Biomarkers Of Oocyte Quality Research Articles

P-158 Quantitative metabolomic profiling of follicular fluid identifies oocyte quality related biomarkers from infertile patients

Abstract Study question Is whether there is a metabolomic signature in the follicular fluid depending on oocyte quality and whether infertility may impact on this quality? Summary answer 46 metabolites were quantified via nuclear magnetic resonance spectroscopy in the follicular fluid, and each metabolite that was a biomarker of oocyte quality was pathology-specific. What is known already Literature confirmed that metabolomics had been valuable for exploring follicular fluid and the search for nutritive and non-nutritive biomarkers. There are currently no data in the literature concerning the homeostasis of this follicular fluid, and two options are possible: this fluid is a continuum of blood plasma, or it has its own homeostasis. Studies have highlighted follicular fluid qualitative and quantitative alterations in the metabolome due to one specific infertility pathology footprint and correlated in some instances to outcome measures of in vitro fertilization success but samples are pooled and there are no information concerning quality and competence oocyte assessment. Study design, size, duration 61 infertile patients who underwent in vitro fertilization (IVF) treatment with intracytoplasmic sperm injection (ICSI) from Femme Mère Enfant hospital (Bron, France) between 6th april 2022 and 12th may 2022 were included. Participants/materials, setting, methods 61 follicular fluids were collected individually and used for metabolomic profiling by nuclear magnetic resonance spectroscopy. A total of 46 metabolites were quantified. Metabolite concentrations in follicular fluid were compared with serum concentrations.Oocyte was considered mature if it has its first polar body and competent if it was able to develop into a blastocyst after culturing. In vitro fertilized oocytes were cultured and monitored with time-lapses incubation technology until day 5 or 6. Main results and the role of chance For 23 metabolites out of 46, follicular fluid exhibited lower concentrations than serum. Glucose and glutamine, which are among the most abundant metabolites, decreased by 54% (not significant) and 47% (P < 0.05) respectively. 7 metabolites exhibited higher concentrations in the follicular fluid than in the serum. An 85%-increase in lactate and a more-than-tenfold increase in pyruvate were noted (P < 0.05). The reverse ratios of lower substrates such as glucose and glutamine and higher waste products like lactate and pyruvate imply that the follicular fluid functions as a homeostatic compartment, being more influenced by exchange within follicular cells than by serum equilibration. The detected metabolic signatures of oocyte maturity and competence were all specific to the patient infertility-related pathology. The follicular fluid concentrations of aspartate, glucose, methionine and methylmalonate were significantly correlated to oocyte maturity in the control group (P < 0.05). For patients having endometriosis, asparagine concentration was significantly decreased by oocyte maturity (P < 0.05). For patients with diminished ovarian reserve, acetone concentration was significantly decreased by oocyte maturity (P < 0.05). The concentration of aspartate, that correlated with oocyte maturity, was also slightly decreased by oocyte competence, and for the patients with endometriosis the concentration of threonine was decreased by 30% by oocyte competence (P < 0.05). Limitations, reasons for caution Although 46 metabolites were well quantified, each metabolite that was a biomarker of oocyte maturity or competence was pathology specific. One major reason would be the high metabolite concentration variation observed between the different infertilities. This study indicates that the diagnosis of patients according to their infertility must be prior. Wider implications of the findings This study underlines the potential of follicular fluid metabolomics as a diagnostic avenue for oocyte-related becoming in order to enhance the selection of oocytes and improve outcomes in assisted reproductive and preservation fertility techniques. Trial registration number not applicable

P-161 Mechanical phenotyping of mammalian oocytes using Atomic Force Microscopy (AFM) to evaluate their developmental potential

Abstract Study question Can we measure the mechanical properties of mammalian oocytes with AFM and use them as a biomarker to identify oocytes with the best development potential? Summary answer We characterized mammalian oocytes’ mechanical properties using AFM, correlated them with oocyte cortex organization, and explored these parameters in contexts critical for oocyte quality. What is known already Oocyte production is crucial for reproduction but prone to errors, yielding a significant number of poor-quality oocytes detrimental to fertility and offspring development. Indeed, oocyte quality determines embryo health post-fertilization. Current selection methods for oocytes rely on subjective morphological analyses. Remarkably, human and mouse oocyte quality correlates with mechanical properties, particularly cortical tension (CT). CT defects in oocytes are rather frequent and impair early embryonic development after fertilization. However, they do not induce any visible oocyte morphological alteration. Mechanical properties could, therefore, be used as a biomarker of oocyte quality. Study design, size, duration We designed an AFM protocol for the measurement and analysis of mammalian oocytes’ mechanical properties, including a new elasto-capillary model describing oocyte mechanics. Concomitantly, we analyzed oocyte cortex organization using spinning disk microscopy in live. We measured mouse oocytes at various stages of meiotic maturation (prophaseI, meiosisI, meiosisII), mutant mouse oocytes with known cortical tension defects, oocytes coming from young and aged mice, and human oocytes at various stages of meiotic maturation (prophaseI, meiosisI, meiosisII). Participants/materials, setting, methods Mouse oocytes were collected from 11-week-old and 44-56-week-old OF1 female mice. Human oocytes were collected from patients who underwent ovarian stimulation for Assisted Reproduction Technologies (ART) as part of their protocol. Only oocytes that were in prophaseI or meiosisI after ovarian punction and thus not suitable for the ART procedure were used in this study. All patients gave informed consent (agreement CNIL number 22_5725). Main results and the role of chance Our AFM approach allows measuring various mechanical parameters, including oocyte cortical tension, elasticity, and capillary indentation rate, quantifying whether the oocyte behaves predominantly as a droplet with surface tension or as an elastic material. We find that these parameters correlate to specific oocyte cortex organization, showing that a thin actin cortex enriched in myosin II is associated with high tension, high elastic modulus, and low capillary indentation rate. In contrast, a thick actin cortex with few myosin II is associated with low cortical tension, low elasticity, and high capillary indentation rate. We go further and show that these parameters can predict cortex organization. Indeed, oocytes from aged mice display altered mechanical parameters, reflecting specific changes in their cortex organization. Finally, we transferred our approach to human oocytes. We show that the mechanical parameters of mouse and human oocytes are in the same range. Still, in humans, they evolve differently from those of mice during meiotic maturation, again reflecting specific changes in the organization of their cortex. Thus, mechanical parameters correlate with oocytes quality, and can predict cortex organization in mouse and human. Limitations, reasons for caution AFM is hardly usable for clinical application, we are thus working in parallel on developing non-invasive devices to perform high-throughput measurements of oocyte mechanical properties in a non-invasive manner, to serve as a mechanical biomarker to identify oocytes with the best developmental potential for ART. Wider implications of the findings Ultimately, this project could improve ART procedures by increasing their success rate while decreasing treatment cycles and risks for the patient and newborn, and optimize protocols for oocyte freezing for fertility preservation. Trial registration number not applicable

The effect of L-carnitine on oocyte mitochondrial health and biomarkers on cyclophosphamide chemotherapy drug in mice

Improving oocyte competence during chemotherapy is widely known as a contributing factor to increasing the probability of fertility. Additionally, the role of cumulus cells in oocyte quality is of utmost importance. Therefore, this study was designed to simultaneously probe into the relative gene expression of oocytes and cumulus cells as biomarkers of oocyte quality with cyclophosphamide and L-carnitine treatment. A total of 60 adult NMRI mice were divided into four groups: control, L-carnitine (LC), cyclophosphamide (CP), and cyclophosphamide+L-carnitine (CP+LC). The relative mRNA expression levels of oocyte quality genes including growth differentiation factor 9 (Gdf9), hyaluronan synthase 2 (Has2), and mitochondrial sirtuin 3 (Sirt3) in oocytes, and genes involved in bilateral communication between cumulus cells and between the oocyte and its neighboring cumulus cells including connexin 37 (Cx37) and connexin 43 (Cx43) were detected by Real-time-PCR. DCFH-DA staining analyzed the level of intracellular ROS in oocytes. Under the influence of L-carnitine, Gdf9, Has2, Cx43, and Cx37 were significantly up-regulated (p ≤ 0.05). However, cyclophosphamide considerably reduced the expression of all these genes (p ≤ 0.05). The expression of the Sirt3 gene in the CP group increased significantly compared to the other groups (p ≤ 0.05). Analysis of fluorescent images revealed that the level of intracellular ROS in the cyclophosphamide group was significantly increased compared to the other groups (p ≤ 0.05), while it plummeted in the L-carnitine group (p ≤ 0.05). L-carnitine as an antioxidant can reduce the destructive effects of cyclophosphamide and enhance bilateral communications between oocytes and cumulus cells, and it may ultimately lead to an increase in the fertility rate.

Relation of serum and follicular level of BMP15 with Oocyte quality, embryo grading and pregnancy rate

Objectives: Use of serum and follicular fluid concentration of oocyte secreted factors BMP15 as biomarkers of oocyte quality, embryo quality and it's relation to pregnancy rate.
 Methods: Eighty eight women were included in this study; they were selected from those undergoing intra-cytoplasmic sperm injection.
 Results: Positive pregnancy was achieved by 14 women accounting for 19.0 % (total number of women that reach embryo transfer was 72). No significant difference in mean serum BMP15 between pregnant and non- pregnant women, but the level of follicular fluid BMP15 was significantly higher in pregnant women. MI oocyte count was not significantly correlated to serum or follicular fluid BMP15 (p > 0.05). MII oocyte count showed highly significant positive correlation to serum and follicular fluid BMP15 (p < 0.01). Grade1 embryo count showed highly significant positive correlation to serum and follicular fluid BMP15 (p < 0.01), Grade 2 embryo count showed significant positive correlation to serum BMP15 (p = 0.032), but the grade 2 embryo count showed non- significant correlation to follicular BMP15 (p > 0.05). Also grade 3 embryo count showed non- significant correlation to serum and follicular BMP15 (p > 0.05).
 Conclusions: The current study revealed that serum and follicular BMP15 could be used as indicator for oocyte maturity, and serum BMP15 could be used as indicator of good quality embryos.

Transcriptomic analysis of cumulus cells shows altered pathways in patients with minimal and mild endometriosis

Endometriosis is a chronic inflammatory disorder that is highly associated with infertility. This association seems to be related to oocyte impairment, mainly in the initial stages of endometriosis (minimal and mild), where no distortions or adhesions are present. Nonetheless, invasive oocyte analyses are not routinely feasible; thus, indirect assessment of oocyte quality is highly desirable, and, in this context, cumulus cells (CCs) may be more suitable targets of analysis. CCs are crucial in oocyte development and could be used as an index of oocyte quality. Therefore, this prospective case–control study aimed to shed light on the infertility mechanisms of endometriosis I/II by analyzing the CCs’ mRNA transcription profile (women with endometriosis I/II, n = 9) compared to controls (women with tubal abnormalities or male factor, n = 9). The transcriptomic analyses of CCs from patients with minimal and mild endometriosis revealed 26 differentially expressed genes compared to the controls. The enrichment analysis evidenced some altered molecular processes: Cytokine-cytokine receptor interactions, Chemokine signaling, TNF signaling, NOD-like receptor signaling, NF-kappa B signaling, and inflammatory response. With the exception of CXCL12, all enriched genes were downregulated in CCs from patients with endometriosis. These findings provide a significant achievement in the field of reproductive biology, directing future studies to discover biomarkers of oocyte quality in endometriosis.

Cumulus Cell DNA Damage as an Index of Human Oocyte Competence

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for ICSI to determine a CC DNA fragmentation index (CCDFI) of each individual oocyte using a variant of the chromatin dispersion test. One hundred and thirty oocytes were selected and studied from two Spanish fertility clinics, 90 of which were fertilized and developed to embryos. Significant differences were found between the CCDFI of unfertilized and fertilized oocytes (p < .001) and between the CCDFI of embryos that were discarded and those that developed suitable for transfer or cryopreservation (p < .001). Oocyte quality was negatively correlated with CCDFI (Spearman’s rho = − 0.45; p < .001). Receiver operator characteristics curves (ROC) suggested that a cut-off value of 24% CCDFI was able to discriminate the capacity of the gametes to result in syngamy with a sensitivity and specificity of 75.6% and 65%, respectively. This cut-off supports the application of CCDFI as potential index for the evaluation of the reproductive potential of oocytes prior to fertilization.

Oocyte Morphometric Assessment and Gene Expression Profiling of Oocytes and Cumulus Cells as Biomarkers of Oocyte Competence in Sheep.

Simple SummaryIn vitro production (IVP) of embryos is an essential element of many reproductive biotechnologies. Although some remarkable achievements have been obtained for ovine IVP, the overall efficiency is still low. A limiting factor of the technique is the great variability of the oocyte quality used in IVP. The current selection criterion of oocytes is based on a morphological factor, which is insufficient to determine oocytes’ competence. Therefore, the identification of biomarkers for the selection of oocytes of high developmental competence is a major research goal. The purpose of this study was to evaluate the morphometric parameters of oocytes and the relative gene expression of oocytes and cumulus cells (CCs) as biomarkers of oocyte quality after individually culturing them in order to increase the final efficiency of the IVP technique in sheep. We observed that higher blastocyst rates were obtained from oocytes classified as intermediate and large according to total diameter, oocyte diameter, and zona pellucida thickness. Moreover, the expression of genes related to oocyte quality was higher in oocytes classified as large and in CCs from oocytes classified as large and thus able to reach the blastocyst stage. Oocyte morphometric assessments and gene expression in CCs may be used as biomarkers of oocyte quality.Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter (p < 0.001), oocyte diameter (p < 0.05), and ZP thickness (p < 0.001). Furthermore, the relative gene expression of BAX, BCL2, GDF9, and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX, STAR, and PTGS2, while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.

Anti-Müllerian hormone and embryo quality as determined by time-lapse imaging.

There is conflicting evidence as to whether serum anti-Mullerian hormone (AMH) is a biomarker of oocyte quality in addition to its known role in assessing ovarian reserve. This study aims to examine the relationship between AMH and embryo potential as assessed by time-lapse imaging (TLI). A total of 106 embryos from 67 patients were included in the study. All subjects were women with recorded pre-treatment AMH levels who underwent in vitro fertilization using a TLI embryo incubator. Exclusion criteria included cases of donor oocytes, rescue-ICSI, and >2 embryos transferred. Individual time measures, presence of multinucleation (MN), and composite TLI score were analyzed in relation to patient AMH. Linear regression was used to model AMH among embryo TLI parameters while controlling for age as a continuous covariate. There was no statistically significant difference in the mean AMH levels between patients in the normal and abnormal time frames for CC2, S2, and T5. Similarly, there was no significant difference in AMH levels based on composite TLI score or presence/absence of multinucleation. The lack of association between AMH levels and embryo TLI variables persisted after controlling for age (Grade P=0.19, CC2 P=0.47, S2 P=0.52, t5 P=0.34, MN P=0.92). Serum AMH is not predictive of embryo quality as assessed by TLI standardized time intervals, composite score, and presence of MN. From a clinical perspective, these findings suggest that diminished ovarian reserve alone does not imply poorer quality of individual embryos.

A paracrine interaction between granulosa cells and leukocytes in the preovulatory follicle causes the increase in follicular G-CSF levels

ObjectiveFollicular granulocyte colony-stimulating factor (G-CSF) is a new biomarker of oocyte quality and embryo implantation in in vitro fertilization (IVF) cycles. Its role in reproduction is poorly understood. Our study aimed to investigate the mechanisms and cells responsible for G-CSF production in the preovulatory follicle.DesignLaboratory research study.SettingSingle-center study.InterventionsGranulosa cells and leukocytes were isolated from the follicular fluids (FF) or the blood of women undergoing IVF and from the blood of a control group of women with spontaneous ovulatory cycles to perform cocultures.Main outcome measureG-CSF-secreted protein was quantified in the conditioned media of cocultures.ResultsG-CSF secretion was considerably increased in cocultures of granulosa cells and leukocytes. This effect was maximal when leukocytes were isolated from the blood of women in the late follicular phase of the menstrual cycle or from the FF of women undergoing IVF. The leukocyte population isolated from the FF samples of women undergoing IVF had a higher proportion of granulocytes than that isolated from the corresponding blood samples. Leukocytes induced the synthesis and secretion of G-CSF by granulosa cells. Among a range of other FF cytokines/chemokines, only growth-regulated oncogene alpha (GROα) was also increased.ConclusionThe notable rise in G-CSF at the time of ovulation coincides with the accumulation of follicular granulocytes, which stimulate G-CSF production by granulosa cells via paracrine interactions. High follicular G-CSF concentrations may occur in follicles with optimal granulosa–leukocyte interactions, which could explain the increased implantation rate of embryos arising from these follicles.

To assess serum Anti Mullerian Hormone (AMH) level as a biomarker for oocyte quality

Introduction: Oocyte competence is a factor that affects ART outcomes. Oocyte morphology assessment and scoring are laborious, have inter-observer variations and lack consensus on parameters to be assessed. Role of anti mullerian hormone (AMH) as a biomarker for oocyte quality and competence is investigated. Aim and Objectives: To evaluate day 2/3 serum AMH as a predictor of oocyte quality, embryo quality and fertilization rate in a stimulated ART cycle. Methods: Single centre, prospective observational study. All patients undergoing IVF-ICSI between August 2018 and February 2019 included. Total Oocyte Score calculated for all oocytes. and subsequently assessed for fertilization, cleavage and day 3 embryo grading. Correlations between AMH levels and TOS, PTOS, Fertilization and Cleavage rates assessed using appropriate statistical methods. Results: Of 86 patients and 780 oocytes studied,639 underwent ICSI with fertilization rate 86% (550/639) and cleavage rate 90.7%(499/550). Embryo grades were A-50.6%, B-38.9% and C-10.5%. Mean TOS score and PTOS scores were 0.3±1.99 and 0.44±1.21.TOS was predictive of fertilization [ROC-AUC=0.598(P = 0.004)]. Mean AMH levels were 3.66 ± 3.96 ng/ml [0.69±0.21ng/ml 75th centile] and decreased with increasing age. AMH levels correlated with TOS, PTOS, fertilization and cleavage rates. The r2 values were low suggesting poor clinical significance. AMH levels were not predictive of fertilization and embryo grade. Conclusion: AMH levels show weak correlation with oocyte quality and are poor predictor of oocyte competence.

Mediator in the Embryo-endometrium Cross-talk: Granulocyte Colony-stimulating Factor in Infertility.

Successful implantation requires a receptive endometrium and a good quality egg. The challenges a physician encounters with regard to this in assisted reproductive technology are obtaining good quality embryo, achieving optimal endometrial thickness (EMT), and subsequently implantation, which is denotive of a receptive endometrium. Granulocyte colony-stimulating factor (G-CSF) has been observed to be a biomarker of oocyte quality and has been shown to enhance EMT and implantation because of its immunological effects. A systematic search for all relevant articles on G-CSF in follicular fluid and its therapeutic benefit in thin endometrium and recurrent implantation failure was performed, and peer-reviewed, full-text articles related to humans were included in the study. As a tool to determine the potentiality of oocyte, G-CSF shows promise with its predictability increasing in combination with morphological embryo scoring or interleukin 15. For the thin endometrium, G-CSF is especially useful in patients who are refractory to other treatment modalities. In recurrent implantation failure (RIF), G-CSF showed potential in a subset of patients with immunological deficiency lacking killer cell immunoglobulin-like receptor genes. This review highlights the various forms of usage of G-CSF and the effectiveness of G-CSF in infertility. G-CSF equips embryologists with a tool to determine the potentiality of oocyte and physicians with therapy for thin endometrium and RIF, especially since the available treatment options are ineffective.

The Influence of Pentraxin 3 on the Ovarian Function and Its Impact on Fertility.

Follicular development is a highly coordinated process that in humans takes more than 6 months. Pituitary gonadotropins and a variety of locally produced growth factors and cytokines are involved in determining a precise sequence of changes in cell metabolism, proliferation, vascularization, and matrix remodeling in order to obtain a follicle with full ovulatory and steroidogenic capability. A low-grade inflammation can alter such processes leading to premature arrest of follicular growth and female reproductive failure. On the other hand, factors that are involved in inflammatory response as well as in innate immunity are physiologically upregulated in the follicle at the final stage of maturation and play an essential role in ovulation and fertilization. The generation of pentraxin 3 (PTX3) deficient mice provided the first evidence that this humoral pattern recognition molecule of the innate immunity has a non-redundant role in female fertility. The expression, localization, and molecular interactions of PTX3 in the periovulatory follicle have been extensively studied in the last 10 years. In this review, we summarize findings demonstrating that PTX3 is synthesized before ovulation by cells surrounding the oocyte and actively participates in the organization of the hyaluronan-rich provisional matrix required for successful fertilization. Data in humans tend to confirm these findings, indicating PTX3 as a biomarker of oocyte quality. Moreover, we discuss the emerging evidence that in humans altered PTX3 systemic levels, determined by genetic variations and/or low-grade chronic inflammation, can also impact the growth and development of the follicle and affect the incidence of ovarian disorders.

The role of mitochondrial activity in female fertility and assisted reproductive technologies: overview and current insights.

Mitochondria have been implicated as key factors regulating female reproductive processes. Notable progress has been made in determining the role of mitochondria with respect to oocyte maturation, fertilization and early embryo development. In addition, mitochondrial function and dysfunction has been the subject of various studies in ovarian ageing and metabolic stress models. However, the overall mitochondrial impact on female fertility is yet to be uncovered. The mitochondrial DNA content of granulosa, cumulus and trophectoderm cells is being explored as a biomarker of oocyte quality and embryo viability. As growing evidence suggests that embryo potential could be related to the ability of oocyte mitochondria to generate energy, efforts have been made to investigate the possibility of improving mitochondrial capacity in women with poor outcomes after treatment with assistedreproductive technologies. Thus far, therapeutic attempts have focused mainly on using nutrients to restore mitochondrial function and transferring mitochondria from autologous germline precursor cells. Moreover, new perspectives on optimizing infertility treatments have arisen with modern mitochondrial replacement therapies, which are being applied in women with mitochondrial disease-causing mutations. This review explores aspects of the distinctive contribution of mitochondria to reproductive processes and discusses current and emerging clinical implications.

Reproductive failure in mice expressing transgenic follicle-stimulating hormone is not caused by loss of oocyte quality.

Human female reproductive aging features declining ovarian follicle reserve and oocyte quality, and rising levels of circulating follicle-stimulating hormone (FSH). We determined the effects of elevated FSH on oocyte-embryo development in mature mice exhibiting premature infertility caused by progressively rising transgenic human FSH (TgFSH) levels. Oocyte-embryo developmental competence and quality were examined using oocyte maturation and aneuploidy rates, biomarkers of oocyte quality, and reciprocal embryo transfers assessed for implantation and pregnancy. In vitro maturation suggested that TgFSH exposure only hindered oocyte developmental competence in old females, as significantly more oocytes from ≥12-month-old TgFSH females remained at germinal vesicle stage compared with age-matched control oocytes. Aneuploidy rates were equivalent in oocytes from aging TgFSH compared with wildtype females. Cumulus cell expression levels of candidate biomarker Inhba, Egfr, and Rgs2 transcripts were elevated in associated aneuploid vs euploid oocytes from both TgFSH and wildtype females. In vivo, embryos transferred from subfertile 6-month-old TgFSH females to wildtype recipients yielded normal implantation rates and more pups born compared with controls. Transfer of wildtype embryos rescued the fertility of 6-month-old TgFSH-recipient females, although pup birth weight was reduced in TgFSH vs wildtype recipients. Our current findings show that elevated FSH had minimal disruption of either embryo developmental capacity or uterine function when examined in isolation, and the subfertility of TgFSH female mice was not caused by altered oocyte aneuploidy or quality.

Follicular fluid biomarkers for human in vitro fertilization outcome: Proof of principle.

BackgroundHuman follicular fluid (FF) is a unique biological fluid in which the oocyte develops in vivo, and presents an optimal source for non-invasive biochemical predictors. Oocyte quality directly influences the embryo development and hence, may be used as a predictor of embryo quality. Peptide profiling of FF and its potential use as a biomarker for oocyte quality has never been reported.MethodsThis study screened FF for peptide biomarkers that predict the outcome of in vitro fertilization (IVF). Potential biomarkers were discovered by investigating 2 training datasets, consisting both of 17 samples and validating on an independent experiment containing 32 samples. Peptide profiles were acquired by nano-scale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS).ResultsFrom the training datasets 53 peptides were found as potential biomarker candidates, predicting the fertilization outcome of 24 out of the 32 validation samples blindly (81.3% sensitivity, 68.8% specificity, AUC = 0.86). Seven potential biomarker peptides were identified. They were derived from: insulin-like growth factor binding protein-5, alpha-2-antiplasmin, complement component 3, inter-alpha-trypsin inhibitor heavy chain H1, serum albumin, protein diaphanous homolog 1 and plastin-3.ConclusionsThe MS-based comprehensive peptidomic approach carried out in this study, established a novel panel of potential biomarkers that present a promising predictive accuracy rate in fertilization outcome, and indicates FF as an interesting biomarker resource to improve IVF clinic routine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0106-9) contains supplementary material, which is available to authorized users.

Cytokines, apoptosis and implantation

The follicle provides a unique micro-environment for the oocyte to develop and mature to a fertilizable gamete, within. Intrafollicular components, such as follicular fluid and cellular material are a potential resource for the noninvasive prediction of oocyte developmental potential. Using the cow as a model, we have established in vitro embryo production (IVP) methods that facilitate the individual tracking of oocytes and embryos, permitting a retrospective analysis of retained follicular components according to thedevelopmental outcomeof the oocyte during IVP. We have investigated the relationship between a range of parameters (including intrafollicular steroids and metabolomic profiles in follicle fluid and gene expression in follicular and cumulus cells) and oocyte development in vitro. Intrafollicular steroid concentrations in fluid from 3to 8-mm antral follicles were not predictive of oocyte developmental competence. However, metabolomic profiling of the same follicular fluid was highly predictive; several amino acids and polyunsaturated fatty acids positively correlated with blastocyst formation. Metabolomic analyses of follicular fluid from alternative bovine models of fertility have confirmed the predictive value of this approach. Employing both candidate and global gene expression analysis of transcript abundance in follicular and cumulus cells, we identified differentially expressed transcripts that were positively associated with blastocyst formation. However, many candidate genes are only predictivewithin themodel inwhich theywerefirst identified. Recently, we have focused on the role of progesterone (P4) during oocyte maturation. P4 has an anti-apoptotic effect on the cumulus–oocyte complex during maturation that is mediated via the nuclear P4 receptors. We have identified P4-responsive genes that have similar expression profiles in both the oocyte and their cumulus cells, implying their potential as biomarkers of oocyte quality. In particular, the pro-survival and cell cycle factor AVEN, whose dynamic regulation by P4 is reflected in both the oocyte and surrounding cumulus cells could potentially be exploited not just as an indicatorof oocytequality, but also as an indicator of IVCconditions. In summary, follicular components are an excellent resource for thenon-invasivepredictionofoocyte developmental potential; however, not all parameters are predictive across all models of oocyte development.

High expression of GSTT1 in ovarian granulosa cells of younger women predicts lower ratio of fertilization of IVF

We previously reported that Glutathione S-transferase theta 1 (GSTT1) was highly up-regulated in aged human ovarian granulosa cells, suggesting that GSTT1 may be a biomarker for oocyte quality in age-related infertility. The purpose of this study was to investigate the correlations between the expression level of GSTT1 in granulosa cells and the IVF outcomes in younger patients.

ANTIMüLLERIAN HORMONE IN FOLLICULAR FLUID OF NORMOGONADOTROPHIC WOMEN UNDERTAKING IVF/ICSI cycles: a candidate biomarker of oocyte quality and embryo implantation potential

ObjectiveTo investigate the impact of follicular fluid (FF) proteomic composition vs AMH levels as biomarkers of oocyte quality and embryo implantation potential.Design270 patients included into a two-arms randomized open-lable prospective study. A fixed antagonist protocol + αr-FSH stimulation was used. r-hCG was used to trigger ovulation.Materials and MethodsInclusion criteria: FSH d3 ≤10 IU/mL, BMI >20 and < 27 kg/m2, Age <38.Exclusion criteria: PCOS, endometriosis, endocrine, genetic, systemic inflammatory-immunological disorders.135 patients with N°3 Follicles with FF AMH level ≥4,0 ng/ml. 135 patients with N°3 Follicles with FF AMH level ≤1,0 ng/ml. Proteomic analyses were carried out by 2D isoelectric focusing by the direct sample rehydratation method followed by Sypro Ruby staining and comparison with PDQuest software. Non parametric comparison, χ2, Mann-Whitney test were used. Logistic regression was used to calculate the likelihood of live birth in relation to the protein spot of interest.Results15 potential FF protein were identified: αHaptoglobin, mitochondrial integrity genome, apolipoprotein H, transferrin, lyzozymeC,αfibrinogen,immunoglobulin heavy chain V-III were increased in the live birth group,whereas γand β chains fibrinogen, antithrombin, vitamin D-binding protein, and complement 3 and 4 were decreased. Serological and FF AMH levels influence FF soluble proteome. In the high FF AMH levels 84% were down-regulated (transferrin, immunoglobulin light chain, albumin,CO4 complement) and 16% up-regulated (immunoglobulin unknown chain, clusterin,α-1-antitrypsin,α-2-HS-glycoprotein,antithrombin,β-fibrinogen,apolipoprotein A2.Haptoglobin was either down- or up-regulated.Conclusion2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity. Proteomic evaluation of follicular fluid is able to identify potential biomarkers of good versus poor prognosys patients. AMH levels influence oocyte quality and embryo implantation potential. ObjectiveTo investigate the impact of follicular fluid (FF) proteomic composition vs AMH levels as biomarkers of oocyte quality and embryo implantation potential. To investigate the impact of follicular fluid (FF) proteomic composition vs AMH levels as biomarkers of oocyte quality and embryo implantation potential. Design270 patients included into a two-arms randomized open-lable prospective study. A fixed antagonist protocol + αr-FSH stimulation was used. r-hCG was used to trigger ovulation. 270 patients included into a two-arms randomized open-lable prospective study. A fixed antagonist protocol + αr-FSH stimulation was used. r-hCG was used to trigger ovulation. Materials and MethodsInclusion criteria: FSH d3 ≤10 IU/mL, BMI >20 and < 27 kg/m2, Age <38.Exclusion criteria: PCOS, endometriosis, endocrine, genetic, systemic inflammatory-immunological disorders.135 patients with N°3 Follicles with FF AMH level ≥4,0 ng/ml. 135 patients with N°3 Follicles with FF AMH level ≤1,0 ng/ml. Proteomic analyses were carried out by 2D isoelectric focusing by the direct sample rehydratation method followed by Sypro Ruby staining and comparison with PDQuest software. Non parametric comparison, χ2, Mann-Whitney test were used. Logistic regression was used to calculate the likelihood of live birth in relation to the protein spot of interest. Inclusion criteria: FSH d3 ≤10 IU/mL, BMI >20 and < 27 kg/m2, Age <38. Exclusion criteria: PCOS, endometriosis, endocrine, genetic, systemic inflammatory-immunological disorders. 135 patients with N°3 Follicles with FF AMH level ≥4,0 ng/ml. 135 patients with N°3 Follicles with FF AMH level ≤1,0 ng/ml. Proteomic analyses were carried out by 2D isoelectric focusing by the direct sample rehydratation method followed by Sypro Ruby staining and comparison with PDQuest software. Non parametric comparison, χ2, Mann-Whitney test were used. Logistic regression was used to calculate the likelihood of live birth in relation to the protein spot of interest. Results15 potential FF protein were identified: αHaptoglobin, mitochondrial integrity genome, apolipoprotein H, transferrin, lyzozymeC,αfibrinogen,immunoglobulin heavy chain V-III were increased in the live birth group,whereas γand β chains fibrinogen, antithrombin, vitamin D-binding protein, and complement 3 and 4 were decreased. Serological and FF AMH levels influence FF soluble proteome. In the high FF AMH levels 84% were down-regulated (transferrin, immunoglobulin light chain, albumin,CO4 complement) and 16% up-regulated (immunoglobulin unknown chain, clusterin,α-1-antitrypsin,α-2-HS-glycoprotein,antithrombin,β-fibrinogen,apolipoprotein A2.Haptoglobin was either down- or up-regulated. 15 potential FF protein were identified: αHaptoglobin, mitochondrial integrity genome, apolipoprotein H, transferrin, lyzozymeC,αfibrinogen,immunoglobulin heavy chain V-III were increased in the live birth group,whereas γand β chains fibrinogen, antithrombin, vitamin D-binding protein, and complement 3 and 4 were decreased. Serological and FF AMH levels influence FF soluble proteome. In the high FF AMH levels 84% were down-regulated (transferrin, immunoglobulin light chain, albumin,CO4 complement) and 16% up-regulated (immunoglobulin unknown chain, clusterin,α-1-antitrypsin,α-2-HS-glycoprotein,antithrombin,β-fibrinogen,apolipoprotein A2.Haptoglobin was either down- or up-regulated. Conclusion2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity. Proteomic evaluation of follicular fluid is able to identify potential biomarkers of good versus poor prognosys patients. AMH levels influence oocyte quality and embryo implantation potential. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity. Proteomic evaluation of follicular fluid is able to identify potential biomarkers of good versus poor prognosys patients. AMH levels influence oocyte quality and embryo implantation potential.

Developmental competence in oocytes and cumulus cells: candidate genes and networks

Common aspects of infertility can be seen across several species. In humans, dairy cows, and mares there is only a 25-35% chance of producing a live offspring after a single insemination, whether natural or artificial. Oocyte quality and subsequent embryo development can be affected by factors such as nutrition, hormonal regulation, and environmental influence. The objective of this study was to identify genes expressed in oocytes and/or cumulus cells, across a diverse range of species, which may be linked to the ability an oocyte has to develop following fertilization. Performing a meta-analysis on previously published microarray data on various models of oocyte and embryo quality allowed for the identification of 56 candidate genes associated with oocyte quality across several species, 4 of which were identified in the cumulus cells that surround the oocyte. Twenty-one potential biomarkers were associated with increased competence and 35 potential biomarkers were associated with decreased competence. The upregulation of Metap2, and the decrease of multiple genes linked to mRNA and protein synthesis in models of competence, highlights the importance of de novo protein synthesis and its regulation for successful oocyte maturation and subsequent development. The negative regulation of Wnt signaling has emerged in human, monkey, bovine, and mouse models of oocyte competence. Atrx expression was linked to decreased competence in both oocytes and cumulus cells. Biological networks and transcription factor regulation associated with increased and decreased competence were also identified. These genes could potentially act as biomarkers of oocyte quality or as pharmacological targets for manipulation in order to improve oocyte developmental potential.

Identification of Candidate Genes and Networks Associated with Developmental Competence in Oocytes and Cumulus Cells: A Cross Species Comparison of Gene Expression in Models of Increased and Decreased Competence.

There are a number of aspects of infertility that are conserved across several species. In many mammalian species there is only a 25-35% chance of producing a live offspring after a single insemination, whether natural or artificial. Oocyte quality and developmental competence can be affected by a number of environmental factors such as nutrition and hormonal regulation. The objective of this study was to identify candidate genes expressed in mammalian oocytes and/or their cumulus cells which may be indicative of oocyte developmental competence. We performed a meta-analysis on previously published Affymetrix microarray data from various models of oocyte competence from several mammalian species (Bovine, Monkey, Mouse and Human), with the specific objective to identify candidate biomarkers of oocyte competence expressed in oocytes and or their cumulus cells. We identified 64 genes, 21 genes which were associated with increased competence including Mitd1, Kif12, and Gkap1. 43 genes were associated with decreased competence including Gdf9, Emi1, and one gene, Atrx, was linked to decreased competence in both oocytes and cumulus cells. Of the 64 genes identified, 5 (Hmga1, Foxm1, Map3k12, Tgfbr3, and Sfrp1) were identified by the Ingenuity Pathway Analysis-Biomarker program (http://www.ingenuity.com/products/ipa-biomarker.html) as being the most promising and relevant biomarker candidates. Only one of the genes identified by this program, Hmga1, was associated with increased competence. Hmga1 indirectly regulates the G2/M transition and is also involved in apoptosis, DNA binding, regulation of transcription and chromatin remodeling, which are key events in oocyte meiotic maturation. Additionally we carried out Bioinformatic analysis of the Affymetrix gene lists using Metacore from GeneGo (http://www.genego.com/metacore.php) to identified overpopulated networks. The regulation of Wnt signaling, mRNA processing and protein synthesis emerged as key networks associated with oocyte competence. We identified several transcription factors which regulate a large proportion of the differentially expressed transcripts in several of the competence models, which may themselves be reflective of the oocyte's competence. For example Nanog and Heat Shock Factor 1 were found to regulate 34 and 38 transcripts, respectively, which were associated with increased oocyte developmental competence. In conclusion we have identified a number of genes, pathways and regulatory networks which could potentially act as biomarkers of oocyte quality or as pharmacological targets for the improvement of in vitro embryo production systems. This work is funded by Science Foundation Ireland Grant # 07/SRC/B1156: Reproductive Biology Research Cluster. (platform)