MII Rate Research Articles

Positive effects of amphiregulin on human oocyte maturation and its molecular drivers in patients with polycystic ovary syndrome.

Does use of medium containing amphiregulin improve meiotic maturation efficiency in oocytes of women with polycystic ovary syndrome (PCOS) undergoing in vitro maturation (IVM) preceded by a capacitation culture step capacitation IVM (CAPA-IVM)? Use of medium containing amphiregulin significantly increased the maturation rate from oocytes retrieved from follicles with diameters <6 or ≥6 mm pre-cultured in capacitation medium. Amphiregulin concentration in follicular fluid is correlated with human oocyte developmental competence. Amphiregulin added to the meiotic trigger has been shown to improve outcomes of IVM in a range of mammalian species. This prospective, randomized cohort study included 30 patients and was conducted at an academic infertility centre in Vietnam from April to December 2019. Patients with PCOS were included. In the first stage, sibling oocytes from each patient (671 in total) were allocated in equal numbers to maturation in medium with (CAPA-AREG) or without (CAPA-Control) amphiregulin 100 ng/ml. After a maturation check and fertilization using intracytoplasmic sperm injection (ICSI), all good quality Day 3 embryos were vitrified. Cumulus cells (CCs) from both groups were collected at the moment of ICSI denudation and underwent a molecular analysis to quantify key transcripts of oocyte maturation and to relate these to early embryo development. On return for frozen embryo transfer (second stage), patients were randomized to have either CAPA-AREG or CAPA-Control embryo(s) implanted. Where no embryo(s) from the randomized group were available, embryo(s) from the other group were transferred. The primary endpoint of the study was meiotic maturation efficiency (proportion of metaphase II [MII] oocytes; maturation rate). In the per-patient analysis, the number of MII oocytes was significantly higher in the CAPA-AREG group versus the CAPA-Control group (median [interquartile range] 7.0 [5.3, 8.0] versus 6.0 [4.0, 7.0]; P = 0.01). When each oocyte was evaluated, the maturation rate was also significantly higher in the CAPA-AREG group versus the CAPA-Control group (67.6% versus 55.2%; relative risk [RR] 1.22 [95% confidence interval (CI) 1.08-1.38]; P = 0.001). No other IVM or embryology outcomes differed significantly between the two groups. Rates of clinical pregnancy (66.7% versus 42.9%; RR 1.56 [95% CI 0.77-3.14]), ongoing pregnancy (53.3% versus 28.6%; RR 1.87 [95% CI 0.72-4.85]) and live birth (46.7% versus 28.6%; RR 1.63 [95% CI 0.61-4.39]) were numerically higher in the patients who had CAPA-AREG versus CAPA-Control embryos implanted, but each fertility and obstetric outcome did not differ significantly between the groups. In the CAPA-AREG group, there were significant shifts in CC expression of genes involved in steroidogenesis (STAR, 3BHSD), the ovulatory cascade (DUSP16, EGFR, HAS2, PTGR2, PTGS2, RPS6KA2), redox and glucose metabolism (CAT, GPX1, SOD2, SLC2A1, LDHA) and transcription (NRF2). The expression of three genes (TRPM7, VCAN and JUN) in CCs showed a significant correlation with embryo quality. This study included only Vietnamese women with PCOS, limiting the generalizability. Although 100 ng/ml amphiregulin addition to the maturation culture step significantly improved the MII rate, the sample size in this study was small, meaning that these findings should be considered as exploratory. Therefore, a larger patient cohort is needed to confirm whether the positive effects of amphiregulin translate into improved fertility outcomes in patients undergoing IVM. Data from this study confirm the beneficial effects of amphiregulin during IVM with respect to the trigger of oocyte maturation. The gene expression findings in cumulus indicate that multiple pathways might contribute to these beneficial effects and confirm the key role of the epidermal growth factor system in the stepwise acquisition of human oocyte competence. This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED; grant number FWO.106-YS.2017.02) and by the Fund for Research Flanders (FWO; grant number G.OD97.18N). L.N.V. has received speaker and conference fees from Merck, grants, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring. T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme and Ferring. J.S. reports speaker fees from Ferring Pharmaceuticals and Biomérieux Diagnostics and grants from FWO Flanders, is co-inventor on granted patents on CAPA-IVM methodologies in USA (US10392601B2), Europe (EP3234112B1) and Japan (JP 6806683 registered 08-12-2020) and is a co-shareholder of Lavima Fertility Inc., a spin-off company of the Vrije Universiteit Brussel (VUB, Brussels, Belgium). NA, TDP, AHL, MNHN, SR, FS, EA and UDTH report no financial relationships with any organizations that might have an interest in the submitted work in the previous three years, and no other relationships or activities that could appear to have influenced the submitted work. NCT03915054.

P–239 In vitro maturation of human oocytes: a systematic review and data analysis

Abstract Study question Based on published studies, how effective is in vitro maturation (IVM) in different patient groups, and how does the maturation rate correlate with culture conditions? Summary answer Clinical IVM is most effective when patients receive only hCG trigger prior to oocyte collection. Multiple additional parameters influencing the outcome were identified. What is known already Despite being used for more than fifty years, the overall efficacy of human IVM has not yet been determined, and results are often conflicting. Indeed, IVM is still perceived skeptically by many embryologists and doctors and not widely used in clinical practice. This review aims to collect all available data in the literature regarding the efficacy of IVM analyzing characteristics of patients, treatment, or laboratory conditions that may influence the MII-rate (MR). Study design, size, duration: A systematic search was performed in the PubMed database following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search was limited to studies in the English language published before October 2020 using the following keyword: “oocyte in vitro maturation». Participants/materials, setting, methods Inclusion criteria for studies were: reporting data obtained on immature human oocytes, which transitioned to the MII stage after IVM. The requirement was that the numbers of cultured and matured oocytes were reported. If available, additional data were collected including patients’ characteristics (for example PCOS), hormonal stimulation prior to the procedure (administration of some FSH or hCG trigger or both), oocyte freezing before or after IVM, type of culture medium and supplements, etc. Main results and the role of chance A total of 350 publications were selected from 6866 search results, 436 abstracts, and 422 full read articles. Selected studies cover 21153 patients and 157420 immature oocytes cultured. It has been demonstrated that oocytes collected in vivo from adult, non-PCOS patients, who received only hCG trigger prior to the procedure had a statistically higher MII rate (66%) than oocytes from patients who received no gonadotropins or some FSH, or a combination of some FSH and hCG trigger (59%, 60% and 58% respectively). The same was valid for PCOS patients: MR in the trigger only cohort (66%) was significantly different from other cohorts. MR for in vivo collected oocytes (61%) from adult non-PCOS patients was significantly different from ex vivo collected oocytes (33%). MI stage oocytes at the moment of collection matured with a statistically higher rate (N = 4322, 73%), than GV oocytes (N = 3328, 54%). When in vitro matured oocytes were vitrified, their average survival rate was 81% (data from 50 studies on 1701 oocytes). Additionally, immature oocytes survived vitrification with a 75% rate (data from 30 studies on 4457 oocytes). Overall, ICSI fertilization rate for IVM oocytes was 69% (N = 59914). A total of 747 babies born from IVM were reported. Limitations, reasons for caution Among selected publications only 2 were randomized controlled trials and therefore the main challenge of this review is striking differences in setups among included studies. However, despite not being a meta-analysis, this study calculated MR for the most frequent treatment modalities and additional individual factors, which might influence MR. Wider implications of the findings: This review provides data regarding IVM efficiency in different cohorts of patients, performed under different culture conditions. Additional laboratory parameters influencing MR have been identified. Based on this new data, target groups benefiting the most were identified, and prognosis regarding the success of their treatment with IVM might be estimated. Trial registration number n/a

P–155 Oocyte recovery 39 hours (from 39h to 41h) after administration of follicular maturation trigger does not affect clinical results

Abstract Study question What is the clinical outcome of oocytes recovered after 39 hours from ovulation inducing drug administration? Summary answer Oocytes obtained after 39 hours from follicular maturation triggering are equally viable to those obtained at the standard time of 36 hrs. What is known already In the clinical setting of ART, ovum pick-up (OPU) is generally performed around 36 hours after the administration of ovulation inducing drugs (OID). However, there are cases where OPU cannot be performed at this time often due to long operating lists. As the time elapsed between the administration of ovulation inducing drugs and OPU becomes longer, there is a concern about time-related oocyte aging. Nevertheless, there are few reports of clinical results of OPU after 36 hours from OID. Study design, size, duration We conducted a review of 1187 cycles and 1951 patients in which OPU and embryo transfer was performed in 2017–2018. All cycles underwent a ‘freeze-all’ of embryos and the transfer cycle was in the thawed embryo transfer cycle for all cases. Participants/materials, setting, methods The time from the administration of OID to the end of OPU was divided into 36h group and over 39h group and the MII and normal fertilization rate of oocytes obtained from OPU after ovarian stimulation were compared. After confirmation of fertilization, the D3 good-quality embryo and the D5 and 6 good-quality blastocyst rates of embryos that continued to be cultured and the pregnancy and miscarriage rates of cleavage-stage embryos and blastocyst transfers were compared. Main results and the role of chance The MII rate in the 36h and &amp;gt;39h groups was 78.1% vs. 80.0%, and the normal fertilization rate was 77.9% vs. 78.1% (ICSI) and 65.4% vs. 67.6% (Conventional-IVF). The D3 good-quality embryo rate (good-quality embryos are embryos with less than 5% fragmentation in 7–9 cells and compaction with more than 50% adhesion between split spheres) was 21.8% vs. 25.3%, the D5 good-quality blastocyst rate (at least 3BB according to Gardner classification) was 33.6% vs. 40.1%, and the D6 good-quality blastocyst rate was 31.1% vs. 37.5%, all of which were not significantly different. The pregnancy rate for cleavage-stage embryo transfer was 26.6% vs. 6.7%, and the miscarriage rate was 25.3% vs. 42.9%, both of which were not significantly different. The pregnancy rate for blastocyst transfer was 45.4% vs. 50.0%, and the miscarriage rate was 22.2% vs. 20.0%, both of which were not significantly different. (The significance difference test was a χ-square test) Limitations, reasons for caution The study was a retrospective study. Wider implications of the findings: Even if OPU is conducted after 36h of the administration of OID, to the extreme range of 39h–41h, oocyte aging does not seem apparent and pregnancy outcomes are similar to the standard time interval of 36 hours. Trial registration number ‘not applicable’

O-175 Impact of female chromosomal polymorphic variants on ovarian reserve markers and fertility treatments prognosis

Abstract Study question Could the presence of chromosomal polymorphisms in women undergoing fertility treatments influence ovarian reserve, oocyte preservation or IVF clinical outcomes? Summary answer Polymorphic variants of chromosomes seem to adversely influence the Anti-Müllerian Hormone (AMH) serum levels and the post-thawing oocyte survival rate. What is known already Infertile couples have been reported to have a higher incidence of karyotype abnormalities than the general population, which could lead to lower fertility rates. However, few studies have demonstrated the controversial relationship between these karyotype alterations in women and the specific involvement of these variants and their combinations in an IVF cycle. Therefore, there is a growing interest in categorizing chromosomal polymorphic variants and relating them to the subsequent evolution in ART cycles. Study design, size, duration Retrospective evaluation of a cohort of women undergoing IVF cycles in a private fertility center. The sample included 280 cycles performed between July 2017 and December 2020. The study explored the correlation between karyotype polymorphisms and IVF outcomes in terms of: Anti-Müllerian Hormone (AMH) level, Antral Follicle Count (AFC), Oocyte Maturity (MII), Oocyte Survival after Thawing (TS), Fertilization (FZ), Useful Embryos (UE), Biochemical (BP) and Clinical Pregnancy (CP), Miscarriage (M) and Live Birth (LB) rates. Participants/materials, setting, methods Women with karyotype performed before an IVF cycle. Chromosome analysis was carried out according to the International System for Human Cytogenetic Nomenclature guidelines (ISCN). Only own eggs cycles were included, and testicular sperm cycles were excluded. The normality of the distribution of the variables was assessed using the Shapiro-Wilk test. The association between IVF cycle parameters and the presence of polymorphisms was assessed by T-Student (parametric) or U-Mann-Whitney (non-parametric). Main results and the role of chance From a total of 280 IVF cycles, 198 met the inclusion criteria: Control Group (CG) with normal karyotype (94) and Study Group (SG) with presence of polymorphisms (104) were analyzed. Cycles with fresh (136) and warmed (62) oocytes were included. Mean female age was 37.66 ± 3.99 (CG) and 36.61 ± 3.72 (SG). The overall outcome rates were: 75.82% MII, 81.39% TS, 72.78% FZ, 49.07% UE on day 5, 21.82% BP, 78.18% CP, 14.53% M and 63.65% LB. Statistically significant differences were found between the presence of polymorphisms and mean AMH serum level between CG (2.36 ng/mL) and SG (1.74 ng/mL) (p = 0.04), especially when the type “qh+” was detected (1.01 ng/mL) (p = 0.02). Furthermore, statistically significant differences were found regarding oocyte thawing survival rate, which decreased in the SG (78.94%) compared to the CG (93.69%) (p = 0.02), mainly when the type “ps+” was detected (75.13%) (p &amp;lt; 0.01). No statistically significant differences were found between the presence of polymorphism and the AFC (p = 0.25), MII (p = 0.10), FZ (p = 0.93) or UE (p = 0.52) rate on day 5. In the same way, from 106 embryo transfers (ET) performed, no significant differences were found for BP, CP, M and LB rates (p &amp;gt; 0.05). Limitations, reasons for caution Larger prospective studies including homogeneous cohorts are needed in order to corroborate our initial results. Wider implications of the findings Our findings may represent a practical tool in order to advise these patients about their reproductive success, especially, in terms of post-thawing oocyte survival prognosis. Therefore, it could be provided more personalized prognostic information before embarking on IVF treatments. Trial registration number Not Applicable

Evaluating in vitro fertilization outcomes of patients with low body mass index following frozen-thawed embryo transfer.

To determine the relationship between patients with a low body mass index (BMI; calculated as weight in kilograms divided by the square of height in meters) and in vitro fertilization (IVF) outcomes following frozen-thawed embryo transfer (FET). Retrospective cohort study including 12618 women aged 20-46years with an underweight (<18.5) or normal weight (18.5-24.9) BMI who underwent controlled ovarian stimulation for IVF in a private and academic IVF center between August 2002 and December 2019. Anti-Müllerian hormone, peak estradiol levels, number of MII oocytes, and fertilized oocytes were greater in the underweight group compared with the normal weight group. The total required gonadotropin dose was lower in the underweight patients compared with the normal weight patients. MII, fertilization, blastulation, and euploid rates did not differ before and after adjusting for confounders between BMI groups. In a cohort of 316 patients who underwent preimplantation genetic testing for aneuploidy and single euploid FET, pregnancy loss, pregnancy, clinical pregnancy, and live birth rates before and after controlling for covariates were similar between groups. Although there are known fetal growth or obstetrical issues associated in patients with a low BMI, it is reassuring that these risks do not extend to embryologic or clinical outcomes from IVF treatment.

Comparison of Oocyte and Embryo Quality Between Random Start and Controlled Ovarian Stimulation Cycles in Cancer Patients Undergoing Fertility Preservation.

Conventional assisted reproductive technology (ART) cycles may delay cancer treatment and compromise survival, and also increase patients' psychological burden as a result of delayed chemotherapy. The aim of this study was to compare the success rates of random start and conventional start GnRH antagonist protocols in terms of oocyte and embryo outputs in cancer patients. Data of 111 patients with a newly diagnosed cancer who underwent ART for fertility preservation at a university-based infertility clinic between January 2010 and September 2019 were reviewed. The study group underwent random start controlled ovarian hyperstimulation (RS-COH) and the control group underwent conventional start COH (CS-COH). The main outcome measures were the number of total oocytes, MII oocytes, and embryo yield. A total of 46 patients (41.5%) underwent RS-COH and 65 (58.5%) underwent CS-COH. Baseline characteristics were similar between the groups. The most common cancer type in both groups was breast cancer (60.9% vs. 52.3%, respectively). The median duration of stimulation was significantly longer in RS-COH than in CS-COH (12 vs. 10days; P = 0.005). The median number of MII oocytes was significantly higher in RS-COH than in CS-COH (7 vs. 5 oocytes, respectively; P = 0.020). The MII/AFC ratio was significantly higher in the RS-COH group compared to the CS-COH group (74% and 57% respectively; p = 0.02). In the linear regression analyses, RS-COH protocol did not have a significant impact on MII/AFC (standardized ß coefficient - 0.514; P = 0.289 {adjusted R2 for the model = 0.779}), oocyte yield (standardized ß coefficient - 0.070; P = 0.829 {adjusted R2 for the model = 0.840}), and MII rate (standardized ß coefficient - 0.504; P = 0.596 {adjusted R2 for the model = 0.271}). In conclusion, RS-COH protocol is as effective as CS-COH protocols for fertility preservation in cancer patients.

1 Effect of cytokine-supplemented maturation medium on bovine somatic cell nuclear transfer embryo development

Invitro maturation is an important process in the invitro production of embryos. It has been shown recently that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), increase the efficiency of IVM, blastocyst production, and invivo development following somatic cell nuclear transfer (SCNT) in pigs (Yuan et al. 2017 PNAS 114, E5796-E5804). This study was designed to assess the effect of these cytokines on IVM in bovine oocytes, their consecutive development to blastocyst, and pregnancy rate when used in SCNT. Cumulus–oocyte complexes (COCs) were retrieved from abattoir-derived ovaries, matured for 21h in either standard maturation medium [TCM-199 (Gibco/Life Technologies), 10% fetal bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 100UmL−1 penicillin/streptomycin] or maturation medium supplemented with 20ngmL−1 human LIF, 20ngmL−1 IGF1, and 40ngmL−1 FGF2. After IVM, the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.28M sorbitol fusion medium (0.1mM calcium, 0.5mM magnesium, 0.5mM HEPES, 1gmL−1 bovine serum albumin) by a single pulse of 1.75 kV/cm for 22ms. Reconstructed embryos were activated by exposure to 5μM ionomycin for 5min, followed by 4h of incubation in 2mM 6-(dimethylamino)purine and 10μgmL−1 cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. Blastocysts were transferred to oestrus synchronized recipients and initial pregnancy rates assessed at Day 40 after embryo transfer. Statistical analysis was performed using one-way ANOVA or chi-squared test. Data are presented as mean±s.e.m. The MII rate was significantly higher in maturation medium supplemented with cytokines compared with control medium (80.2±2.33%, n=885 vs. 66.8±1.82%, n=822; P&amp;lt;0.05, 7 replicates, one-way ANOVA). No statistical difference was found in the cleavage rate of SCNT embryos between treatment and control groups (94.2±1.34%, n=259 vs. 90.9±1.22%, n=208; P&amp;gt;0.05, 8 replicates, one-way ANOVA), respectively. However, a significant increase in blastocyst rate was observed in the treatment group compared with the control group (40.6±5.1%, n=446 vs. 24.3±2.9%, n=300; P&amp;lt;0.05, 8 replicates, one-way ANOVA). SCNT embryos derived from the treatment group also resulted in a significant increase in initial pregnancy rates (50.3±20.9%, n=48 vs. 29.0±20.6%, n=31; P&amp;lt;0.05, 4 replicates, chi-squared). Full-term pregnancy rates are pending. In conclusion, addition of FGF2, LIF, and IGF1 to maturation medium improves bovine IVM and SCNT blastocyst development and initial pregnancy rates. The effect on full-term pregnancy success is yet to be determined. This research was supported by UAES project (1343) and RFBR (18-29-07089).

Inhibition of miR-152 during In Vitro Maturation Enhances the Developmental Potential of Porcine Embryos.

Simple SummaryMiR-152 is a highly conserved miRNA across different species and plays a role in the regulation of cell differentiation, proliferation, and survival. However, the exact role of miR-152 in oocyte and embryo development is not yet known. In this study, we specifically manipulated the expression level of miR-152 in porcine cumulus-oocyte complexes (COCs) and monitored their developmental competence until the blastocyst stage. We mainly found that a suppressed expression of miR-152 during oocyte maturation significantly improved the blastocyst rate. Our results indicate that this negative correlation between miR-152 during oocyte maturation and the blastocyst rate in pigs could be through targeting IGF system components during oocyte development. These results provide more insights into the role of miRNAs during oocyte and embryonic development that could improve the in vitro production system for mammalian embryos. Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.

The aim of the present study was to improve the in vitro maturation (IVM) procedure using oocytes from surplus ovarian tissue after fertility preservation. Twenty-five patients aged 17-37years were included in the study. Maturation was compared between oocytes collected in HEPES-buffered medium or saline, and we determined whether transport on ice prior to oocyte collection affected maturation. Two different IVM media were used that were supplemented with and without recombinant human midkine. Mature oocytes were assessed for aneuploidy using next-generation sequencing (NGS). On average, 36 immature oocytes were collected from each patient (range 7-90, N = 895). Oocytes recovered from HEPES-buffered medium matured at a higher rate than oocytes recovered from saline (36% vs 26%, p < 0.01). Ovarian transportation on ice prior to the procedure negatively affected maturation compared with non-transported samples (42% vs 27%, p < 0.01). The addition of midkine improved maturation rate (34% vs 27%, p < 0.05). On average, 11 MII oocytes were obtained per patient (range 1-30). NGS of 53 MII oocytes and their first polar bodies indicated that 64% were euploid. The study demonstrated unexpectedly high number of immature oocytes collected from surplus ovarian tissue without any stimulation. The overall MII rate was one in three, resulting in a total number of MII oocytes that was similar to the number obtained after ovarian stimulation. If these MII oocytes prove suitable for IVF, they will provide a substantial improvement in fertility preservation for patients and advance IVM as an interesting platform for further improvements in assisted reproduction.

199 Strategies to improve canine oocyte invitro maturation

Canine oocyte invitro maturation (IVM) is one of the challenges of animal reproduction because of low maturation and high degeneration rates. In the bitch, after ovulation, oocytes remain in an immature stage and acquire their competence in the intra- and extrafollicular (oviductal) environments. Oxidative stress and reactive oxygen species affect canine oocytes, which can be related to the high amount of lipids they contain. Therefore, the use of antioxidants such as insulin-transferrin-selenium (ITS) and lower oxygen tension during IVM could be beneficial for oocyte maturation and survival. The purpose of this study was to determine an optimum IVM culture medium and to evaluate the effect of ITS and lower oxygen tension in canine IVM. In experiment 1, TCM-199 and synthetic oviductal fluid (SOF) media were evaluated for their ability to promote nuclear maturation at 72 and 48h of culture. Also, two protein sources were used: 8% bovine serum albumin (BSA) and 2.5% fetal bovine serum (FBS), and media were supplemented with hormones. The results revealed that SOF with FBS and BSA had similar results to TCM-199 supplemented with FBS after 72 and 48h of IVM (MII rates of 7% and 4% for the 72-h group, and 4% and 10% for the 48-h group). Synthetic oviductal fluid supplemented with BSA but without FBS produced significantly higher degeneration rates compared with SOF with FBS and BSA (44% and 23%, respectively). Forty-eight hours of IVM decreased degeneration rates, with higher MII rates compared with 72h of IVM. In experiment 2, SOF medium supplemented with FBS and BSA was chosen. Oocytes were cultured in SOF with FBS and BSA supplemented at two concentrations of ITS (1 and 10μLmL−1 ITS). Supplementation with 1μLmL−1 ITS demonstrated a beneficial effect by improving maturation rates up to 20%, compared to control and 10μLmL−1 supplemented group (4% and 6% MII, respectively) after 72h of IVM. For experiment 3, oocytes were cultured in SOF medium with or without ITS (0 and 1μLmL−1 ITS) under two oxygen tensions (5% and 20% O2) for 48h. Results from this experiment demonstrated that the combination of low oxygen tension and ITS (5% O2 and 1μLmL−1 ITS) improved maturation rates up to 26.2%, although there were no statistically significant differences compared with high oxygen and ITS (20% O2 and 1μLmL−1 ITS) and low oxygen without ITS (5% O2 and 0μLmL−1 ITS) groups. These treatments were able to increase MII rates compared with the control group (20% O2 and 0μLmL−1 ITS). Parthenogenetic activation was performed on the low oxygen with or without ITS supplemented groups. The untreated group generated higher degeneration rates after 7 days of culture, and cleavage rates were low for both groups. Nevertheless, an oocyte at the 8-cell stage was obtained in the ITS-supplemented group. Taken together, these results indicate that ITS supplementation and low oxygen tension during IVM improve canine oocyte maturation.

190 Effect of TCM-199 and synthetic oviductal fluid medium supplemented with varying hormone concentrations on invitro maturation of canine oocytes

Invitro oocyte maturation (IVM) in the domestic canine is yet to be optimized, with low rates of cumulus-oocyte complexes (COCs) reaching MII. This limits the progression of assisted reproductive technologies, which could benefit breeding programs for assistance dogs and endangered Canidae. Canine oocyte maturation differs from that in other mammals, with the ovulation of a COC in the germinal vesicle stage and nuclear maturation occurring in the oviduct. Because of this, the environment in which a canine COC matures is unlike that of other mammals, meaning that IVM protocols cannot be readily adapted. The aim of the current work was to determine (1) the effects of varying concentrations of FSH, human chorionic gonadotrophin (hCG), and oestradiol (E2) during IVM on meiotic resumption and nuclear maturation of canine COCs; and (2) the optimal medium base, either synthetic oviductal fluid (SOF) or tissue culture medium-199 (TCM). Reproductive tracts of bitches (6 months to 7 years of age) were collected from veterinary clinics within 2h of routine spaying. Ovaries were sliced using a scalpel blade, releasing the COCs into aspiration medium. The COCs were randomly allocated to a maturation medium consisting of one of the hormones at two concentrations (FSH: 5 or 10µgmL−1; hCG: 5 or 10IUmL−1; E2: 1 or 5µgmL−1) and for both SOF and TCM base. Each hormone was tested individually for a replicate of eight animals per hormone (total of 12 experimental groups; 24 animals). The COCs were cultured for 72h in their allocated medium and then denuded and stained with Hoechst 33258. Fluorescence microscopy was used to determine nuclear maturation stage. Nuclear maturation rates to MII were analysed using a general linear model with pairwise comparison (SPSS version 25; SPSS Inc./IBM Corp.) with each individual animal acting as a replicate. Canine COCs matured in a SOF-based media had higher rates of meiotic resumption (MI and MII) (SOF: 38.68%, n=515; TCM: 25.78%, n=542; P&amp;lt;0.05) and number reaching MII (SOF: 7.54%; TCM: 4.39%; P&amp;lt;0.05) compared with TCM-based medium. Resumption of meiosis and nuclear maturation to MII did not differ between media with differing E2 or hCG concentrations. The use of FSH at 10µgmL−1 in SOF medium decreased resumption of meiosis (8.57%) and MII rates (0%) compared with 5µgmL−1 FSH in SOF (29.41% and 3.92%, respectively; P&amp;lt;0.05). In summary, our data indicated that higher concentrations of FSH during IVM have a negative effect on meiotic resumption and maturation to MII, whereas canine COCs resume meiosis and mature to MII in higher rates in a SOF-based medium compared with a TCM base. An IVM medium that replicates the invivo environment in which canine COCs mature is beneficial. However, rates of IVM canine oocytes reaching maturity are low, signifying that future research must investigate a greater range of hormone concentrations and combinations to better mimic invivo conditions to assess the possible benefits for canine IVM.

180 Glucose versus fatty acids: Different energy supplies for the bovine oocyte

Studies show that optimal concentrations of fatty acids (FA) and glucose are crucial for proper oocyte development, maturation, and further embryo quality support (Sutton McDowall et al. 2010 Reproduction 139, 685-95, https://doi.org/10.1530/REP-09-0345; Sutton McDowall et al. 2014 Theriogenology 82, 95-103, https://doi.org/10.1016/j.theriogenology.2014.03.011). A balance between metabolism of FA and glucose in oocytes needs to be maintained; however, it is not clear what the preferable pathway of energy production is and how it affects the oocyte. The aim of the experiment was to selectively block pathways of either glucose or FA metabolism during IVM of bovine oocytes to reveal changes within oocyte lipid droplets (LD) under crucial changes in energy metabolism. Cumulus-oocyte complexes were matured under standard conditions (Warzych et al. 2007Mol. Reprod. Dev. 74, 280-289; https://doi.org/10.1002/mrd.20610) without FA and glucose supplementation. The experimental groups were (1) control (IVM in basic medium), (2) group with inhibited glucose metabolism [supplementation with 1.5 µM iodoacetate (IO, inhibitor of glycolysis) and 150 µM dehydroepiandrosterone (DHEA, inhibitor of pentose phosphate pathway)], and (3) group with inhibited FA metabolism (150 µM etomoxir supplementation, ETO). Oocytes after 24h of IVM were stained with boron-dipyrromethene (BODIPY) 493/503 dye (lipid droplets) and 4′,6-diamidino-2-phenylindole (DAPI; chromatin) and analysed using a confocal microscope (LSM 880 AiryScan FAST; Zeiss). Obtained data were analysed using the Kruskal-Wallis test. The MII rate decreased to 60.5% for IO+DHEA (P&amp;lt;0.05) and to 78% for the ETO group compared with the control (83.8%). The average LD area (% of total oocyte area) significantly decreased in both experimental groups (IO+DHEA 6.72±2.5, ETO 6.28±3.2; P&amp;lt;0.01) compared with control (8.7±3.6). Total lipid content (intensity of the fluorescence), was significantly lower in experimental groups (IO+DHEA: 1.17×106±3×105, ETO: 7.1×104±2×104 vs. control: 1.68×106±4.6×105; P&amp;lt;0.01). With regard to the total number of LD, only in the IO+DHEA group were significantly fewer LD noted (987±343; P&amp;lt;0.05), whereas the ETO group did not differ significantly (1146±414) compared with control (1148±357). The ETO group had significantly lower total lipid content (P&amp;lt;0.01) and higher total LD number (P&amp;lt;0.05) compared with IO+DHEA. The area of lipid droplets did not differ between the experimental groups. These results show that blocking glucose metabolism strongly affects the nuclear maturation process of the oocyte, limiting the number of oocytes that reach the MII stage. A significant decrease in total lipid content in the ETO group may suggest strong utilisation of lipids during the maturation process, when de novo synthesis of lipids is blocked. Accumulation of lipids in LD before maturation allows this process to be passed even under the applied FA metabolism inhibitory conditions. In contrast, inhibition of glucose metabolism negatively affects oocyte development strictly after inhibition conditions, indicating the greater significance of glucose for proper oocyte maturation. Further studies are being conducted to investigate the effect of inhibitory systems on lipids in bovine cumulus cells and further embryos. Funding for this study was provided by National Science Centre, Poland (project no 2017/27/B/NZ9/00904).

In vitro embryos production of oocytes selected based on follicular size and by brilliant cresyl blue staining

We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2 &lt; 4 mm), large (4–8 mm) or mixed (2–8 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 ?M of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p &gt; 0,05) BCB+ rates, but both differed (p &lt; 0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB – oocytes, there was no difference (p &gt; 0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p &gt; 0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p &lt; 0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p &lt; 0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos.

Influence of Different Gonadotropin-releasing Hormone Agonist Administration Methods on Pregnancy Outcomes of Patients Undergoing In-vitro Fertilization-embryo Transfer

This study aimed to investigate the effect of different gonadotropin-releasing hormone agonist (GnRH-a) administration methods on pregnancy outcomes of patients undergoing in-vitro fertilization-embryo transfer (IVF-ET). Clinical data of 5217 patients who underwent IVF-ET were retrospectively analyzed. Patients were divided into the long-acting GnRH-a group (n=1330) and the short-acting GnRH-a group (n=3887) based on their various treatment plans. The clinical and laboratory embryo data and clinical pregnancy outcomes were compared between the two groups. The results showed that there were no significant differences in the age, infertility, primary/secondary infertility rate, IVF rate, body mass index (BMI), antral follicle counting (AFC), follicle-stimulating hormone (FSH) level, and the number of transplanted embryos between the two groups (P>0.05). There were no significant differences in the oocyte numbers, MII rate, fertilization rate, cleavage rate and blastocyst formation rate (P>0.05) between the two groups. The gonadotropin (Gn) using days, Gn dose and endometrial thickness were significantly greater in the long-acting GnRH-a group than those in the short-acting GnRH-a group (P<0.01). Additionally, the estradiol (E2) levels, blastocyst freezing rate, embryo utilization rate, transplant cancellation rate and abortion rate were significantly lower in the long-acting GnRH-a group than those in the short-acting GnRH-a group (P<0.01). The clinical pregnancy rate and embryo implantation rate were significantly higher in the long-acting GnRH-a group than in the short-acting GnRH-a group (P<0.01). It was concluded that use of long-acting GnRH-a can effectively reduce the transplant cancellation rate and improve the clinical pregnancy rate of the fresh cycle.

Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes.

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72hr in a supplemented TCM199-Hepes medium as (a) Control group; (b) GDF-9 antibody (Ab); (c) BMP-15 Ab; (d) recombinant human (rh) GDF-9; (e) rh BMP-15 or (f) rh BMP-15 and GDF-9. Data were evaluated by ANOVA. The Abs against GDF-9 or BMP-15 had a negative impact on meiotic development. Higher (p<0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF-9 Ab (64.4±2.1%) or BMP-15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4±7.8%). In contrast, more (p<0.05) oocytes in control group reached MI (37.4±1.3%) and MII stages (10.2±2.1%) comparing to those groups with GDF-9 Ab (23.1±4.7% MI; 0.0% MII) or BMP-15 Ab (16.4±2.4%MI; 5.9% ± 2.1 MII). Higher rates (p<0.05) of oocytes in control group stayed still arrested at GV (19.9±8.6%) in comparison to those cultured with either rhGDF-9 (3.7±0.4%) or rhBMP-15 (10.9±0.7%). However, there were no differences in MII rates between oocytes cultured with GDF-9 (14.7±3.1) and BMP-15 (7.8±2.5) separately. But, more oocytes (p<0.05) reached the MII stage (20.5±3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs.

163 Effect of melatonin and its receptors on bovine oocyte maturation and cumulus cell gene expression after heat shock in vitro: preliminary results

The aim of this study was to assess the effect of melatonin (MLT) during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COC) under normal and heat stress conditions, on oocyte nuclear maturation and expression of genes related to antioxidant, apoptotic, and expansion activities in cumulus cells (CC), and whether effects are mediated by melatonin receptors (MT1, MT2, or both). The COC were submitted to IVM (25-30/group) in TCM-199 supplemented with 3mg mL−1 BSA, 0.5µg mL−1 FSH, 11µg mL−1 sodium pyruvate and 10µg mL−1 gentamicin. Melatonin (10−6 M) was used alone or associated with luzindole (10−6 M; LUZ) or 4-phenyl-2-propionamidotetralin (10−6 M; 4-P-PDOT). Groups were matured under normal conditions for 24h (38.5°C, control, and 38.5°C+MLT) and heat shock (cultured for 14h at 41°C, 41°C+MLT, 41°C+MLT+LUZ, and 41°C+MLT+4-P-PDOT), and then incubated for the remaining 10h at 38.5°C. The CC were collected and evaluated by real-time quantitative PCR for GPX4, SOD1, and SOD2 (antioxidant enzymes); BAX, CASP3, and P53 (apoptosis-related); and HAS1, HAS2, and PTX3 (expansion-related) transcripts. Gene expression data were normalized by the geometric mean of housekeeping genes ACTB, GAPDH, and PPIA. Relative expression levels were calculated by the comparative method of 2−ΔΔCt (Livak and Schmittgen 2001 Methods, 25, 402-408). Maturation rates (metaphase II; MII) were determined by staining oocytes with Hoechst 33342. Data were analysed by ANOVA followed by Tukey’s test to compare effects of treatments on maturation rate (3 replicates/treatment). Significance level was 5% (GraphPrism software, GraphPad Inc., San Diego, CA, USA). After 24h of IVM, the MII rate decreased (P&amp;lt;0.05) for the 41°C group (30±14.1%, 21/70) compared with the control at 38.5°C (76.1±14.7%, 51/67; P&amp;lt;0.05) and addition of MLT to the 41°C group reversed the decrease (41°C+MLT, 55.3±7.1%, 26/47) and was similar to that of the control (P&amp;gt;0.05). When the heat stress with MLT group was associated with MT inhibitors, MII rates decreased relative to control (P&amp;lt;0.05) and were similar to 41°C without MLT and to each other (41°C+MLT+LUZ, 31.6±8.9%, 24/76; and 41°C+MLT+4-P-PDOT, 30±9.0%, 21/70; P&amp;gt;0.05). Addition of MLT did not differ from control without the hormone (38.5°C+MLT, 90.5±6.7%, 38/42; P&amp;gt;0.05). For gene expression in CC, only the 41°C+MLT+LUZ group increased (P&amp;lt;0.05) transcripts for CASP3 compared with control. There was no difference for antioxidants (GPX4, SOD1, and SOD2), expansion (HAS1, HAS2, and PTX3), or apoptotic (BAX and P53) transcripts for any of the groups (P&amp;gt;0.05). In conclusion, MLT reversed the negative effects on nuclear maturation caused by heat stress conditions during IVM; this effect was probably mediated by the MT2 receptor. However, MLT did not influence the antioxidant enzymes, apoptosis, or expansion-related transcripts in CC, but inhibition of MT1/MT2 receptors increased CASP3 transcripts, suggesting a possible role of the receptors on apoptosis in these cells. Further studies are necessary to improve our knowledge on the role of MLT in heat stress during IVM. The authors acknowledge FAPESP for funding (HF-2016/24884-3; CLVL-2015/20379-0).

Cilostamide affects in a concentration and exposure time-dependent manner the viability and the kinetics of in vitro maturation of caprine and bovine oocytes

This study investigated: 1) the kinetics of oocyte chromatin configuration during in vitro maturation (IVM) of caprine and bovine oocytes; and 2) the effect of in vitro pre-maturation (IVPM) with cilostamide with or without association of the follicular wall (FW) on the same parameters. In experiment I, cumulus-oocyte complexes (COCs) were cultured in vitro in a standard maturation medium for 6, 12, 18 or 30 h. For experiment II, the COCs were cultured for 30 h, either in a standard IVM medium or in IVPM containing cilostamide (10 or 20 μM) and FW alone or in combination, for 6 or 12 h before the onset of maturation. The MII rate was similar (P > .05) between 18 and 30 h of maturation, both of which were higher (P < .05) than 6 and 12 h IVM in both species (Experiment I). Contrary to caprine, all IVPM treatments presented a higher (P < .05) percentage of bovine oocytes arrested at the GV stage than the control treatment after 6 h of culture. The percentage of MII oocytes after 30 h (IVPM+IVM) of culture in bovine oocytes treated with 10 μM cilostamide associated with FW and FW alone cultured for 6 h presented MII percentages similar to the control. However, in caprine, these treatments significantly reduced the percentages of MII in relation to the control treatment (Experiment II). In conclusion, the combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both species.

Optimizing treatment of tauroursodeoxycholic acid to improve embryonic development after in vitro maturation of cumulus-free oocytes in mice.

Cumulus-free in vitro maturation (IVM) provides a powerful tool to manipulate immature oocytes, but IVM oocytes lead to poor development after fertilization. Supplementation of the culture medium with tauroursodeoxycholic acid (TUDCA), a bile acid, has been reported to improve the development of embryos derived from in vivo fertilized (IVF) embryos after in vitro culture (IVC) by attenuating endoplasmic reticulum stress. However, it remains unclear if TUDCA can improve development of IVM-IVF embryos. Here, we examined whether TUDCA treatment could improve embryonic development during or after IVM. Immature GV oocytes collected from ovaries of ICR female mice that were free from cumulus cells were subjected to IVM in αMEM containing 5% FBS for 16 h. TUDCA was added to the media at varying concentrations (0–1000 μM) during IVM and IVC. TUDCA treatment during IVM reduced both MII and pronuclear (PN) rates but did not affect blastocyst rates of fertilized embryos. In contrast, TUDCA treatment during IVC significantly increased blastocyst formation rates in a concentration dependent manner. Finally, embryo transfer after TUDCA treatment revealed a significant improvement in the rates of offspring production (15% with 1000 μM TUDCA vs. 6.0% control). These results show that treatment with 1000 μM of TUDCA significantly can improve poor embryonic development of cumulus-free IVM-IVF embryos.

168 The Effect of Prematuration Culture Using Epidermal Growth Factor Receptor Kinase Inhibitor on Nuclear Maturation of Bovine Oocytes and Cumulus Cell Gene Expression

Epidermal growth factor receptor (EGFR) activation is an essential step in triggering LH-induced meiotic resumption. Here, we used an EGFR kinase inhibitor (AG1478) to assess the blockage and reversal of meiosis block in bovine oocytes, the competence of such oocytes for the embryonic development, and the gene expression of cumulus cells (CC). Cumulus-oocyte complexes (COC, n = 583) were pre-matured (PM) during 8 h in TCM-199 with 1 μM AG1478 (EGFR-group). Next, COC were washed from meiotic inhibitor and cultured for 22 h in in vitro maturation (IVM) medium (TCM-199 with bicarbonate, 0.5 mg mL−1 FSH, 100 IU mL−1 hCG, and 10% FCS). The control group was only cultured for 22 h in IVM medium. Meiosis progression in oocytes was evaluated after PM (blocked_GV) and after IVM of PM oocytes (blocked_MII) or immediately after follicle aspiration (immature control = control_GV) and IVM (matured control = control_MII); oocytes were classified as immature (germinal vesicle; GV) or mature (metaphase II; MII). Another sample of matured oocytes was fertilized and cultured to the blastocyst stage. Abundance of 81 transcripts was evaluated by qPCR using a microfluidic platform (BioMark HD System™, Fluidigm®, South San Francisco, CA, USA) in CC collected at the same time as oocyte evaluation. Relative mRNA abundance was calculated by ΔCt (target genes were normalized by 2 reference genes: PPIA and RPL15). Data were analysed by ANOVA followed by Tukey’s test (P ≤ 0.05). The GV rates after 8-h PM differed (P ≤ 0.05) between 0-h oocytes (96.4 ± 2.2%) and EGFR- (59.8 ± 2.2%). After 22-h IVM, meiosis block was fully reversed and there was no difference (P ≥ 0.05) in MII rates between treatments (84.8%, averaged). Blastocyst rates (38.2%, averaged) were unaffected by treatment (P ≥ 0.05). Abundance of several transcripts was modulated by PM culture (blocked_GV v. control_GV), including up-regulation of genes that control the expansion of CC/COC competence markers (GREM1 and VCAN), cell proliferation/survival control (IGF1R and EGFR) and maintenance of cellular structural integrity/prediction of embryo quality (KRT8 and GATM), and down-regulation of genes associated with failures in pregnancy establishment/glucose metabolism (AKR1B1), endoplasmic reticulum stress (ATF4), meiotic arrest (ADCY3 and NOS2) and cell survival(HSPA1A). After IVM (blocked_MII v. control_MII), it was found an up-regulation in the relative abundance of PTX3 and PTGS2 (expansion of CC), RGS2 (regulator of G-protein signalling), LUM (embryo quality) and FOXO3 (apoptosis and oxidative stress resistance), whereas relative abundance of PFKP (glucose metabolism) was down-regulated. Our results indicate that blockade of meiosis with the EGFR kinase inhibitor before IVM is reversible and does not affect subsequent embryonic development. The gene expression profile of CC indicates a possible improvement in the quality of COC; however, more studies will be necessary to evaluate whether these improvements will be maintained until the embryonic stage. Financial support provided by FAPESP (#2015/06733-5 and #2012/50533-2) and CNPq (#307416/2015-1).

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P &lt; 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P &gt; 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P &lt; 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P &gt; 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&amp;M University.