDBP; assisted reproductive technology: ART.

Intra- and inter-individual variability of fatty acid composition of the follicular fluid in a cohort of 23 women undergoing assisted reproductive treatment.

PURPOSE: To examine the intra- and inter-individual variability in fatty acid composition of follicular fluid (FF) of 23 patients undergoing assisted reproductive treatment. METHODS: The average coefficient of variation within each patient (CVw) and intra-class correlation coefficient (ICC) values of FF fatty acid composition as well as correlation between the fatty acid composition of individual, pooled or first-punctured follicles, were assessed. RESULTS: The proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 20:5n-3, total MUFA and n-3 PUFA showed good reproducibility (CVw < 10%). Although CVw values of 18:3n-3 and 20:3n-6 exceeded 10%, variation between patients exceeded intra-individual variation as indicated by elevated ICC values (0.61 and 0.66, respectively). Nevertheless, 20:4n-6 and 22:6n-3 showed non-negligible intra-patient variation. With the exception of some minor fatty acids (< 0.30 g/100 g), strong relationships were demonstrated between the average proportion in individually analysed follicles and the proportion determined in pooled samples and in the first, largest follicle. CONCLUSION: The CVw and ICC values of proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 18:3n-3, 20:5n-3, total MUFA and n-3 PUFA showed limited intra-individual variation and moderate to good reliability. However, this is not the case for some other PUFA, such as 20:4n-6 and 22:6n-3. Nevertheless, for all of these fatty acid(s) (groups), calculated average fatty acid proportions were highly correlated with proportions determined in pooled samples and in the first, largest follicle. This implies that single or pooled follicle aspiration suffices to assess intra-individual variation in the FF of these fatty acids.

Cell-free nucleic acids and melatonin levels in human follicular fluid predict embryo quality in patients undergoing in-vitro fertilization treatment.

Despite many advances in assisted reproductive technology (ART), the most viable embryo selection remains a challenge for infertility treatment. This study was designed to investigate whether intra-follicular circulating cell-free DNA (cfDNA) fragments and Melatonin levels predict embryo quality in patients undergoing IVF treatment. A total of eight hundred and ninety-five follicular fluid (ff) samples were collected from 325 infertile patients undergoing IVF treatment. Patients were enrolled from August 2017 to December 2018 in the infertility center of a tertiary care hospital. A clear non-hematic follicular fluid was aspirated after the removal of eggs from the dominant follicles (>18mm) of each patient. Melatonin and E2 levels in each follicular sample were estimated by immune-chemiluminescence using commercially available kits. ALU-qPCR evaluated cfDNA levels in individual follicular fluid samples. Our study presented a significant and negative relationship between intra-follicular cfDNA and melatonin concentration (-0.541: P<0.001). Each individual follicle contains measurable copy number of cfDNA [mean: 1.85±2.98ng/µl (median; 1.86ng/µl (95% Cl: 0.96-2.87)]. In pregnant women cfDNA copy number was significantly decreased in follicular fluid samples(ff) aspirated from matured oocytes than in immature ones [p<0.01; ß = -0.42±0.49; median; 1.45ng/ml (95% Cl: 0.36-2.97) vs. 3.57ng/µl (95% Cl: 0.37-4.01) respectively. While melatonin concentration in ff samples corresponding to mature oocytes was significantly higher than in ff samples related to immature oocytes (p<0.001). Moreover, in pregnant women cfDNA level was significantly lower in ff samples related to oocytes which produces top-quality embryos versus low quality embryos [p<0.001; ß=1.81±0.91; median; 1.25ng/µl (95% Cl: 0.35-1.97)] vs. [(median; 3.65ng/ml (95% Cl: 1.23-6.36)] respectively. Likewise, in non-pregnant women melatonin levels were significantly decreased in ff samples related to embryos with high fragmentation rate (≥25%) than embryos with low fragmentation rate (<25%; p<0.001). Conclusively, this study indicates that Intra-follicular cfDNA and melatonin concentration possibly a new supplemental tool that supports to establish an advanced non-invasive early prognostic test for the patients undergoing IVF/ICSI procedure.

Identification of potential metabolic biomarkers of polycystic ovary syndrome in follicular fluid by SWATH mass spectrometry.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex disorder associated with multiple metabolic disturbance, including defective glucose metabolism and insulin resistance. The altered metabolites caused by the related metabolic disturbance may affect ovarian follicles, which can be reflected in follicular fluid composition. The aim of this study is to investigate follicular fluid metabolic profiles in women with PCOS using an advanced sequential window acquisition of all theoretical fragment-ion spectra (SWATH) mass spectrometry. MATERIALS AND METHODS: Nineteen women with PCOS and twenty-one healthy controls undergoing IVF/ET were recruited, and their follicular fluid samples were collected for metabolomic study. Follicular fluid metabolic profiles, including steroid hormones, free fatty acids, bioactive lipids, and amino acids were analyzed using the principal component analysis (PCA) and partial least squares to latent structure-discriminant analysis (PLS-DA) model. RESULTS: Levels of free fatty acids, 3-hydroxynonanoyl carnitine and eicosapentaenoic acid were significantly increased (P < 0.05), whereas those of bioactive lipids, lysophosphatidylcholines (LysoPC) (16:0), phytosphingosine, LysoPC (14:0) and LysoPC (18:0) were significantly decreased in women with PCOS (P < 0.05). Additionally, levels of steroid hormone deoxycorticosterone and two amino acids, phenylalanine and leucine were higher in the PCOS patients (P < 0.05). CONCLUSION: Women with PCOS display unique metabolic profiles in their follicular fluid, and this data may provide us with important biochemical information and metabolic signatures that enable a better understanding of the pathogenesis of PCOS.

Characterization of follicles in girls and young women with Turner syndrome who underwent ovarian tissue cryopreservation.

OBJECTIVE: To characterize ovarian follicles of girls and young women with Turner syndrome (TS) who underwent ovarian tissue cryopreservation (OTC). DESIGN: Retrospective case-control study. SETTING: University hospital. PATIENT(S): Fifteen girls and young women with TS aged 5-22 years at OTC were included, together with 42 control girls and young women aged 1-25 years who underwent OTC because of cancer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle density (follicles/mm3), morphology, and health were assessed in ovarian cortex biopsies from TS patients and compared with controls. Hormone concentrations were measured in serum and follicle fluids. Immature cumulus oocyte complexes were obtained and matured in vitro. RESULT(S): Follicles were found in 60% of the biopsies (9 of 15) from TS ovaries. In 78% of the ovaries (7 of 9) with follicles, the follicle density was within the 95% confidence interval of the control group. There was a high rate of abnormal follicle morphology. Six follicle-specific proteins were expressed similarly in TS and control ovaries. However, apoptosis and zona pellucida protein expression were found to be abnormal in TS. Turner syndrome follicle fluid from small antral follicles had lower concentrations of estrogen and testosterone and higher concentrations of antimüllerian hormone than controls. Thirty-one cumulus oocyte complexes were collected from one patient and cultured for 48 hours in vitro, resulting in five metaphase II oocytes (maturation rate 16%, degeneration rate 19%). CONCLUSION(S): The benefits of OTC may be limited to a highly selected group of TS mosaic patients in whom a sizeable pool of normal follicles is present at OTC.

Evidence for the involvement of FXR signaling in ovarian granulosa cell function.

Farnesoid X receptor (FXR) is mainly present in enterohepatic tissues and regulates cholesterol, lipid, and glucose homeostasis in coordination with target genes such as SHP and FABP6. Although FXR has been revealed to be expressed in reproductive tissues, FXR function and expression levels in the ovary remain unknown. In this study, we investigated FXR expression in mouse ovaries and its target genes in ovarian granulosa cells. In situ hybridization and immunohistochemical staining showed that FXR was mainly distributed in secondary and tertiary follicles. The agonist-induced activation of FXR in cultured granulosa cells induced the expression of SHP and FABP6, while siRNA targeting of FXR decreased CYP19a1 and HSD17b1 expression. Upon examination of the roles of SHP and FABP6 in granulosa cells, we found that SHP overexpression significantly decreased StAR, CYP11a1, and HSD3b gene expression. In addition, siRNA targeting of FABP6 decreased CYP19a1 and HSD17b1 expression, while FABP6 overexpression increased CYP19a1 expression. In conclusion, the present study demonstrates the presence of FXR signaling in the ovary and reveals that FXR signaling may have a role in function of granulosa cells.

Presence and function of kisspeptin/KISS1R system in swine ovarian follicles.

Kisspeptin and its receptor KISS1R are involved in the neuroendocrine regulation of mammalian reproduction and their role on follicular development and function can be hypothesized. The present work was designed to confirm the immunopresence of kisspeptin and its receptor in the ovary of swine and to study the effects of kisspeptin 10 and its antagonist, kisspeptin 234, on main functional parameters of granulosa cells (i.e. cell proliferation, steroid production, and redox status) as well as their modulatory action on angiogenesis. The immunopresence of kisspeptin and KISS1R were detected in granulosa cells. Kisspeptin 10 stimulated progesterone in vitro production, thus indirectly suggesting that it can have a role in the luteinization process of granulosa cells. Kisspeptin 10 displayed potentiating effects on non-enzymatic scavenging activity, thus supporting its involvement in the control of the antioxidant defense system of ovarian follicles. In addition, results from the angiogenesis bioassay suggest that kisspeptin may have a role in the physiological development of new ovarian vessels. Additional studies are needed to confirm the functional significance of the kisspeptin/KISS1R system within the swine ovary.

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.

Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. BIOLOGICAL SIGNIFICANCE: Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases.

Oocyte maturation in the toad Rhinella arenarum (Amphibia, Anura): Evidence of cAMP involvement in steroid production and action.

In this work, we describe the participation of the adenylate cyclase/3'-5'-cyclic adenonsine monophosphate (cAMP) pathway in the seasonal follicular secretion of progesterone (P4 ) and testosterone (T), and its relationship with the maturation of Rhinella arenarum oocytes. Under gonadotropin stimulation, P4 secretion was the dominant steroid produced during the reproductive period, resulting in 100% germinal vesicle breakdown (GVBD) in oocytes in vitro; in contrast, T and estradiol (E2 ) secretion increased (∼16 nM/20 follicles and ∼80 pM/20 follicles, respectively) during the non-reproductive period, but only yielded 50% GVBD. Treatment of the follicles with dibutyryl-cAMP or forskolin induced a significant increase in T secretion during both periods, but P4 secretion did not significantly change and GVBD did not occur. These results suggest that high cAMP levels in the oocyte maintain meiotic arrest and prevent the induction effect of follicular steroids. An increase in cAMP levels in denuded oocytes, however, negatively regulated T-induced maturation since treatment with increasing db-cAMP or forskolin inhibited their maturation. Therefore, we hypothesize that an elevation in T during the non-reproductive period favors its aromatization to E2 , leading to follicle growth. During the reproductive period, P4 production might promote oocyte maturation when environmental conditions are favorable for reproduction. Together, the results indicate that steroidogenesis is seasonal and depends on gonadotropic activity in R. arenarum.

Frozen section with improved H&E staining for follicular morphometric analysis of mouse ovary in oestrus cycle.

Careful analysis of follicular morphology and numbers of different follicular maturation stages, in combination with the measurements of gonadotropic and sex hormone profiles, provide an accurate and rapid evaluation system of the ovarian function. The aim of this study is to improve the existing methods of ovarian tissue section preparation and staining methods, and to establish a fast and easy method to observe and evaluate follicular maturation stage and numbers using mouse ovary samples. Ovaries were collected at menstrual phases of proestrus, oestrus, metestrus and diestrus from C57BL/6J female mice. Then the ovaries were fixed in 4% paraformaldehyde, dehydrated with graded sucrose, embedded with OCT, frozen-sectioned at 7 µm thick, and subjected to quick haematoxylin & eosin staining for observation. The results showed that the present method was able to distinguish secondary, preantral, antral, and preovulatory follicles. Although our method was unable to discriminate and distinguish primordial follicles and primary follicles, the results were comparable to those from more complicated techniques. We conclude that this improved and quick method can be used in combination with hormone analysis to investigate ovarian development and function in different mouse models.

Protein profile of mouse ovarian follicles grown in vitro.

STUDY QUESTION: Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage? SUMMARY ANSWER: From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages. WHAT IS KNOWN ALREADY: The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells. STUDY DESIGN, SIZE, DURATION: The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics. PARTICIPANTS/MATERIALS, SETTING, METHODS: SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 µg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 µg and 170 µg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. LARGE SCALE DATA: Data are available via ProteomeXchange with identifier PXD006227. LIMITATIONS, REASONS FOR CAUTION: The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. WIDER IMPLICATIONS OF THE FINDINGS: This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report.