Design and optimization of an experimental maintenance system for yellow clam broodstock Amarilladesma mactroides (Reeve, 1854)

The yellow clam is a sand-burrowing bivalve that inhabits the dissipative beaches from southern Brazil to the north coast of Argentina. In the last decades, populations of this species have been impacted by mass mortality events, overfishing and other anthropogenic activities. The production of juveniles in captivity would allow feasibility studies to be carried out to restore the natural stock as well as the production in aquaculture systems. Given the scarcity of studies on the maintenance of this species in captivity, a culture system and a management protocol were developed and tested. Wild-caught clams (total length ≥50 mm) were used in a series of 14 day-long trials. Survival was higher in clams that were allowed to bury into the sand. A permanent ink marker covered with a thin layer of a quick-hardening adhesive proved to be a reliable method to tag clams. The maintenance of yellow clams in this system resulted in high survival and growth, increases in the condition factor and oocyte diameter, and a relative advancement of gonadal development.

Evidences for Red Pigment Concentrating Hormone (RPCH) and Beta-Pigment Dispersing Hormone (β-PDH) Inducing Oocyte Meiotic Maturation in the Chinese Mitten Crab, Eriocheir sinensis

Red pigment concentrating hormone (RPCH) and pigment dispersing hormone (PDH) are crustacean neuropeptides involved in broad physiological processes including body color changes, circadian rhythm, and ovarian growth. In this study, the full-length cDNA of RPCH and PDH were identified from the brain of the Chinese mitten crab Eriocheir sinensis. The deduced RPCH and PDH mature peptides shared identical sequence to the adipokinetic hormone/RPCH peptides family and the β-PDH isoforms and were designated as Es-RPCH and Es-β-PDH, respectively. Es-RPCH and Es-β-PDH transcripts were distributed in the brain and eyestalks. The positive signals of Es-RPCH and Es-β-PDH were localized in the neuronal clusters 6, 8, 9, 10, and 17 of the brain as revealed by in situ hybridization. The expression level of Es-RPCH and Es-β-PDH mRNA in nervous tissues were all significantly increased at vitellogenic stage, and then decreased at the final meiotic maturation stage. The administrated with synthesized Es-RPCH peptide results in germinal vesicles shift toward the plasma membrane in vitellogenic oocyte, and significant decrease of the gonad-somatic index (GSI) and mean oocyte diameter as well as the expression of vitellogenin mRNA at 30 days post injection in vivo. Similar results were also found when injection of the Es-β-PDH peptide. In vitro culture demonstrated that Es-RPCH and Es-β-PDH induced germinal vesicle breakdown of the late vitellogenic oocytes. Comparative ovarian transcriptome analysis indicated that some reproduction/meiosis-related genes such as cdc2 kinase, cyclin B, 5-HT-R and retinoid-X receptor were significantly upregulated in response to Es-RPCH and Es-β-PDH treatments. Taken together, these results provided the evidence for the inductive effect of Es-RPCH and Es-β-PDH on the oocyte meiotic maturation in E. sinensis.

The proteome, not the transcriptome, predicts that oocyte superovulation affects embryonic phenotypes in mice

Superovulation is the epitome for generating oocytes for molecular embryology in mice, and it is used to model medically assisted reproduction in humans. However, whether a superovulated oocyte is normal, is an open question. This study establishes for the first time that superovulation is associated with proteome changes that affect phenotypic traits in mice, whereas the transcriptome is far less predictive. The proteins that were differentially expressed in superovulated mouse oocytes and embryos compared to their naturally ovulated counterparts were enriched in ontology terms describing abnormal mammalian phenotypes: a thinner zona pellucida, a smaller oocyte diameter, increased frequency of cleavage arrest, and defective blastocyst formation, which could all be verified functionally. Moreover, our findings indicate that embryos with such abnormalities are negatively selected during preimplantation, and ascribe these abnormalities to incomplete ovarian maturation during the time of the conventional superovulation, since they could be corrected upon postponement of the ovulatory stimulus by 24 h. Our data place constraints on the common view that superovulated oocytes are suitable for drawing general conclusions about developmental processes, and underscore the importance of including the proteins in a modern molecular definition of oocyte quality.

Characteristics of the cytoplasmic halo during fertilisation correlate with the live birth rate after fresh cleaved embryo transfer on day 2 in minimal ovarian stimulation cycles: a retrospective observational study

Background Information regarding the influence of cytoplasmic events during fertilisation on the clinical outcome remains limited. The cytoplasmic halo is one of these events. A previous study that used time-lapse technology found an association of the presence and morphokinetics of the cytoplasmic halo with cleavage patterns, development to the blastocyst stage, and the ongoing pregnancy rate after blastocyst transfer. Therefore, the cytoplasmic halo may be a useful predictor of the pregnancy outcome after cleaved embryo transfer. This study evaluated the ability of the cytoplasmic halo to predict a live birth after fresh cleaved embryo transfer on day 2, and sought to identify factors potentially influencing the presence and morphokinetics of the halo. Methods A total of 902 embryos cultured in the EmbryoScope+® time-lapse system and subjected to single fresh cleaved embryo transfer were retrospectively analysed. The presence and duration of a cytoplasmic halo were annotated. The initial positions of the pronuclei were also observed. The correlation between the cytoplasmic halo and live birth was evaluated and the association of the cytoplasmic halo with patient, cycle, and embryonic characteristics was determined. Results Absence of a cytoplasmic halo was associated with a significant decrease in the likelihood of a live birth after fresh cleaved embryo transfer. Prolongation of the halo, especially the duration of central repositioning of cytoplasmic granules, had an adverse impact on the live birth rate. The characteristics of the cytoplasmic halo were not affected by the ovarian stimulation method used, female age, the serum steroid hormone level on the day of trigger, or semen quality. However, the cytoplasmic halo was significantly affected by male age, oocyte diameter, and the initial position of the male pronucleus. Conclusions Absence or prolongation of the cytoplasmic halo was negatively correlated with the live birth rate after fresh cleaved embryo transfer. The characteristics of the cytoplasmic halo were strongly associated with oocyte diameter, male age, and the initial position of the male pronucleus. These findings indicate that the characteristics of the cytoplasmic halo can be used to select more competent embryos for transfer at the cleavage stage.

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

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.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility. Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10 mg/kg/day/body weight through their drinking water continuously for 4 weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

Evaluation of the reproductive parameters of female neotropical migratory fish from a lotic and lentic environment of a dammed river

The fragmentation of watercourses caused by dams is considered to be one of the main threats to aquatic biodiversity worldwide, especially for ichthyofauna. Several studies have shown that the environmental modifications caused by dammed water bodies can change the various reproductive parameters of freshwater fish. Therefore, the present study aims to comparatively analyze the reproductive potential of female Megaleporinus reinhardti, a migratory species, sampled in the lentic environment of the Três Marias Reservoir and the lotic environment of the São Francisco River. Biometric data were obtained from 79 females and the biological indices were subsequently calculated. Additionally, the microscopic analysis of the gonads was performed, and the follicular atresia index was compared. The results of the study show that fish from the lotic environment presented higher gonadal volume, fecundity, and oocyte diameter, and a lower follicular atresia index when compared to fish from the lentic environment. In summary, the data suggest that environmental changes, i.e. from a lotic to lentic environment, caused by river damming, may negatively affect the reproductive process of migratory fish, such as M. reinhardti, and impair the maintenance of the population of this species in lentic environments

The Proteome, Not the Transcriptome, Predicts that Oocyte Superovulation Affects Embryonic Phenotypes in Mice

Superovulation is the epitome for generating oocytes for molecular embryology in mice, and it is used to model medically assisted reproduction in humans. Yet whether a superovulated oocyte is normal, is an open question. This study establishes for the first time that superovulation is associated with proteome changes that bear on phenotypic traits in mice, whereas the transcriptome is far less predictive. The proteins that were differentially expressed in superovulated mouse oocytes and their derived embryos relative to their naturally ovulated counterparts were enriched in ontology terms describing abnormal mammalian phenotypes: a thinner zona pellucida, a smaller oocyte diameter, increased cleavage arrest, and defective blastocyst formation, which we could all verify functionally. Moreover, our findings indicate that embryos with such abnormalities are negatively selected during preimplantation, and ascribe these abnormalities to incomplete ovarian maturation during the time of the conventional superovulation, since they could be corrected upon postponement of the ovulatory stimulus by 24 h. Our data place constraints on the common view that superovulated oocytes are suited to draw conclusions of general validity about developmental processes, and underscore the importance of including the proteins in a modern molecular definition of oocyte quality.

P–245 Machine learning predicting oocyte’s fertilization and blastocyst potential based on morphological features

Study question Can machine learning (ML) predict oocyte’s fertilization and blastocyst development potential based on morphological features extracted from single static images? Summary answer AI accurately predicted 70.4% of fertilization and 60.4% of blastocyst development outcomes from a database of 1000 oocytes. What is known already Some morphological features of the oocyte have been associated with IVF-related outcomes, such as size, shape, and coloration of zona pellucida, polar body, perivitelline space, cytoplasm, and the meiotic spindle. Based on these characteristics, clinics might discard the low-quality oocytes according to a subjective assessment. AI-based algorithms could reduce the subjectivity and improve prediction on IVF outcomes such as successful fertilization and blastocyst development. Study design, size, duration Non-intervention study based on a cohort of 1000 oocytes’ micrographs collected between January 2019 and December 2020 from two IVF clinics. The inclusion criteria were known fertilization and blastocyst development outcome, and patient’s age between 25 and 45 years old undergoing IVF/ICSI treatment. Different features were considered for this study including metadata from oocyte’s (e.g. age, source), as well as manually extracted morphological features from the oocytes’ images (e.g. diameters, shape, granularity, presence/absence of spindle). Participants/materials, setting, methods We trained three machine-learning (ML) classifiers (i.e. Support Vector Machine, logistic regression, and neural networks) to predict successful fertilization and blastocyst development. For the training process we used a 10-fold cross validation approach to assess the model’s generalization capabilities. Besides we tested the statistical difference of each feature among groups (i.e. fertilized and no fertilized) using a two sided Student’s t-test for numerical and Z-test for categorial features (significance of p &lt; 0.01). Main results and the role of chance Our database showed 68.2% of successful fertilization and 34.8% of blastocyst formation. To balance the training data (50% per training class), we aleatory selected 318 and 348 samples per branch of successful/unsuccessful fertilization and blastocyst formation, respectively. From all ML classifiers, the neural network obtained the best results with an accuracy of 0.70 (AUC of 0.74) for predicting fertilization; and an accuracy of 0.60 (AUC of 0.62), for predicting blastocyst formation. We found that spherical shape, presence of meiotic spindle, clear coloration, larger oocyte diameter, thicker zona pellucida, and smaller vacuoles are statistically associated with both successful outcomes. As expected, we also found a strong association between age groups and outcome. The younger group (&lt;35 years) demonstrated to have a larger proportion of successful fertilization compared to the rest of the age groups (36–37, 38–39, 40–42, &gt;42). For the blastocyst formation we observed a similar association. Limitations, reasons for caution It is relevant to note that all cycles were performed under a mini-IVF protocol. Oocytes extracted through conventional stimulation might show the same associations, but it would need further testing. Wider implications of the findings: The present study revealed that our system can predict fertilization success and blastocyst development potential based on metadata and morphometric features extracted from single digital micrographs of oocytes, offering a novel, adaptable and robust integration into clinical practice. Trial registration number CONBIOETICA–09-CEI–001–2017–0131

P–273 Effects of ovulation induction with GnRH Agonist (GnRHa) on oocyte and embryo quality at the mitochondrial level: A retrospective and experimental study

Study question Does the GnRHa trigger improve oocyte and embryo quality in patients younger than 40, and do mtUPPR have a role? Summary answer GnRHa trigger improves oocyte nuclear/cytoplasmic maturation, blastocyst utilization and downregulates HSP60 levels and upregulates ATF5 levels compared to hCG trigger. GnRHa trigger suppresses mitochondrial stress. What is known already hCG has been used for decades to achieve final oocyte maturation and, thereby, correct oocyte retrieval timing in connection with ovarian hyperstimulation protocols. As an alternative to hCG, a GnRH agonist has been used to trigger the endogenous release of LH (and FSH) in a fashion resembling the mid-cycle surge of gonadotrophins. GnRHa is as effective as hCG for the induction of ovulation. It has been very well known that the GnRHa trigger improves oocyte nuclear maturation, embryo quality, and implantation rate, but the underlying mechanism remains unknown. Study design, size, duration 3054 women younger than 40; oocytes retrieved more than 10 (up to 20) analyzed. Male infertility was excluded. Ovulation triggered either by hCG (n = 1368) or GnRHa (1668). Female mice were divided into three groups as control, hCG-treated and GnRHa-treated group. Superovulation was performed by FSH + hCG or GnRHa. Oocytes were collected 13 hours after hCG/GnRHa injection. ATF5, BiP, and HSP60 levels were analyzed by Western blot. Statistical analysis was performed using Student’s t-test. Participants/materials, setting, methods This study has two parts. i) RCT and ii) Experimental. In the experimental part, three months old female BALB/C mice (25–30 g) were used and divided into three groups (n = 20/group) as control, hCG-treated and GnRHa-treated group. Superovulation was performed by administering an injection of 5 IU FSH (i.p.) and hCG (i.p.) or GnRHa (20 mg/kg) i.m. Oocytes were collected 13 hours after hCG/GnRHa injection. ATF5, BiP, and HSP60 levels were analyzed by Western blot. Main results and the role of chance The mean age (34.8 vs. 35.2 years), total gonadotropin dose (2176 vs. 2230 IU), and the number of oocytes picked up (14.9 vs. 13.4) were not statistically different among GnRHa and hCG group, respectively. No LH rise or any OHSS was noticed in any groups. Oocyte maturation (79.8% vs. 75.9%), oocyte diameter (as a marker of cytoplasmic maturity) (10198 µm2 and 9474 µm2), fertilization rate (78% vs. 72%), and embryo utilization rate (52% vs. 47.2%) were significantly higher in GnRHa group compared to hCG group, respectively. HSP60 level (activated by mtUPR) was statistically higher in the hCG group compared to the GnRHa group (55% vs. 22%, p &lt; 0.05 respectively). On the other hand, the ATF5 level was significantly higher in the GnRHa group than the hCG group (p &lt; 0.0001). Limitations, reasons for caution The limitation is that this is a proof-of-concept study to reveal the mechanism of good embryo quality with GnRHa trigger. Wider implications of the findings: This application offers convenience and simplifies the IVF protocol with a better oocyte and embryo quality while reducing Ovarian Hyperstimulation Syndrome (OHSS) risk during IVF care Trial registration number Not applicable