Vivo-matured Oocytes Research Articles

Assessing the developmental competence of oocytes matured following rescue in vitro maturation: a systematic review and meta-analysis.

To assess the developmental competence of oocytes matured following rescue in vitro maturation (IVM). PubMed, EmBASE, and SCOPUS were systematically searched for peer-reviewed original papers using relevant keywords and Medical Subject Heading terms. Study quality was assessed using the Newcastle-Ottawa Scale. Odds ratios with a 95% confidence interval were calculated by applying a random effects model. The primary outcomes were fertilization and blastulation rates. Secondary outcomes included abnormal fertilization, cleavage, euploidy, clinical pregnancy, and live-birth rates. Twenty-four studies were included in the meta-analysis. The oocytes matured following rescue IVM showed significantly reduced fertilization, cleavage, blastulation, and clinical pregnancy rates compared to sibling in vivo-matured oocytes. No significant differences were found for the euploidy and live-birth rates in euploid blastocyst transfer. In poor responders, a reduced fertilization rate was observed using in vitro-matured GV but not with in vitro-matured MI. A reduced cleavage rate in MI matured overnight compared to < 6 incubation hours was found. Our results showed compromised developmental competence in oocytes matured following rescue IVM. However, in poor responders, rescue IVM could maximize the efficiency of the treatment. Notably, our data suggests using in vitro MI matured within 6 incubation hours. CRD42023467232.

In Vivo-Matured Oocyte Resists Post-Ovulatory Aging through the Hub Genes DDX18 and DNAJC7 in Pigs.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.

P-251 Denudation time is crucial to increase the proportion of top-quality embryos in egg donation cycles

Abstract Study question How do different denudation times affect in the efficiency of the egg donation cycles? Summary answer Undetected in vitro matured oocytes, resulting from delayed denudations, contribute to a decreased top-quality blastocyst ratio. What is known already While the timeframe for sperm microinjection in ICSI cycles is widely agreed upon (2-6 hours post-oocyte retrieval), no definitive denudation time preference has been established. Published studies diverge on the advantages of early or late denudation, assessing the impact of trigger-to-denudation time (T-D time) on oocyte maturation. Advocates of early denudation contend it prevents oocyte aging, reducing in vitro culture and enhancing outcomes. These studies, often using data from cycles with own oocytes, exhibit high variability and inconsistent conclusions. The present work introduces an alternative hypothesis on the benefits of early denudation, employing a more stable study model. Study design, size, duration This retrospective study encompasses oocyte donation cycles, employing a short antagonist protocol and ovum pick-up 35.5 hours post-trigger, conducted from January 2019 to July 2023 including 1,559 stimulations. Additionally, 1,275 ICSI cycles without detected sperm pathologies derived from these stimulations were examined to evaluate the influence of T-D time on the reproductive outcomes. Six comparable groups were established by segmenting the sample according to T-D time, annotated in 30-minute intervals (36.5h to 39h). Participants/materials, setting, methods Rates of MII, MI, and nonviable oocytes were examined among 1,559 stimulations resulting in 29,671 oocytes (36.5h:187-stimulations/3,178-oocytes; 37h:226/3,836; 37.5h:614/11,921; 38h:329/6,884; 38.5h:119/2,322; 39h:84/1,530). Additionally, in 1,275 ICSI cycles involving 11,333 MII-oocytes (36.5h:117-ICSI cycles/1,027-MII; 37h:130/1,133; 37.5h:502/4,426; 38h:316/2,925; 38.5h:120/1,041; 39h:90/781), comparisons were made for fertilization, abnormal fertilization, ICSI degeneration, useful day 5/6 blastocyst (transferred or cryopreserved; &amp;gt;3BB-top and ≤3BB-good quality by Gardner score), pregnancy, and live birth rates. Statistics were ANOVA, Chi-square test and Pearson’s correlation. Main results and the role of chance Oocyte maturation profile assessment reveals that groups with later denudation exhibit a higher proportion of MII-oocytes (36.5h:80.71%; 37h:81.04%; 37.5h:81.26%; 38h:80.83%; 38.5h:84.84%; 39h:85.29%; r:0.8475; p &amp;lt; 0.05), reaching significance from 38.5h onward (p &amp;lt; 0.05). This is complemented by a strong negative correlation between T-D time and MI rate (36.5h:5.51%; 37h:5.32%; 37.5h:4.64%; 38h:4.17%; 38.5%h:3.27%; 39h:2.94%; r:-0.9881; p &amp;lt; 0.05), with significant declines at 38h and 38.5h (p &amp;lt; 0.05). No correlation between useless oocytes proportion and T-D time was observed (r:-0.5983; p = 0.21). This illustrates that certain MI-collected oocytes progressively mature during in vitro culture and subsequently, upon denudation, are classified as MII. This overestimates in vivo-matured oocytes. No differences attributed to T-D time were observed regarding fertilization, abnormal fertilization, or ICSI degeneration rates. While time does not affect the rate of useful embryos achieved (36.5h:49.4%; 37h:46.4%; 37.5h:44.6%; 38h:48.0%; 38.5h:50.6%; 39h:47.2%; r:0.1342; p = 0.80), it has an impact on the blastocysts quality. The proportion of top-quality blastocysts (&amp;gt;BB) progressively decreases with T-D time (36.5h:86.2%; 37h:85.2%; 37.5h:85.4%; 38h:81.2%; 38,5h:80.5%; 39h:80.5%; r:-0.925; p &amp;lt; 0.05). Out of 1,794 single embryo transfers, 1,477 have conclusive outcome. No implication of T-D time was detected in pregnancy rate (36.5h:59.88%; 37h:64.07%; 37.5h:61.54%; 38h:65.02%; 38.5h:59.55%; 39h:62.26%; r:0.0489; p = 0.93) or live birth rate (36.5h:40.31%; 37h:51.85%; 37.5h:39.12%; 38h:45.04%; 38.5h:41.83%; 39h:37.96%; r:-0.375; p = 0.46). Limitations, reasons for caution This study has inherent limitations as a retrospective analysis. Additionally, the potential effects of a denudation earlier than 36.5 hours post-trigger, not yet standardized in the laboratory, were not assessed. Wider implications of the findings Delayed denudation may benefit autologous IVF by fostering in vitro oocyte maturation. In egg donation cycles, early denudation is preferable for stringent selection of in vivo matured oocytes, yielding superior results. Additionally, shortening or eliminating incubation periods would enhance the automated sequence of diverse processes during an IVF cycle. Trial registration number not applicable

Supplementation of SkQ1 Increases Mouse In Vitro Oocyte Maturation and Subsequent Embryonic Development by Reducing Oxidative Stress.

In vitro oocyte maturation (IVM) technology is important for assisted animal and human reproduction. However, the maturation rates and developmental potential of in vitro-matured oocytes are usually lower than those of in vivo-matured oocytes. Oxidative stress is a main factor that causes the lower maturation rates and quality of in vitro-matured oocytes. The purpose of this study was to investigate the effects of treatment with SkQ1, a mitochondria-targeted antioxidant, on mouse IVM and subsequent embryonic development. The results demonstrated that the supplementation of SkQ1 during IVM improves the maturation rates of mouse oocytes and the subsequent developmental competence of in vitro-fertilized embryos. The addition of SkQ1 to the IVM medium also decreased oxidative stress and apoptosis, and increased mitochondrial membrane potential in matured mouse oocytes. This study provides a new method through which to enhance the maturation rates and the quality of in vitro-matured mouse oocytes, thus promoting the application and development of assisted animal and human reproductive technology.

Prolonged maturation reduces cleavage and blastocyst rates after ICSI

Most equine assisted reproduction laboratories use overnight room-temperature holding of immature oocytes, followed by in vitro maturation (IVM) for ∼30 hours. The period of maturation may vary according to laboratory workload and shipment schedules. Prolonged IVM (36 h) has been reported to increase blastocyst rates, and in vivo-matured oocytes are typically cultured until 40 to 46 h after gonadotrophin administration before ICSI is performed. There is no information on whether prolonged IVM (48 h) of immature oocytes supports acceptable blastocyst production. We compared maturation, cleavage and blastocyst rates for oocytes held overnight before being cultured for 30 or 48 h before ICSI. We noted that after IVM, some oocytes appeared mature (intact plasmalemma, heterogenous cytoplasm) but lacked a visible polar body, and we assessed whether these oocytes could produce blastocysts. Oocytes from slaughterhouse-derived ovaries were held at room temperature overnight (17 to 20 h) then randomly divided into two groups (n = 115 each): T30, 30 hours IVM culture; and T48, 48 hours IVM culture. After IVM, oocytes were divided into those with a visible polar body (MII) and those that appeared mature but without a polar body (PB-). Frozen-thawed semen from a single stallion was used for ICSI.The injected oocytes were cultured in vitro using a sequential mix of human embryo culture medium, DMEM/F-12, and FBS. Differences between groups were analyzed by Fisher's exact test. There was no difference (P > 0.1) between T30 and T48 in percentage of MII oocytes (37.4% and 32.3%, respectively), percentage of PB- oocytes (8.7% and 15.7%, respectively) or total percentage of oocytes with intact cytoplasm (MII+PB-; 46.1% and 47.8%, respectively). Within maturation duration, there were no significant differences in cleavage or blastocyst rate between MII and PB- oocytes (e.g. for T30, 27.9% and 20% blastocysts, respectively), so data for MII and PB- were combined. The cleavage rate was significantly higher for T30 than T48 (69.8% vs. 41.8%, p = 0.004). Similarly, the blastocyst rate per injected oocyte (26.4% vs. 5.5%, p = 0.003) and the blastocyst rate per cleaved oocyte (38% vs. 13%; p = 0.045) were significantly higher for T30 than T48. We conclude that increasing the duration of IVM to 48 hours does not increase the number of mature oocytes obtained, and indeed significantly decreases oocyte developmental competence. However, we were able to obtain blastocysts after 48 hours IVM, which has not been reported previously. After 30 h IVM, oocytes with a mature morphology but without a visible polar body may be used effectively for ICSI.

Alternative Culture Systems for Bovine Oocyte In Vitro Maturation: Liquid Marbles and Differentially Shaped 96-Well Plates.

In vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment.

Comparison of pregnancy outcomes following the transfer of early-developmental stage embryos and blastocysts produced by somatic cell nuclear transfer in Camelus dromedarius

The embryonic stage, site of embryo transfer in the reproductive tract of the surrogate, and embryo transfer method are important for the successful production of offspring. In the present study, there was comparison of pregnancy rates in camels following the surgical transfer of early-developmental stage embryos at Day 2 and transvaginal transfer of blastocysts at Day 7. Embryos were produced by somatic cell nuclear transfer using in vivo-matured oocytes and ear fibroblasts as donor cells. A total of 305 oocytes were collected from 27 donors, among which 275 oocytes were in metaphase II. In Group A, 110 oocytes were reconstructed, 78 fused oocytes were cultured for 2 days, and 37 early-developmental stage embryos were transferred into 13 surrogates. In Group B, 165 oocytes were utilized, 117 fused oocytes were cultured for 7 days, and 24 blastocysts were trans-vaginally transferred into 12 surrogates. Pregnancy was determined when there was an increase in serum progesterone concentrations and was confirmed using real-time ultrasonography. Microsatellite analysis was performed to confirm the parentage of offspring. Two live births occurred in Groups A and B (live birth rate of 15.4% and 16.7%, respectively). Results indicate both early-developmental stage embryos and blastocysts produced by somatic cell nuclear transfer using in vivo-matured oocytes can lead to live births in camel with similar efficiency. It, therefore, is recommended that trans-vaginal blastocyst transfer be utilized for camels considering the pregnancy and live birth rates, ease of the transfer procedure and comfort and safety of surrogates.

CHROMOSOME ANOMALIES DO NOT INCREASE IN RESCUE-IVM OOCYTES: ANALYSIS OF TROPHECTODERM BIOPSIES BY NEXT-GENERATION SEQUENCING FOR PREIMPLANTATION GENETIC TESTING

The in vitro maturation of GV oocytes retrieved from gonadotrophin-stimulated IVF cycles (rescue-IVM) is generally not recommended because of the lower developmental competence, lower implantation rate, and higher rate of chromosomal anomalies than in vivo-matured oocytes. However, most of the relevant studies analyzed cleavage stage embryos and may therefore have included some that were developmentally arrested. Additionally, few studies have examined rescue-IVM oocytes and embryos from mild stimulation cycles such as the clomiphene cycle and the letrozole cycle. We analyzed 41 blastocysts from 8 patients under clomiphene or letrozole cycles from March to December 2020. For all patients, blastocysts were obtained from both in vitro- and in vivo-matured oocytes. Mean patient age was 35.5 ± 5.3 years (range 26 to 41 years). Blastocysts derived from in vitro- and in vivo-matured oocytes were classified as rescue-IVM (n = 9) and control (n = 32) groups, respectively. n the rescue-IVM group, GV oocytes were cultured for 27 hours in IVM medium and matured oocytes were used for ICSI. In the control group, MII oocytes were subjected to IVF or ICSI. Prospective zygotes were cultured in a time-lapse incubator (EmbryoScope+) from day 0 to the day of trophectoderm biopsy. Trophectoderm biopsy was performed at day 5 or 6 and next-generation sequencing was used to analyze numerical chromosome anomalies in rescue-IVM and control embryos. The rates of aneuploidy in the rescue-IVM and control groups were 44.4% (4/9) and 50.0% (16/32), respectively. There was no significant difference between the groups. One of the 8 patients obtained a euploid embryo only from rescue-IVM. We show that rescue-IVM embryos derived from GV oocytes from a mild stimulation cycle have a similar rate of chromosomally normal embryos to that of in vivo-matured oocytes.

Development and pregnancy rates of Camelus dromedarius-cloned embryos derived from in vivo- and in vitro-matured oocytes.

ObjectiveThe present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius.MethodsCamelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method.ResultsThe collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitro-matured oocytes.ConclusionIn this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

A Comparison of Embryonic Development and Clinical Outcomes between In vitro Oocytes Maturation Using Micro-Vibration System and In vivo Oocytes Maturation in Polycystic Ovarian Syndrome Patients

Background/Objectives: Mechanical micro-vibration remains insufficient for improving embryo culture conditions in human immature oocytes. This study compared the clinical outcomes and embryo development between germinal vesicle (GV) oocytes with the micro-vibration culture (MVC) system in in vitro maturation (IVM) cycles and in vivo-matured oocytes in controlled ovarian hyperstimulation (COH) cycles in polycystic ovarian syndrome (PCOS) patients. Methods: This study investigated 152 PCOS patients who underwent 159 fresh embryo transfer cycles, including IVM cycles with embryos derived from GV oocytes and the COH cycles with embryos derived from in vivo-matured oocytes. The IVM cycles were divided into groups according to the culture system used: static culture (SC) and MVC: In the IVM-S group (n = 47), SC was applied during both IVM and in vitro culture (IVC), whereas in the IVM-MV group (n = 44), MVC was applied during both IVM and IVC. For the COH cycles, in the COH-S group (n = 68), SC was applied during IVC. Results: The number of in vitro-matured oocytes was similar in the IVM-S and IVM-MV groups, but the good-quality embryo (GQE; ≥6-cells) rate was significantly higher in the IVM-MV group (p < 0.01). The GQE rate and clinical outcomes of the COH-S group were significantly better than those of the IVM-S group (p < 0.05) but similar to those of the IVM-MV group. Conclusion: Compared with the SC system, the MVC system in IVM cycles improves the embryonic quality of GV oocytes and clinical outcomes, resulting in development of potential equivalent to in vivo-matured oocytes.

Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes.

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

Comparison of pregnancy rates with transfer of in vivo produced embryos derived using multiple ovulation and embryo transfer (MOET) with in vitro produced embryos by somatic cell nuclear transfer (SCNT) in the dromedary camel (Camelus dromedaries)

In the present study, there was comparison of pregnancy rates with transfer of in vivo-produced embryos using multiple ovulation and embryo transfer (MOET) with in vitro-produced embryos by somatic cell nuclear transfer (SCNT) in dromedary camels. In vivo-produced embryos were collected from donors after super-stimulation of follicular development on day 7 after ovulation, while in vitro-derived embryos were produced using SCNT from in vivo-matured oocytes collected from camels after follicular development super-stimulation. As a result of estrous synchronization, all recipient camels for both groups were 1 day earlier in stage of estrous cycle than developmental status of embryos at the time of transfer. The animals into which embryos were transferred were monitored at 7-day intervals after embryo transfer for signs of pregnancy based on response to presence of a male and there was ultrasonic confirmation on days 35 and 60 subsequent to day of estrus in recipient animals. A greater proportion of recipients (P < 0.05) were considered pregnant based on response to male presence when there was transfer of MOET-(76.8 ± 3.2) compared with SCNT- (26.4 ± 2.4) derived embryos on day 14. There was no difference in pregnancy losses in subsequent weeks until day 60 between groups. There were also no differences in calving rates of females in which MOET- (91.7%) and SCNT- (93.3%) derived embryos were transferred. These results indicate pregnancies at day 60 with SCNT-derived embryos are sustained for the remainder of gestation periods similar to when there was transfer of MOET-derived embryos in dromedary camels.

The Domestic Dog Embryo: In Vitro Fertilization, Culture, and Transfer.

Advances in embryo technologies in the domestic dog have made significant strides in the past decade. This progress has been spurred by interests in taking advantage of the dog as a biomedical research model for human and companion animal medicine, developing assisted reproductive technologies to manage genetic diversity in endangered canids maintained ex situ, and improving breeding in rare or working breeds of dogs. Here, we focus on recent advancements and techniques for collection of in vivo-matured oocytes, in vitro fertilization (IVF), in vitro culture of early (≤8-cell) and advanced stage (≥16-cell) embryos, and embryo transfer.

136 Efficient in vitro embryo production system using in vivo-matured oocytes from superstimulated Japanese black cows

In this study, we examined whether in vivo matured oocytes collected by ovum pickup (OPU) from superstimulated Japanese black cows can improve the productivity and quality of in vitro-produced embryos. Cows in the stimulated group received an intravaginal progesterone-releasing device (Day 0), administration of 100μg of GnRH on Day 5, a single administration of 30 Armour units of FSH on the evening of Day 8 and prostaglandin F2α on the evening of Day 10. The progesterone device was removed on the morning of Day11, and then 100μg of GnRH was administered on the morning of Day 12 (0 h). The OPU and IVF were conducted at 25~26 and 30h, respectively. Cows in the control group received no treatment before OPU, and collected oocytes were subjected to in vitro maturation followed by IVF. The cortical granules distribution of oocytes at metaphase II stage, the cleavage pattern of embryos at the first cell cycle, the developmental rate, and the quality of blastocysts were compared between the stimulated and control groups. Oocytes with cortical granules distributing cortical cytoplasm were classified as normal distribution. The cleavage pattern was evaluated at 28h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the one-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The developmental rate to blastocyst stage was measured on Day 9 of culture. The morphological quality of blastocysts was evaluated based on the IETS manual. All data were obtained from more than 3 replicates. In vitro development and cortical granules distribution data were analysed using chi-squared test. Other data were analysed using Student’s t-test. Normal cortical granules distribution rate in the stimulated group was higher than that in the control group (90.3v. 23.1%; P&amp;lt;0.01). Although no differences in the developmental rate to blastocyst stage (51.5v. 58.6%) was observed, the normal cleavage rate (73.4v. 51.2%) and the transferable embryo rate (98.3v. 88.0%) in the stimulated group were significantly higher (P&amp;lt;0.01) than those in the control group. The ratio of embryos from normal cleavage among the transferable embryos in the stimulated group was also significantly higher than in the control group (82.2v. 57.2%; P&amp;lt;0.01). In addition, the freezable embryo ratio (71.7v. 58.1%; P&amp;lt;0.072) and the total production number of embryos per head (28.0v. 15.5; P&amp;lt;0.106) showed a tendency to be higher in the stimulated group than in the control group. These results suggest that high quality embryos can be efficiently produced by the use of in vivo matured oocytes collected by OPU from superstimulated Japanese black cows.

127 Temporal regulation of molecular pathways, metabolic enzymes and growth factor receptors during bovine oocyte maturation in vitro

Although great efforts have been made to improve in vitro oocyte maturation (IVM) medium, we have yet to achieve competence equivalent to in vivo-matured oocytes. The failure in development of culture conditions for IVM yielding high quality eggs is attributed to an incomplete understanding of molecular pathways regulating oocyte and cumulus cell metabolism. The objective of the present study was to characterise the expression and functional activity of cell signalling pathways (mTOR, AKT, 4EBP1, ERK1/2), metabolic enzymes (PKM2, PDH, LDHA, AMPK), and growth factor receptors (IGF1R, IGFIIR, EGFR, FGFR1) in bovine oocytes and cumulus cells before and after in vitro maturation. In vitro-derived cumulus-oocyte complexes were collected at germinal vesicle (GV) and metaphase II (MII) stages (20 cumulus-oocyte complexes per stage; n=3 replicates) and subjected separately to Western blot analysis using antibodies against both phosphorylated (p) and total (t) protein abundance; the ratio p:t was used to determine the activity of each pathway. Results demonstrate increased (P&amp;lt;0.05) mTOR and ERK1/2 signalling, with no change in AKT and 4EBP1 activity, in oocytes during IVM. We observed increased (P&amp;lt;0.05) abundance of oocyte t-ERK from the GV to MII stage, but total expression of AKT, mTOR and 4EBP1 did not change. In cumulus cells, there was a significant (P&amp;lt;0.05) reduction in mTOR and 4EBP1, an increase in AKT, and no significant change in ERK activity. Analysis of metabolic enzymes in oocytes demonstrated increased (P&amp;lt;0.05) PDH, reduced AMPK, and unchanged PKM2 and LDHA phosphorylation during IVM. However, increased expression of t-PKM2 abundance was observed from the GV to MII stage. In cumulus cells, tAMPK abundance was reduced (P&amp;gt;0.05), but no significant change was observed in the activity of other metabolic enzymes analysed during IVM. Finally, we observed abundant expression of IGF2R in the oocyte compared with other growth factor receptors analysed, although IGF2R was significantly (P&amp;lt;0.05) reduced from GV to MII oocytes. In cumulus cells, both IGF1R and IGF2R were highly abundant compared with EGFR and FGFR but did not change during IVM. Data were analysed using one-way ANOVA. Results suggest that regulatory mechanisms including AKT/mTOR/4EBP1 and ERK are entirely different in oocytes and cumulus cells during maturation. An increase in the inhibitory phosphorylation of oocyte PDH (S293) toward the end of maturation suggests low metabolism of pyruvate via the Krebs cycle at that time. Similarly, dephosphorylation of AMPK (T172) suggests reduced AMPK activity and reduced fatty acid oxidation in mature oocytes. In addition, temporal regulation of IGF1R in the oocyte and EGFR in cumulus cells suggests an important role for these growth factor receptors during maturation and that these growth factors could be used to improve IVM medium in the bovine. Collectively, these results increase our understanding of the molecular pathways regulating oocyte metabolism during maturation and provide a strategy to improve the IVM environment for assisted reproductive technology.

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&amp;lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&amp;lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&amp;lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

Do immature and mature sibling oocytes recovered from stimulated cycles have the same reproductive potential?

How can laboratory and clinical outcomes of spontaneously, early maturing germinal-vesicle oocytes and sibling in-vivo-matured (metaphase II [MII]) oocytes be quantified and compared? A prospective, non-randomized intra-cohort study of oocytes from women aged 38 years or younger, with six or fewer MII oocytes and four or more germinal vesicles retrieved. No indication was identified for genetic tests or oocyte or embryo cryopreservation. The study was carried out at IVIRMA-Valencia. Early maturing germinal vesicles were selected for reproductive purposes. In vitro- and in-vivo MII oocytes were fertilized. After time-lapse culture, hatching blastocysts from germinal vesicles were biopsied for aneuploidy screening and vitrified. Laboratory and clinical outcomes were compared according to oocyte origin. Almost 70% of germinal vesicles had matured early and spontaneously, and had comparable in vitro-outcomes and morphokinetics to sibling in vivo-matured oocytes. Fifty per cent of biopsied blastocysts were euploid. Germinal-vesicle rescue increased the number of MII oocytes per cycle to 3.9, finally adding one extra-blastocyst per cycle. A live birth confirmed the feasibility of this approach. Further data, however, are needed to quantify its real contribution to standard intracytoplasmic sperm injection cycles. Nevertheless, 40% of patients obtained either an immediate advantage (reduction of cancellation rate) or long-term benefit (availability of extra blastocysts of attempts). Germinal-vesicle rescue can be considered as a complementary approach when folliculometry (expected) and number of MII (observed) are unequal.

α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes.

The developmental competence of invitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus-oocyte complexes (COCs) after IVM with 100μM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100μM α-tocopherol was included in the IVM medium, but remained low compared with invivo-matured oocytes. In conclusion, the addition of 100μM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

164 The Origin of Oocyte, In Vitro-Matured Oocyte With/Without Super-Stimulation, and In Vivo-Matured Oocyte Influence the Timing of Cleavage in Early Embryo and Oxygen Consumption of Blastocyst After IVF in Japanese Black Cow

It has been reported that in vitro- and in vivo-matured oocyte obtained from fully growth follicles have high developmental competence. Furthermore, the timing of cleavage in early embryo after IVF affect pregnancy success after embryo transfer. It is still unknown whether origin of oocyte affects the timing of cleavage. In this study, we examined the influence of oocyte origin on cleavage timing of early embryo after IVF. Japanese Black cows were used as donors. Oocytes derived from non-stimulation follicles (control: CON), fully grown follicles after super-stimulation treatment (SST) and follicles just before ovulation after ovulation-induction treatment (in vivo-matured oocyte: VIVO) were obtained by ovum pick-up (OPU). In the CON group, OPU was conducted on arbitrary days except oestrus. In SST group, dominant follicles were aspirated and a CIDR was inserted into the vagina on Day 0, and then FSH was injected twice a day from the evening of Day 1 to the morning of Day 5 with decreasing doses in total 20 AU. In the evening of Day 4, prostaglandin F2α (0.5 mg of cloprostenol) was administered. On Day 6, SST oocytes were collected after CIDR withdrawl. In the VIVO group, the treatment was carried out as SST until prostaglandin F2α administration, and then CIDR withdrawal and administration of gonadotropin-releasing hormone (GnRH, 0.2 mg of fertirelin acetate) performed on the evening of Day 4 and morning of Day 5, respectively. The VIVO oocytes were collected at 25 to 26 h after GnRH. The CON and SST oocytes were inseminated after 20 to 22 h of IVM, and VIVO oocytes were inseminated at 30 h after GnRH, with 3 × 106 sperm mL−1, respectively. After 6 h of IVF, presumptive zygotes were individually cultured for 168 h, using a well-of-the-well dish (Dai-Nippon-Print, Japan) and were observed by time-lapse cinematography (CCM-4MZS; Astec, Japan) to analyse the cleavage timing of embryos. Oxygen consumption (O2) was measured in blastocysts on 168 hpi with a scaning electrochemical microscopy system (HV-405SP; Hokuto Denko, Japan). Statistical analysis was carried out by Steel-Dwass test for the timing of cleavage and Tukey-Kramer test for O2. In CON (n = 15), SST (n = 25), and VIVO (n = 36), the time of first cleavage was 27.5, 29.1, and 26.1 hpi, that of second cleavage was 38.9, 40.3, and 36.0 hpi, and that of third cleavage was 48.5, 46.1, and 45.9 hpi, respectively. These cleavage times were shorter in VIVO than in CON and SST (P &lt; 0.01). The time interval between first and second cleavage (2nd cell cycle) was shorter in VIVO (10.1; P &lt; 0.01) than CON (11.4) and SST (11.2). The time interval between second and third (3rd cell cycle) were shorter (P &lt; 0.01) in SST (9.4) than in VIVO (10.1), and in VIVO than in CON (10.2), respectively. Consumption of O2 was lower (P &lt; 0.01) in CON (0.61 × 10−14 mol s−1) than in SST (0.94 × 10−14 mol s−1) and VIVO (0.94 × 10−14 mol s−1). These results suggest that the origin of oocyte influences the length of cell cycle and O2 consumption of blastocyst producted in vitro.

Dog cloning with in vivo matured oocytes obtaining using serum estradiol levels for predicting time of ovulation

Dog cloning using in vivo-matured oocytes has been carried out for a decade. To obtain mature oocytes, serum progesterone (P4) levels are used to evaluate ovulation. However, the accuracy of these methods is not sufficient. Thus, the aim of the present study was to verify the feasibility of serum estradiol (E2) on canine ovulation determination as assessed by the percentage of dogs yielding mature oocytes. In vivo-matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum P4 and E2 levels were assessed to determine ovulation and oocyte maturation. Canine serum P4 and E2 concentrations during both pro-estrus and estrus were analyzed by electrochemiluminescence immunoassay. The percentage of dogs yielding mature oocytes using each of the two ovulation prediction methods were compared, and correlations between the percentage of each method and temperature were analyzed. Following evaluation, oocytes were collected surgically, and a significantly higher percentage (P < 0.05) of dogs yielding mature oocytes was observed using E2 (56.43%) for ovulation detection as compared with that using P4 (39.60%). The percentage of dogs yielding mature oocytes using P4 significantly lower (P < 0.05) than E2 in autumn (P4, 37.50% vs. E2, 52.00%) and winter (P4, 29.17% vs. E2, 59.09%). Using E2, the percentage was maintained at about 52.00–66.67% regardless of the season and temperature. Correlation analysis showed that the dynamic of percentage of dogs yielding mature oocyte using P4 was highly correlated with environmental temperature (RP4 = 0.862), whereas E2 was not affected by temperature (RE2 = 0.199). To determine whether serum E2 could be used for ovulation prediction for canine cloning, ovulation of 25 and 19 dogs (P < 0.05) were predicted using P4 or E2 methods, respectively and two puppies, one from each ovulation prediction method, were obtained after SCNT and embryo transfer. Thus, compared with the P4 method, E2 was an accurate and reliable method for canine cloning.