Markers Of Oocyte Quality Research Articles

GDF-9 and BMP-15 direct the follicle symphony.

Understanding the physiology underlying the complex dialog between the oocyte and its surrounding somatic cells within the ovarian follicle has been crucial in defining optimal procedures for the development of clinical approaches in ART for women suffering from infertility and ovarian dysfunction. Recent studies have implicated oocyte-secreted factors like growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15), members of the transforming growth factor-beta (TGFβ) superfamily, as potent regulators of folliculogenesis and ovulation. These two factors act as biologically active heterodimers or as homodimers in a synergistic cooperation. Through autocrine and paracrine mechanisms, the GDF-9 and BMP-15 system has been shown to regulate growth, differentiation, and function of granulosa and thecal cells during follicular development playing a vital role in oocyte development, ovulation, fertilization, and embryonic competence. The present mini-review provides an overview of recent findings relating GDF-9 and BMP-15 as fundamental factors implicated in the regulation of ovarian function and discusses their potential role as markers of oocyte quality in women.

Diminished ovarian reserve and poor response to stimulation in patients

Do infertile women aged <38 years with quantitative evidence of diminished ovarian reserve and/or poor response to stimulation also exhibit poor oocyte quality as measured by blastulation rates, aneuploidy rates, and live birth rates? Young women with evidence of accelerated follicular depletion, either by precycle ovarian reserve testing or postcycle evidence of low oocyte yield, exhibit equivalent blastulation rates, aneuploidy rates and live birth rates per euploid embryo transfer as age-matched controls with normal precycle and postcycle parameters. Previous studies are conflicted as to whether women with evidence of diminished ovarian reserve and/or poor ovarian response are also at increased risk of exhibiting evidence of poor oocyte quality. Most prior studies have failed to adequately control for the confounding effect of female age on typical markers of oocyte quality in poor responders. The rate of follicular depletion occurs at around 38 years on average; thus, evidence of quantitative depletion before this would indicate a premature diminution of ovarian reserve and allow evaluation of whether markers of oocyte quality are tied to quantitative markers. This was a retrospective cohort study at a single center between 2012 and 2016. This time frame was specifically chosen as all embryos were cultured to the blastocyst stage at this center during the study period (no cleavage stage transfers were performed). Two comparisons were made: precycle assessment of ovarian reserve (based on anti-mullerian hormone (AMH) level) and postcycle oocyte yield results. For each comparison, patients in <10th percentile were compared to patients in the interquartile range (IQR) with respect to blastulation rate, aneuploidy rate and live birth rate. A mixed effects model was created to control for female age (in the <38 year old range) and correlation among oocytes from a given cohort. For the precycle blastulation analysis, only patients with AMH data available were included (345 patients with AMH in the <10th percentile versus 1758 patients with AMH in the 25th to 75th percentile (IQR)). To compare aneuploidy rates, the subset of these patients who pursued preimplantation genetic testing for aneuploidy (PGT-A) was then analyzed (124 patients in the <10th percentile versus 782 patients in the IQR). For the postcycle blastulation analysis, all patients who proceeded to retrieval (whether or not they also had AMH data available) were included (535 patients with oocyte yield in the <10th percentile versus 2675 patients in the IQR). To compare aneuploidy rates, the subset of these patients who pursued PGT-A was then analyzed (156 patients in the <10th percentile versus 1100 patients in the IQR). The adjusted odds of a given fertilized oocyte developing to a blastocyst, being aneuploid or leading to a live birth after euploid transfer were no different if the oocyte was retrieved from a cycle with ovarian reserve parameters or oocyte yield in the <10th percentile compared to an oocyte retrieved in a cycle with those parameters in the 25-75th percentile. An AMH level in the <10th percentile did more commonly result in cycle cancellation prior to retrieval and after retrieval prior to transfer due to global arrest of embryos. The timing of retrieval in patients with fewer oocytes may be more optimal given the greater ability to discern the overall maturity of the cohort, thus enhancing performance per retrieved oocyte. Analyses included only first cycles. Subsequent adjustment of protocol due to prior performance may mean that some patients in the <10th percentile for oocyte yield are actually better prognosis patients than their first cycle indicates. Data on whether or not patients were on oral contraceptives at time that AMH level drawn was not available. Other unknown biases are also likely to be present given retrospective nature of the study. While young women with evidence of quantitative depletion of ovarian reserve have lower live birth rates per stimulation cycle, this not attributable to poor oocyte quality because the blastulation rate per fertilized oocyte and live birth rate per embryo transfer are equivalent to that in women with normal quantitative markers of ovarian reserve. Thus, the pathophysiology mediating a premature quantitative decline in ovarian reserve appears different than that which mediates markers of oocyte quality, such as aneuploidy. Young poor responders may use this information to help guide embryo accumulation strategies when considering their family building plans. None. N/A.

Corrigendum

Garcia-Velazco JA, Motta L, Rodriguez S, et al. Decreased concentrations of AMH in follicular fluid of women with endometriosis: a hypothetical new marker of oocyte quality. J Endometr Pelvic Pain Disord 2009; 1(1): 52–56. DOI: 10.1177/228402650900100108. In this article, the second author’s name was misspelled. It should have appeared as Leticia Motta. This has been corrected in the online version of the article.

Early lethality of embryos derived from transgenic Xenopus females is associated with reduced ovarian grem1 expression

The grem1 gene codes a protein that inhibits the action of multiple members of a growth factor family known as bone morphogenetic proteins (BMPs). Certain members of this BMP family can regulate both fecundity and fertility in mammals via their action on oocyte (egg) development, and grem1 has been identified as a marker of oocyte quality in humans. The model amphibian Xenopus laevis is far more fecund than mammals, producing thousands of eggs in a clutch. However, female transgenic frogs carrying grem1 under the control of a stress inducible hsp70 promoter (G frogs) produce very few viable offspring. Here, we show that this is not due to reduced fecundity or fertilization rate, but results from a significant reduction in subsequent survival over the first day of development. Embryos that successfully survive for the first day were found to go on to develop normally when compared to their peers. Both the morphology and stage distribution of oocytes from G females appears normal, and oocytes develop at expected rates, although stage VI oocytes were found to have a lower response to in vitro progesterone treatment. Unexpectedly, levels of grem1 mRNA were found to be consistently lower in the female ovaries from four independent G transgenic lines than in wild type ovaries. Both transgenic and wild type offspring were equally affected, confirming a maternal effect. Our study shows that transgenic females with the lowest levels of grem1 transcripts in the ovary have the lowest rates of survival past the first day of amphibian embryogenesis, equivalent to pre-implantation staged mammalian embryos. The reduced expression of grem1 in the oocytes of transgenic females suggests transgene supression of an endogenous locus may occur in the Xenopus female germline, an unexpected finding.

MicroRNA-574 suppresses oocyte maturation via targeting hyaluronan synthase 2 in porcine cumulus cells.

MicroRNAs (miRNAs) have been established as important regulators of gene expression in the mammalian ovary. A previous screen of small RNA in the porcine ovary identified the downregulation of miR-574 during oocyte maturation, although its role during this process was not established. Here, we found that miR-574 directly targets the transcript for hyaluronan synthase 2 protein (HAS2), a key enzyme in the production of extracellular matrix by the surrounding cumulus cells. Inhibiting this miRNA during in vitro maturation (IVM) increased HAS2 levels along with several markers of oocyte quality. Furthermore, inhibiting miR-574 increased oocyte meiotic progression. We then stably overexpressed miR-574 using a lentiviral vector to transduce cumulus cells during IVM. This gain-of-function approach resulted in a 50% decrease in HAS2 expression and nearly 20% reduction in oocyte progression through meiosis. To confirm the specific targeting of HAS2 by miR-574, we constructed several luciferase vectors harboring the HAS2 3'-untranslated region. Cotransfection of the reporter and miR-574 attenuated luciferase activity. After mutating the putative miR-574 binding site, however, this effect was abolished and luciferase activity remained high. Our results show that the direct targeting of HAS2 by miR-574 negatively impacts oocyte quality during IVM and that inhibiting miR-574 derepresses HAS2 expression and subsequently improves oocyte maturation. Taken together, we help to elucidate a mechanism of posttranscriptional regulation by miRNA in the mammalian ovary.

Efficacy of Dehydroepiandrosterone (DHEA) to overcome the effect of ovarian ageing (DITTO): A proof of principle double blinded randomized placebo controlled trial

ObjectiveTo evaluate the effect of DHEA supplementation on In-Vitro Fertilisation (IVF) outcome as assessed by ovarian response, oocyte developmental competence and live birth rates in women predicted to have poor ovarian reserve (OR). The feasibility of conducting a large trial is also assessed by evaluating the recruitment rates and compliance of the recruited participants with DHEA/placebo intake and follow-up rates. Study designA single centre, double blinded, placebo controlled, randomized trial was performed over two years with 60 women undergoing in-vitro fertilisation (IVF). Subjects were randomized, based on a computer-generated pseudo-random code to receive either DHEA or placebo with both capsules having similar colour, size and appearance. 60 women with poor OR based on antral follicle count or anti-Mullerian hormone thresholds undergoing IVF were recruited. They were randomised to receive DHEA 75mg/day or placebo for at-least 12 weeks before starting ovarian stimulation. They had long protocol using hMG 300 IU/day. Data analysed by “intention to treat”. Ovarian response, live birth rates and molecular markers of oocyte quality were compared between the study and control groups. ResultsThe recruitment rate was 39% (60/154). A total of 52 participants (27 versus 25 in the study and placebo groups) were included in the final analysis after excluding eight. While the mean (standard deviation) DHEA levels were similar at recruitment (9.4 (5) versus 7.5 (2.4) ng/ml; P=0.1), the DHEA levels at pre-stimulation were higher in the study group than in the controls (16.3 (5.8) versus 11.1 (4.5) ng/ml; P<0.01). The number (median, range) of oocytes retrieved (4, 0–18 versus 4, 0–15 respectively; P=0.54) and live birth rates (7/27, 26% versus 8/25, 32% respectively; RR (95% CI): 0.74 (0.22-2.48) and mRNA expression of developmental biomarkers in granulosa and cumulus cells were similar between the groups. ConclusionPre-treatment DHEA supplementation, albeit statistical power in this study is low, did not improve the response to controlled ovarian hyperstimulation or oocyte quality or live birth rates during IVF treatment with long protocol in women predicted to have poor OR.

Temporal expression of cumulus cell marker genes during in vitro maturation and oocyte developmental competence.

Cumulus cells (CC) play important roles in oocyte development and cumulus expressed genes can be used as markers for oocyte quality. Thisstudy aimed to investigate temporal changes in the expression of cumulus marker genes during oocyte maturation as possible biomarkers of embryo developmental competence in ovine. Gene expression was assessed in the CC of the BCB+ (developmentally competent) and BCB- (developmentally poor) oocytes at 0, 12, and 24h of in vitro maturation (IVM). Further, the association between the temporal cumulus gene expression and in vitro oocyte and embryo development was assessed. The maturation and blastocyst formation rates were found significantly greater for the BCB+ than the BCB- oocytes. At the 0h of IVM, a significant upregulation in the expression of PTGS2, STAR, SDC2, LHR, FGF2, BCL2, IL7RA, HSPA1A, and IFNT was observed in the CC of the poor (BCB-) as compared to the competent (BCB+) oocytes. In contrast, it was observed that as maturation progressed, the cumulus expression of most of the favorable genes was reduced and was found significantly downregulated at the completion of IVM in the poor as compared to the competent oocytes. The study revealed noticeable differences in the cumulus gene expression profile at different stages of IVM between ovine oocytes of differential developmental ability. The results indicated that the loss of cumulus gene expression along the maturation period in the poor oocytes was related to their intrinsic poor quality in the ovarian follicle.

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 significanceIntact 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.

OMICS-TECHNOLOGIES TO ANALYZE INDIVIDUAL BOVINE OOCYTES FOR IDENTIFICATION OF DEVELOPMENTAL OOCYTE COMPETENCE BIOMARKERS

Oocyte quality is a capacity to be fertilized and to develop into viable embryo and this is crucial for reproductive biotechnologies in farm animals. Technical progress and possible miniaturization of «omics» technologies made possible application of transcriptomic, proteomic and lipidomic methodologies to single oocyte, and thus to search molecular factors representing possible markers of oocyte quality. Oocyte quality is determined by its follicular environment and affects transcript, protein and lipid composition of an oocyte, which has to progress through maturation — a final step before fertilization, crucial for the acquisition of oocyte developmental competence. Here we describe the examples of «omics» analysis performed on single bovine oocytes and their neighboring cumulus cells through the comparison of the oocytes with different competence to maturate and to develop blastocyst in vitro. In particular, we focus to original technologies of proteomics and lipidomics based on mass spectrometry phenotyping of intact cells and identification of molecular biomarkers.

Regulation of endometrial cell proliferation by estrogen-induced BDNF signaling pathway

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin growth factors family. Recent studies indicated that the level of BDNF in follicular fluid is a marker for oocyte quality and infertility. Here, we intend to further investigate the function of BDNF and its signaling pathway in the regulation of endometrial cells proliferation. We found that BDNF is a critical growth factor in endometrial cells. Activation of signal transducer and activator of transcription 3 signaling pathway is required for BNDF-regulated endometrial cell proliferation. Furthermore, BDNF is an effector of estrogen in endometrial cells. Finally, we investigated the different role of Val66Met, a single-nucleotide polymorphism of the BDNF gene, in regulating endometrial cells proliferation. The results showed that BDNFV66M polymorphism is a loss-of-function polymorphism in the regulation of endometrial cells growth. Given the correlation between endometriosis and infertility, it is important to understand the role of BDNF in regulating endometrial cells proliferation and to develop new therapeutic strategies for the treatment of endometriosis-related infertility.

Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice.

The preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

Estradiol and luteinizing hormone concentrations in the follicular aspirate during ovum pickup as predictors of in vitro fertilization (IVF) outcome

Background: A relationship between ‘oocyte quality’ and follicular fluid hormones is expected, since its formation coincides with the ‘oocyte maturation’ phase. The aim of this study was to find a possible relation between oocyte quality with follicular luteinizing hormone (LH) and estradiol (E2) as hormonal parameters of oocyte quality during ovum pickup for intra-cytoplasmic sperm injection (ICSI). Methods: Concentrations of LH and E2 in individual follicular fluid samples obtained during assisted reproduction treatment were related to oocyte nuclear maturation, fertilization and embryo grading. E2 and LH differences between individual groups of oocytes and embryos were calculated using the paired Student’s t test and ANOVA test. Results: Follicular E2 levels showed a significant positive correlation with oocyte nuclear maturation, fertilization and embryo grading being higher in follicles whose oocytes had matured nucleus (475 ± 142.9 ng/ml vs. 332 ± 76.4 ng/ml, P value <0.001), normally fertilized (502.5 ± 131.3 ng/ml vs. 339.8 ± 78.3 ng/ml, P value <0.001) and developed into good quality embryos (596.9 ± 72.4 ng/ml grade A vs. 511.7 ± 73 ng/ml grade B vs. 310.9 ± 57 ng/ml grade C, P value <0.001). However Follicular LH was only positively correlated with oocyte nuclear maturation. Conclusions: The local follicular environment may play a key role in the observed differences in oocyte quality. Our results suggest that the use follicular E2 may be of value in the assessment of oocyte quality. If there is a marker for oocyte quality, it would be possible to select oocytes rather than embryos, which may improve selection criteria of the best embryo to transfer, therefore increases success rate of ICSI.

Cytokine antibody array profiling in human follicular fluid as a potential marker for oocyte quality

Cytokine antibody array profiling in human follicular fluid as a potential marker for oocyte quality

Impact of sperm DNA damage and oocyte-repairing capacity on trout development.

Zygotic repair of paternal DNA is essential during embryo development. In spite of the interest devoted to sperm DNA damage, its combined effect with defect-repairing oocytes has not been analyzed. Modification of the breeding season is a common practice in aquaculture. This practice reduces developmental success and could affect the both factors: sperm DNA integrity and oocyte repair capacity. To evaluate the maternal role, we analyzed the progeny outcome after fertilizing in-season trout oocytes with untreated and with UV-irradiated sperm. We also analyzed the offspring obtained out of season with untreated sperm. The analysis of the number of lesions in 4 sperm nuclear genes revealed an increase of 1.22-11.18 lesions/10 kb in out-of-season sperm, similar to that obtained after sperm UV irradiation (400 µW/cm(2)5 min). Gene expression showed in out-of-season oocytes the overexpression of repair genes (ogg1, ung, lig3, rad1) and downregulation of tp53, indicating an enhanced repairing activity and reduced capacity to arrest development upon damage. The analysis of the progeny in out-of-season embryos revealed a similar profile tolerant to DNA damage, leading to a much lower apoptotic activity at organogenesis, lower hatching rates and increased rate of malformations. The effects were milder in descendants from in-season-irradiated sperm, showing an enhanced repairing activity at epibolia. Results point out the importance of the repairing machinery provided by the oocyte and show how susceptible it is to environmental changes. Transcripts related to DNA damage signalization and repair could be used as markers of oocyte quality.

A microarray-based analysis of oocyte quality in the European clam Ruditapes decussatus

Abstract A microarray-based analysis was performed with the objective of describing genomic features of oocytes and looking for potential markers of oocyte quality in the economically important European clam, Ruditapes decussatus . Oocytes of 25 females from Ria de Aveiro, western coast of Portugal (40°42′N; 08°40′W) were selected for this study and oocyte quality was estimated by success of D-larval rate under controlled conditions, which appeared to vary from 0 to 95%. By genome-wide expression profiling with a DNA microarray, 526 probes appeared differentially expressed between two groups representing the largest and smallest value of D larval rates, named good (represented by a mean D-larval yield of 57 ± 22%) and poor (9 ± 5%) quality oocytes. Enrichment analysis showed “lysosome” (dre04142) as the single pathway represented in the enriched KEGG pathway terms, with 8 genes coding for putative cathepsins. Furthermore, differentially expressed genes involved in oocyte protection (DnaJ (Hsp40), Hsp70, cyclophilin B, PDI), maturation (Cam-PDE1C, PRDM9, G protein), sperm–egg interaction (PDI, G protein) and apoptosis (TNF) were also identified. From these, the apoptosis pathway was supposed to assume an important role in oocyte quality as the mRNA level of caspase 8 appeared also negatively correlated with the D-larval yield. Finally, a G-protein transcript was identified as more abundant in poor quality oocytes, which underlines the importance of release prophase I block and meiotic maturation in the oocytes of this species. This study provided new highly valuable information on genes specifically expressed by mature oocytes of R. decussatus , for further understanding of the mechanisms of early development in this species. Statement of relevance Understand the mechanisms of early development in the European clam R. decussatus .

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFβ-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence.

Does ovarian reserve predict egg quality in unstimulated therapeutic donor insemination cycles?

To compare reproductive outcomes of patients with very low, low, normal, and high antral follicle counts undergoing unstimulated therapeutic donor insemination (TDI) cycles. Retrospective cohort study. University-affiliated regional fertility clinic. Four hundred fifty-nine patients who had 1,107 TDI treatment cycles from January 2006 to December2013. Unstimulated therapeutic donor insemination. Clinical pregnancy rates and miscarriage rates as surrogate markers for oocyte quality. The overall pregnancy rate per cycle start was 12.46% in the study population. There was no difference in per-cycle or cumulative pregnancy rates among patients with very low, low, average, or high antral follicle counts within each patient age group of ≤35, 36-39, and ≥40years. The overall miscarriage rate per pregnancy was 13.61%. When stratified by patient age, there was no correlation between miscarriage rate and antral follicle count. AFC is not a predictor of pregnancy or miscarriage rates in patients undergoing unstimulated TDI.

Apoptotic Analysis of Cumulus Cells for the Selection of Competent Oocytes to Be Fertilized by Intracytoplasmic Sperm Injection (ICSI)

Oocyte quality is one of the main factors for the success of in vitro fertilization protocols. Apoptosis is known to affect oocyte quality and may impair subsequent embryonic development and implantation. The aim of this study was to investigate the apoptosis rate of single and pooled cumulus cells of cumulus cell–oocyte complexes (COCs), as markers of oocyte quality, prior to intracytoplasmatic sperm injection (ICSI).We investigated the apoptosis rate by TUNEL assay (DNA fragmentation) and caspase-3 immunoassay of single and pooled cumulus cells of COCs. The results showed that DNA fragmentation in cumulus cells was remarkably lower in patients who achieved a pregnancy than in those who did not. Cumulus cell apoptosis rate could be a marker for the selection of the best oocytes to be fertilized by intracytoplasmatic sperm injection.

307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (&lt;3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P &lt; 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P &lt; 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P &lt; 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P &lt; 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P &lt; 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.

Metabolic control of oocyte development: linking maternal nutrition and reproductive outcomes.

Obesity, diabetes, and related metabolic disorders are major health issues worldwide. As the epidemic of metabolic disorders continues, the associated medical co-morbidities, including the detrimental impact on reproduction, increase as well. Emerging evidence suggests that the effects of maternal nutrition on reproductive outcomes are likely to be mediated, at least in part, by oocyte metabolism. Well-balanced and timed energy metabolism is critical for optimal development of oocytes. To date, much of our understanding of oocyte metabolism comes from the effects of extrinsic nutrients on oocyte maturation. In contrast, intrinsic regulation of oocyte development by metabolic enzymes, intracellular mediators, and transport systems is less characterized. Specifically, decreased acid transport proteins levels, increased glucose/lipid content and elevated reactive oxygen species in oocytes have been implicated in meiotic defects, organelle dysfunction and epigenetic alteration. Therefore, metabolic disturbances in oocytes may contribute to the diminished reproductive potential experienced by women with metabolic disorders. In-depth research is needed to further explore the underlying mechanisms. This review also discusses several approaches for metabolic analysis. Metabolomic profiling of oocytes, the surrounding granulosa cells, and follicular fluid will uncover the metabolic networks regulating oocyte development, potentially leading to the identification of oocyte quality markers and prevention of reproductive disease and poor outcomes in offspring.