Markers Of Oocyte Quality Research Articles

AREG expression in cumulus cells as a prognostic marker of oocyte quality

Background. Assisted reproductive technologies (ART) represent the most promising and successful methods of infertility treatment. A personalized approach may enhance its efficacy. One such approach is the development of reliable methods for assessing the quality and selection of embryos for transfer. The quality of the embryo is largely contingent upon the quality of the gametes involved in fertilization; thus, the development of non-invasive methods to assess oocyte quality represents a crucial step in the advancement of personalized ART. It is proposed that molecular and biological characterization of cumulus cells can be utilized to assess oocyte quality and predict the success of implantation of transferred embryos. The aim of the study was to evaluate the expression levels of potential oocyte quality marker genes (AREG, STAR, PTGS2, HAS2 and SCD5) in cumulus cells from healthy donors and patients with primary and secondary infertility. Materials and Methods. Nine donors and 19 patients were enrolled in the study. RNA was isolated from cumulus cells obtained during oocyte preparation for fertilization, and cDNA was synthesized. The cDNA was used as a matrix for real-time PCR with primers for the above-mentioned genes of interest. Results. Significant difference in AREG gene expression was observed between patients with successful (i.e. ended with birth) outcome and with IVF failure. No difference was found for the STAR, HAS2, PTGS2 and SCD5 genes. Conclusion. The method of assessing the expression level of marker genes in cumulus cells by real-time PCR shows considerable promise for the assessment of oocyte quality. The AREG gene is a potential candidate for use as a marker of oocyte quality.

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium)

BackgroundOocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles.MethodsThe growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed.ResultsThe maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF.ConclusionsThis work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

Epigenetic-related transcriptional reprogramming elucidated by identification and validation of a novel reference gene combination for RT-qPCR studies in porcine oocytes of contrasting quality

BackgroundReliable RT-qPCR results are dependent on appropriate normalisation. Oocyte maturation studies can be challenging in this respect, as the stage of development can distinctively affect reference gene transcript abundance. The aim of this study was to validate the use of reference genes in oocyte in vitro maturation RT-qPCR studies, and thereafter, examine the abundance of transcripts supporting histone modification during oocyte and early embryo development in oocytes of contrasting quality.Methods and resultsTotal RNA from oocytes from prepubertal gilts and sows was extracted either directly succeeding follicle aspiration or after 44 h in vitro maturation, followed by RT-qPCR. The stability of YWHAG, HPRT1, ACTB, GAPDH, HMBS and PFKP, was analysed by NormFinder and further cross-validated by assessing results generated following application of different combinations of potential reference genes for normalisation of the RT-qPCR data. Combining ACTB and PFKP generated high stability according to NormFinder and concordant results. Applying this normalisation, gilt derived oocytes displayed significantly higher abundance than oocytes from sows of almost all the epigenetic-related transcripts studied (HDAC2, SIRT1, SALL4, KDM1A, KDM1B, KDM5A), both before and after maturation.ConclusionsThis study identified the combined use of ACTB and PFKP as the optimal normalisation for porcine oocyte RT-qPCR data. In oocytes collected from prepubertal gilts, transcription did not appear to be silenced at the time of aspiration, and accumulation of transcripts supporting histone modification facilitating proper fertilization and further embryo development seemed delayed. The results imply the epigenetic-related transcripts may have potential as markers of oocyte quality.

Role of miRNAs in glucose metabolism of mouse cumulus cells†.

It is known that the oocyte has a limited capacity to acquire and metabolize glucose, and it must rely on cumulus cells (CCs) to take up glucose and produce pyruvate for use to produce ATP through oxidative phosphorylation. We therefore propose that miRNAs might regulate glucose metabolism (GM) in CCs and might be used as markers for oocyte quality assessment. Here, mouse CC models with impaired glycolysis or pentose phosphate pathway (PPP) were established, and miRNAs targeting the key enzymes in glycolysis/PPP were predicted using the miRNA target prediction databases. Expression of the predicted miRNAs was compared between CCs with normal and impaired glycolysis/PPP to identify candidate miRNAs. Function of the candidate miRNAs was validated by transfecting CCs or cumulus-oocyte-complexes (COCs) with miRNA inhibitors and observing effects on glucose metabolites of CCs and on competence of oocytes. The results validated that miR-23b-3p, let-7b-5p, 34b-5p and 145a-5p inhibited glycolysis, and miR-24-3p, 3078-3p,183-5p and 7001-5p inhibited PPP of CCs. Our observation using a more physiologically relevant model (intact cultured COCs) further validated the four glycolysis-targeting miRNAs we identified. Furthermore, miR-let-7b-5p, 34b-5p and 145a-5p may also inhibit PPP, as they decreased the production of glucose-6-phosphate. In conclusion, miRNAs play critical roles in GM of CCs and may be used as markers for oocyte quality assessment. Summary sentence: We identified and validated eight new miRNAs that inhibit glycolysis and/or pentose phosphate pathways in cumulus cells (CCs) suggesting that miRNAs play critical roles in glucose metabolism of CCs and may be used for oocyte quality markers.

An update on Telomeres Dynamics in Reproduction; Specifically in Females &amp; Ageing: A Narrative Review

The oocyte portrays a postmitotic cell having a longer life working as locus of reproductive ageing in women. Female germ cells replication occurs in fetal life which age right through reproductive period. Modes behind oocyte ageing are oxidative injury, mitochondrial impairment and disturbed protein inclusive of cohesion. BobEdwards invented the “generation line” at the time of oogonial proliferation in mouse oogenesis, where the initial oocytes to leave the oogonial proliferation while fetal life are the ones which undergo ovulation first at puberty and the last oocytes to leave the oogonial proliferation are the last to undergo ovulation in adult. Hence Kalmbach K group posited ‘’second hit’’ hypothesis of reproductive ageing for incorporating to plethora of characteristics of the oocyte ageing. The 1st hit being oocyte continuing in the older women pass via more cell cycles while fetal oogenesis. The 2nd hit was oocytes which continued had&gt;accrual of environmental &amp; endogenous oxidative injury right through woman’s life. Telomeres (T’s) might modulate both parts of oocyte ageing. T’s offer a mitotic clock, with T debilitation becoming a granted inimical sequelae of cell division due to end replication cell botheration. The guanine enrichment of the telomeres makes them more vulnerable to oxidative injury, in post mitotic cell also. Since telomerase,/ the reverse transcriptase is more adept in sustenance rather than T lengthening. Furthermore telomerase continues to be inactive in maximum oogenesis &amp; early generation. Thus oocytes possess small T’s on the border of viability. To corroborate this posit mice having induced T debilitation and women having natural telomeropathies have diminished ovarian reserve (DOR), aberrant embryonic generation &amp; infertility. Conversely sperms form all through life of male by ALT, have telomerase active progenitor, spermatogonia, longest body T’s. Numerous queries arise i) does normal T debilitation of human oocytes aid in meiotic nondisjunction (MN)? ii) does recombination dependent T lengthening make embryos more prone to MN / mosaicism? iii) can certain features of T’s work as oocyte quality markers.

Markers of Oocyte Quality to Enhance Human IVF Outcomes: A Bibliographic Review

Markers of Oocyte Quality to Enhance Human IVF Outcomes: A Bibliographic Review

Chronological age enhances aging phenomena and protein nitration in oocyte.

The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging. Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration. At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition. Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.

Cumulus Cell and Oocyte Gene Expression in Prepubertal Gilts and Sows Identifies Cumulus Cells as a Prime Informative Parameter of Oocyte Quality.

Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus-oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.

PSIV-18 Combination of 1.8 Mm Choline and Follistatin Supplementation May Reduce Transcript Abundance of CEPT1 and CPBE4 During In-Vitro Bovine Oocyte Maturation

Abstract One-carbon metabolite supplementation during in vitro embryonic development improves blastocyst quality; however, our understanding of the effects of combining these metabolites during in vitro maturation (IVM) is limited. Supplementation with follistatin improved oocyte quality following IVM. Choline supplementation was beneficial during early embryonic development; however, the role of choline during oocyte maturation is unknown. The objective of this study was to investigate the combination of choline and follistatin during IVM on oocyte quality. We hypothesized that supplementation of choline with follistatin would synergistically improve oocyte quality. Follicles (2 to 7mm) were aspirated from slaughterhouse ovaries to obtain cumulus oocyte complexes for IVM that were treated with choline (0, 1.3, or 1.8 mM) and follistatin (0 or 10 ng/mL) in a 3 x 2 design for 23 hr and visually graded for maturation. Cumulus cells and oocytes were used for qPCR transcript abundance analysis (cumulus cells: CHPT1, CX43, EFGR1, FST and STAR; oocytes: BMP15, GDF9, CEPT1, CPBE4, MTHFR, NANOS, CX43 and COX2). Oocyte maturation was analyzed using the GLIMMIX procedure of SAS and transcript abundance was analyzed using the GLM procedure of SAS with the main effect of choline, follistatin and the interaction as fixed effects. There were no differences in the visual characteristics of the oocytes during maturation (P &amp;gt; 0.1). No differences in transcript abundance were observed with choline or follistatin in the cumulus cells (P &amp;gt; 0.1). Addition of 1.3 mM choline during IVM may increase CPEB4 (P = 0.1) in oocytes compared with controls. Transcript abundance of CEPT1 tended to be reduced in oocytes supplemented with 1.8 mM choline and follistatin compared with control oocytes (P = 0.07). Incorporation of follistatin with 1.8 mM choline supplementation during IVM tended (P = 0.08) to reduce CPEB4 in oocytes compared with controls. Other markers of oocyte quality (BMP15, GDF9, NANOS, CX43 and COX2) were not different between choline or follistatin. In contrast to the hypothesis, the combination of choline and follistatin appeared to reduce overall in vitro oocyte quality. While no statistical differences were observed for the other markers, it appears that either 1.3 mM choline or follistatin supplementation may provide a beneficial impact on oocyte quality. Choline 1.3 mM supplementation may improve cell cycle progression from MI to MII phase through the up-regulation of CBEP4. This further supports the conclusion that choline is beneficial for in vitro oocyte development. Although follistatin appears to antagonize the actions of choline during oocyte maturation, they target different mechanisms (TGFβ signaling and DNA methylation, respectively) and both provide long-term benefits during in-vitro embryonic development. Further research is, therefore, warranted to investigate the antagonizing relationship between choline and follistatin during IVM and long-term impacts on blastocyst development. USDA is an equal opportunity provider and employer.

P-687 Serum Anti-Müllerian hormone to antral follicle count ratio does not predict euploidy rate in young patients who had at least 1 zygote after fertilization

Abstract Study question Does serum Anti-Müllerian hormone to antral follicle count ratio predict euploidy rate in young patients who had at least 1 zygote after fertilization? Summary answer Serum Anti-Müllerian hormone to antral follicle count ratio (AMH/AFC) does not predict euploidy rate in young patients who had at least 1 zygote after fertilization. What is known already AMH is produced by the granulosa cells of the growing follicles. It has been demonstrated that the antral follicles are the main contributors to its serum values. AMH´s role as a quantitative predictor of the ovarian response to stimulation is undeniable. However, studies looking at its correlation with oocyte quality, assessed as embryo ploidy, have yielded conflicting results. All of them include only patients who had at least one blastocyst for biopsy obviating those who had all embryos arrested, which might be genetically abnormal. Besides, no study before has considered the per-follicle production of AMH when evaluating serum AMH levels. Study design, size, duration Retrospective analysis of women undergoing Preimplantation Genetic Testing for Aneuploidy at the blastocyst stage according to standard clinical procedures in a tertiary referral IVF center from April 2017 to August 2022. As linear regression excluded an effect of age on euploidy rate until 35 years, 570 patients below this age were included. AMH/AFC ratio was calculated as a marker of per-follicle AMH production. Patients were classified into quartiles. Euploidy rates/zygote were compared among them. Participants/materials, setting, methods Patients who had at least 1 zygote were included. AMH was measured by Elecsys within 6 months before start of stimulation. Scans were performed by 3 experienced sonographers. Women with an AMH &amp;gt; 5,98 ng/ml, endometriosis, autoimmune disease, non-functional ovarian cyst, hormonal treatment prior to AMH measurement or history of ovarian surgery, men with less than 1 million of sperm/milliliter of ejaculate, and couples with an abnormal karyotype or history or gonadotoxic treatment were excluded. Main results and the role of chance Included patients had a median (+ interquartile range) AMH of 2.73 ng/ml (1.68-3.77), median BMI of 25.7 kg/m2 (22.8-28.9) and median antral follicle count of 14 (11-19). Median number of oocytes collected was 14 (10-20) and median number of metaphase II oocytes was 11 (7-15). Thresholds for p25th and p75th AMH/AFC ratios were 0.13 and 0.24 respectively. The number of patients included in the &amp;lt;p25th, p25th-p75th and &amp;gt;p75th groups were 134, 297 and 139, respectively. Whereas median age and BMI wereńt statistically different among groups, significant differences were found in median AMH (&amp;lt;p25th: 1.38 ng/ml (0.8-2.1); p25th-p75th: 2.75 ng/ml (2-3.7); &amp;gt;p75th: 3.9 ng/ml (3.1-4.8), p &amp;lt; 0.001) and AFC (&amp;lt;p25th: 14 (9.2-20) p25th-p75th: 16 (12-20); &amp;gt;p75th: 13 (10-16), p &amp;lt; 0.001). No significant differences were found in the fertilization rate (&amp;lt;p25th: 0.75 (0.6-0.8); p25th-p75th: 0.75 (0.6-0.9); &amp;gt;p75th: 0.78 (0.7-0.9)) nor in the biopsy rate/zygote (&amp;lt;p25th: 0.64 (0.5-0.8), p25th-p75th: 0.6 (0.4-0.8), &amp;gt;p75th: 0.6 (0.5-0.7)). Euploidy rate/zygote wasńt statistically different among groups: &amp;lt;p25th: 0.36 (0.2-0.5); p25th-p75th: 0.33 (0.2-0.5); &amp;gt;p75th: 0.33 (0.2-0.5). When compared with p25th-p75th, patients with an AMH/AFC &amp;lt;p25th showed a tendency towards a higher risk of not having an euploid embryo, due to culture arrest or aneuploidy (19.4% vs 10.8%, p 0.065). Limitations, reasons for caution The retrospective design of the study might restrict an adequate control of confounding factors. The small size of the lower quartile group might compromise the accuracy of the findings. Wider implications of the findings The association between AMH levels and live birth rate after In Vitro Fertilization remains debated. Whereas this study didńt find a correlation between AMH/AFC ratio and euploidy rate/zygote, further research will have to evaluate other markers of oocyte quality that might by affected by a low per-follicle production of AMH. Trial registration number Not applicable

P-268 Artificial intelligence (AI) image analysis outperforms patient age as a surrogate marker for oocyte quality, demonstrating an increased accuracy in predicting blastocyst development

Abstract Study question Can an AI image analysis tool (VIOLET) provide better predictions of oocyte potential than the current standard, patient age, as it relates to blastocyst development? Summary answer While increasing patient age slightly correlates with decreases in blastocyst development, VIOLET provides personalized assessments of individual oocytes correlated to blastocyst development with improved accuracy. What is known already Increasing patient age correlates to decreases in oocyte competency, leading to greater challenges in successful fertility treatment. Clinically, patient age is used to estimate probabilities of success based on national databases. Such as the case in oocyte cryopreservation cycles, considering only age and number of mature oocytes vitrified. However, oocyte quality may vary widely between patients of the same age, and even within each cohort of oocytes. VIOLET is an AI tool that assesses images of mature denuded oocytes to provide an analysis shown to significantly correlate with subsequent blastocyst development and quality, consistently outperforming embryologists in this task. Study design, size, duration This large-scale retrospective study assessed 9,120 mature denuded oocytes retrieved during 2014-2022, representing 1,384 patients between ages 19-49, attending 7 fertility clinics across 5 countries. The VIOLET prediction model is based on image analysis of mature denuded oocytes, without incorporating clinical variables, such as age. Patient age was used to build a separate predictive model of blastocyst development (10,947 training and 3,750 validation samples) to assess the predictive value of age in comparison to VIOLET. Participants/materials, setting, methods Blastocyst development was determined by embryos achieving a Gardner grade by Day5/6 post-ICSI. Blastocyst rates per oocyte cohort were calculated by number of blastocysts divided by total number of mature oocytes retrieved. Various machine learning techniques were trialed to build the predictive model strictly using age; with Random Forest model providing the best-balanced performance. VIOLET and the age model assessed the images and age of 9,120 mature denuded oocytes, respectively, providing predictions of blastocyst development. Main results and the role of chance Blastocyst development was significantly different between patients &amp;lt;35 years old compared to those ≥35 years old [47% vs 42%; p &amp;lt; 0.05 by Two Proportion Z-test]; however, not when the older age group was stratified further. On a patient level, the cohort of oocytes was evaluated by the blastocyst development ratio. Among four age groups (&amp;lt;35, 35-37, 37-40, &amp;gt;40), the blastocyst development ratios per cohort were very similar, with overlapping distributions of 84-94% using Kernel Density estimates. Thus, patient age group does not provide enough information to explain blastocyst development success for an individual oocyte or within an oocyte cohort. In comparison, VIOLET probability is significantly correlated to blastocyst development (p &amp;lt; 0.05; Welch’s Two sample t-test). And blastocyst development rates display a stepwise positive correlation that is significantly different between VIOLET probability quartiles [24% vs 39% vs 47% vs 53%; p &amp;lt; 0.05 by Two Proportion Z-test], providing meaningful information on individual oocytes. Additionally, the predictive model built with age as the only feature had poor ability to predict blastocyst success of an individual oocyte with a limited area-under-the-curve (AUC) of 0.5—unable to separate positive and negative classes. This was outperformed by VIOLET, which displayed an AUC of 0.62 on the same unseen dataset. Limitations, reasons for caution Increasing maternal age causes increased chromosomal abnormalities in oocytes, which translates to lower efficacy with treatment outcomes; therefore, further research to assess VIOLET correlation with PGT-A and implantation outcomes is needed and underway. Wider implications of the findings Patient age correlates with blastocyst development on a general level; however, it does not provide meaningful insights to distinguish rates of blastocyst success on an individual oocyte or oocyte cohort level. VIOLET augments oocyte understanding over the current standard of care, which can be utilized to support personalized clinical decision-making. Trial registration number Not applicable.

Follicular homocysteine as a marker of oocyte quality in PCOS and the role of micronutrients

PurposeDoes follicular homocysteine predict the reproductive potential of oocytes following FSH stimulation in PCOS women? Can it be modulated by dietary interventions?MethodsThis was a prospective, randomized, interventional clinical study. Forty-eight PCOS women undergoing in vitro fertilization at a private fertility clinic were randomized for a dietary supplementation providing micronutrients involved in homocysteine clearance or no treatment. The supplement was assumed 2 months before stimulation until pick-up day. Monofollicular fluids were collected and frozen. After embryo transfer, the fluids from the follicles generating the transferred embryos were thawed and analyzed.ResultsFollicular homocysteine showed a negative correlation with clinical pregnancy both in the whole population (r = − 0.298; p = 0.041) and in controls (r = − 0.447, p = 0.053). The support achieved a non-significantly lower concentration of follicular homocysteine (median [IQR]–7.6 [13.2] vs 24.3 [22.9]). Supplemented patients required far less FSH for stimulation (1650 [325] vs 2250 [337], p = 0.00002) with no differences in the number of oocytes collected, MII rate, and fertilization rate. Supplemented patients enjoyed higher blastocyst rate (55% [20.5] vs 32% [16.5]; p = 0.0009) and a trend for improved implantation rate (64% vs 32%; p = 0.0606). Clinical pregnancy rates were 58% vs 33% in controls (p = ns).ConclusionFollicular homocysteine is a suitable reporter that might be investigated as a tool for oocyte-embryo selection. A diet enriched with methyl donors may be useful in PCOS and supplements may also help. These findings may be also true for non-PCOS women, which warrants investigation.The study was approved by the Acibadem University Research Ethics Committee (2017–3-42). Clinical trial retrospective registration number ISRCTN55983518.

Ovarian inflammation mediated by Toll-like receptor 4 increased transcripts of maternal effect genes and decreased embryo development†.

Obese women are subfertile and have reduced assisted reproduction success, which may be due to reduced oocyte competence. We hypothesize that consumption of a high-fat/high-sugar diet induces ovarian inflammation, which is a primary contributor to decreased oocyte quality and pre-implantation embryo development. To test this hypothesis, C57BL/6 (B6) mice with a normal inflammatory response and C3H/HeJ (C3H) mice with a dampened inflammatory response due to dysfunctional Toll-like receptor 4 were fed either normal chow or high-fat/high-sugar diet. In both B6 and C3H females, high-fat/high-sugar diet induced excessive adiposity and hyperglycemia compared to normal chow-fed counterparts. Conversely, ovarian CD68 levels and oocyte expression of oxidative stress markers were increased when collected from B6 high-fat/high-sugar but not C3H high-fat/high-sugar mice. Following in vitro fertilization of in vivo matured oocytes, blastocyst development was decreased in B6-high-fat/high-sugar but not C3H high-fat/high-sugar mice. Expression of cumulus cell markers of oocyte quality were altered in both B6 high-fat/high-sugar and C3H high-fat/high-sugar. However, there were no diet-dependent differences in spindle abnormalities in either B6 or C3H mice, suggesting potential defects in cytoplasmic maturation. Indeed, there were significant increases in the abundance of maternal effect gene mRNAs in oocytes from only B6 high-fat/high-sugar mice. These differentially expressed genes encode proteins of the subcortical maternal complex and associated with mRNA metabolism and epigenetic modifications. These genes regulate maternal mRNA degradation at oocyte maturation, mRNA clearance at the zygotic genome activation, and methylation of imprinted genes suggesting a mechanism by which inflammation induced oxidative stress impairs embryo development.

Kuantifikasi Protein HAS2, PTX3, BCL2 dan BAX dalam Sel Kumulus Oosit Matur (MII) dan Imatur (MI) Pada Pasien Bayi Tabung

Oocyte developmental competence is one of the determining factors that in?uence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation and be fertilized. A non-invasive approach using proteomic analysis of oocyte cumulus cells (CCs) can be used as a method to predict oocyte competence and viability that could potentially function as molecular predictors for IVF program prognosis. Our study was aimed at quantifying protein markers (HAS2, PTX3, BCL2 and BAX) that are expressed in human CCs between mature (MII) and immature (MI) oocytes. This research is an analytic observational study. Twelve samples of CCs from mature (MII) and immature (MI) oocytes were collected after ovum pick up from patients undergoing ICSI. CCs samples were isolated using an ultrasonicator and protein expressions were quantified using ELISA method. The Mann-Whitney test was used to compare the protein expressions from CCs between mature and immature oocytes. The results showed HAS2, PTX3 and BCL2 proteins expressions was increase in CCs from mature oocytes but had no significant effect compared to CCs from immature oocytes, however, BAX protein expression was significantly higher (P&lt;0.05) in CCs from immature oocytes which CCs from mature oocytes. This present study shows that BAX proteins could be used as markers for oocyte quality and maturation.&#x0D; Key words: ELISA, maturation, proteins marker, cumulus cells (CCs)

Higher quality rather than superior quantity of oocytes determine the amount of fertilizable oocytes in two outbred Dummerstorf high‐fertility mouse lines

Dummerstorf fertility lines FL1 and FL2 represent two models of enhanced fertility characterized by the doubling of the litter size compared with an unselected control population (ctrl line, Dummerstorf FztDU). Both biodiverse FLs managed to reach this goal by increasing the ovulation rate per cycle, even showing decreased pregnancy rate and irregular oestrous cycle and metabolic hormone levels, compared with ctrl. The aim of the present study was to analyse oocytes in terms of quality and quantity by comparing the entire pool of oocytes per ovary, with those from the antral follicles within the same animal. We performed Brilliant Cresyl Blue staining as a non-invasive marker of oocyte quality in combination with an analysis of additional morphological indicators, e.g. cytoplasm clarity, cumulus cell layers, nuclear anatomy, size and shape. We compared our fertility lines with the unselected control population and with another independent line selected from the same founder population, showing lower litter size (DU6P). Our results suggest that fertility lines show decreased number of oocytes per ovary compared with DU6P but increased number of high-quality oocytes before ovulation. Hence, the raise in the ovulation rate and litter size of those super fertile mouse lines are not associated with an increased number of oocytes per ovary but rather with an increased number of higher quality fertilizable oocytes per cycle. In addition, the most conspicuous method to acquire oocytes with the highest quality in our lines is to assess their morphology, rather than their status after staining. All these discoveries together may be of fundamental importance for further studies in livestock farm animals showing some similar characteristics, e.g. irregular cycle or hormonal misbalances, to improve production while lowering costs, and in humans to increase the possibilities of successful pregnancies for couples undergoing in vitro fertilization (IVF).

P-238 Treatment options to overcome impaired fertilization and embryonic development caused by an oocyte-related deficiency in the Patl2-/- mouse model

Abstract Study question Can assisted oocyte activation (AOA) or spindle transfer (ST) overcome the lower fertilization and blastocyst rates observed in the Patl2-/- mouse model? Summary answer ST, but not AOA, has the potential to restore both normal fertilization and blastocyst rates in a mouse model with an oocyte-related deficiency. What is known already PATL2 is an RNA-transcriptional repressor involved in the regulation of maternal mRNAs during oocyte maturation. Mutations in PATL2 have been detected in patients with oocyte maturation arrest, reduced fertilization and embryo developmental arrest. Consequently, these patients are mostly referred to oocyte donation. Both AOA, inducing artificially calcium oscillations by calcium ionophores, and ST, replacing bad-quality oocyte cytoplasm with healthy cytoplasm, have been proposed to treat oocyte-related infertility. However, there is a lack of studies showing which oocyte-related infertility indications may benefit from which specific treatment. The Patl2-/- mouse model offers a unique opportunity to study the efficiency of these treatments. Study design, size, duration An experimental study using C57BL/6NTac-Patl2tm1a mice was conducted between April to December 2021. Breeding of heterozygous C57BL/6NTac-Patl2tm1a mice resulted in a total of 156 pups, from which 22 were homozygous C57BL/6NTac-Patl2tm1a ( Patl2-/-) females. Metaphase II (MII) oocytes were collected from Patl2-/- (test group) and Patl2+/+ (control group) females and used to evaluate different oocyte quality markers, as well as the efficiency of AOA and ST treatments to improve the oocyte-related subfertility observed in Patl2-/- mice. Participants/materials, setting, methods Four- to 12-week-old mice were subjected to ovarian hyperstimulation and oocyte collection, where oocyte maturation was assessed. Afterwards, MII oocytes were used to evaluate oocyte diameter, spindle abnormalities, activation rate (AR) and blastocyst rate (BR) after PIEZO-ICSI and calcium releasing capacity after SrCl2 exposure. Finally, Patl2-/- MII oocytes were treated with AOA, by direct exposure to SrCl2, or ST treatment, by transferring the Patl2-/- spindle to Patl2+/+ cytoplasm (previously enucleated) followed by SrCl2 exposure. Main results and the role of chance No difference in the number of cumulus-oocyte complexes (COCs) and mature MII oocytes was observed between 6 Patl2-/- mice (128 MII/159 COCs) and 5 Patl2+/+ mice (108 MII/132 COCs). However, Patl2-/- MII oocytes showed a significantly lower cytoplasm diameter (68,11 ± 2,25, n = 79) than Patl2+/+ oocytes (75,19 ± 2,09, n = 77, p-value&amp;lt;0,001), suggesting that Patl2 affects oocyte growth but does not seem to affect oocyte maturation. Nuclear maturity was not compromised either, as a similar percentage of oocytes with normal spindle was observed in Patl2-/- (48,4%, 15/31) and Patl2+/+ MII oocytes (50%, 18/36). Activation and blastocyst rates after PIEZO-ICSI were lower in Patl2-/- (AR = 45,5%, 5/11 and BR = 20,0%, 1/5) than in Patl2+/+(AR = 75,0%, 21/28 and BR = 52,4%, 11/21). In addition, total calcium released during oocyte activation after SrCl2 exposure was not different between Patl2-/- (AxF=6,88 AU, n = 30) and Patl2+/+ MII oocytes (AxF=5,04 AU, n = 28, p-value=0,087). In line with these results, AOA treatment did not improve activation and blastocyst rates in Patl2-/- oocytes (AR = 66,7%, 44/66 and BR = 45,5%, 20/44) compared to Patl2+/+ oocytes (AR = 96,5%, 110/114 and BR = 79,1%, 87/110, p-value&amp;lt;0,001). Nonetheless, when performing ST using Patl2-/- as spindle donors and Patl2+/+ as cytoplasm recipients, activation (8/8, 100,0%) and blastocyst rate (6/8, 75,0%) were restored to normal values. Limitations, reasons for caution This study describes preliminary results and is limited by its small sample size. In addition, we did not identify an increased number of immature oocytes and spindle abnormalities in Patl2-/- female mice in contrast to previously published reports. Wider implications of the findings ST could be offered to treat PATL2- related female-infertility caused by a cytoplasmic deficiency, to increase both fertilization and blastocyst rates. AOA would not be beneficial for these patients as calcium releasing capacity of Patl2-/- oocytes does not seem to be affected, suggesting a deficiency independent from calcium releasing machinery. Trial registration number Not applicable

Glycine and Melatonin Improve Preimplantation Development of Porcine Oocytes Vitrified at the Germinal Vesicle Stage

Lipid-rich porcine oocytes are extremely sensitive to cryopreservation compared to other low-lipid oocytes. Vitrification has outperformed slowing freezing in oocyte cryopreservation and is expected to improve further by minimizing cellular osmotic and/or oxidative stresses. In this study, we compared the effects of loading porcine cumulus-oocyte complexes with glycine (an organic osmolyte) or glycine plus melatonin (an endogenous antioxidant) during vitrification, thawing and subsequent maturation to mitigate osmotic injuries or osmotic and oxidative damages on the developmental potential of porcine oocytes. Our data demonstrated that glycine treatment significantly increased the vitrification efficiency of porcine oocytes to levels comparable to those observed with glycine plus melatonin treatment. It was manifested as the thawed oocyte viability, oocyte nuclear maturation, contents of reactive oxygen species, translocation of cortical granules and apoptotic occurrence in mature oocytes, levels of ATP and transcripts of glycolytic genes in cumulus cells (markers of oocyte quality), oocyte fertilization and blastocyst development. However, the latter was more likely than the former to increase ATP contents and normal mitochondrial distribution in mature oocytes. Taken together, our results suggest that mitigating osmotic and oxidative stresses induced by vitrification and thawing can further enhance the developmental competency of vitrified porcine oocytes at the germinal vesicle stage.

Age-Dependent Metabolomic Profile of the Follicular Fluids From Women Undergoing Assisted Reproductive Technology Treatment.

Female fertility declines with age, and this natural variation culminates in reproductive senescence. Human follicular fluids are rich in low-molecular weight metabolites which are responsible for the maturation of oocytes. The metabolomic approaches are powerful tools to study biochemical markers of oocyte quality in the follicular fluids. It is necessary to identify and quantify the reliable metabolites in follicular fluids reflecting oocyte developmental potential. The goal of this study is to conduct a metabolomic analysis of the follicular fluids in women of different ages and study the metabolomic profile of the follicular fluids in relationship with oocyte quality in assisted reproductive technology (ART) treatment. A total of 30 women seeking for ART treatment at the Women’s Hospital, Zhejiang University School of Medicine from October 2014 to April 2015 were recruited for the present study. Fifteen women aged from 39 to 47 were grouped as advanced maternal age, and the other 15 women aged from 27 to 34, as young controls. Ovarian stimulation and oocyte retrieval were conducted using a regular protocol involving mid-luteal pituitary down-regulation and controlled ovarian stimulation. Follicular fluids from mature follicles were collected and centrifuged for analyses. Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectroscopy (GC-MS) were used to perform the quantitative metabolomic analysis. The follicular fluid levels of 311 metabolites and the metabolic significance were assessed. 70 metabolites showed significant differences between women with young and advanced ages. Follicular fluids from women with advanced age showed significantly higher levels of creatine, histidine, methionine, trans-4-hydroxyproline, choline, mevalonate, N2,N2-dimethylguanosine and gamma-glutamylvaline, as compared to those from the young age group. 8 metabolites were found significantly correlated with maternal age positively. Moreover, 3 metabolites were correlated with the number of oocytes retrieved, and 5 metabolites were correlated with cleaved embryo numbers, both negatively. The follicular fluids from women undergoing ART treatment exhibited age-dependent metabolomic profile. Metabolites associated with oocyte quality were identified, suggesting them as potential biomarkers for oocyte maturation and ART outcomes.

Protein source in maturation media affects gene expression in cumulus cells and embryo development in cattle

We aimed to evaluate if protein source (PS) alterations during IVM affect embryo sex/development and gene expression profile in cumulus cells (CCs). Bovine oocytes were matured and cultured in the presence of FBS or BSA. Then, the PS effect during IVM on gene expression (GPC4, VCAN, GHR, PTGS2, and ALCAM) was determined. CC biopsy was removed before and after IVM treatments. After fertilization and cultured, CCs were grouped according to their fate into CCs from immature COCs, CCs from COCs that did or did not result in embryos (according to PS). Results showed that when the culture was performed in FBS presence, blastocyst rate was higher (p < 0.05) than BSA. However, when embryos were cultured with BSA, no effect (p > 0.05) of PS during IVM was observed. PS used during IVM did not affect embryos sex (p > 0.05) but changed VCAN, GHR, PTGS2, and ALCAM genes expression. No differences (p > 0.05) were observed between immature and mature CCs groups in gene expression, regardless of their fate. Only the GHR gene was related to embryo production but just with FBS on IVM. In conclusion, PS can affect embryo development when using the serum on IVM and IVC, influences CCs gene expression, and has to be considered when studying oocyte quality markers.

The microenvironment of ovarian follicles in fertile dairy cows is associated with high oocyte quality

We hypothesised that heifers and cows with positive genetic merit for fertility would have a follicular microenvironment that resulted in better quality oocytes. To test this, we compared cumulus cell-oocyte complexes (COC) and follicular fluid from preovulatory follicles of 36 Holstein-Friesian nulliparous heifers and 50 primiparous lactating cows with either positive (POS, +5%) or negative (NEG, −5%) fertility breeding values (FertBV). Established gene markers of oocyte quality were measured in individual cumulus cell masses and oocytes, and concentrations of amino acids, steroids, and metabolites were quantified in corresponding follicular fluid and plasma. The timing of visually detectable oestrus in NEG FertBV heifers was inconsistent with their stage of COC maturation. Retrospective analyses of oestrous activity data indicated that NEG FertBV heifers were sampled earlier. Their recovered COC were morphologically less mature and exhibited differential expression of genes that are associated with follicular maturation (lower levels of BMPR2) and protein processing (higher levels of HSP90B1). Despite consistent sampling times being achieved in the lactating cows, lower concentrations of serine, proline, methionine, isoleucine, and non-esterified fatty acids were present in follicular fluid from POS FertBV cows. This was associated with higher expression of gene biomarkers of good oocyte quality (VCAN, PDE8A) in COC recovered from POS FertBV cows. This study supports our hypothesis that the follicular microenvironment in lactating dairy cows with high genetic merit leads to COC with higher metabolic rates and oocytes of superior quality. Moreover, an additional stressor such as lactation is required for this difference to be pronounced.