Conventional In Vitro Maturation Research Articles

Effect of biphasic in vitro maturation (CAPA-IVM) on EGF receptor and embryo development of prepubertal goat oocytes according to follicle size

Oocytes spontaneously resume meiosis following their liberation from follicles, preventing full competence acquisition. Biphasic IVM (CAPA-IVM) maintains oocytes in meiotic arrest to improve developmental competence, and it specially affects poorly developed oocytes. We assessed the effect of CAPA-IVM on oocytes from small (<3mm) and large (>3mm) follicles of prepubertal goats. Oocytes were cultured for 6h in pre-IVM with C-type natriuretic peptide (CNP) and estradiol as meiotic inhibitors, and germinal vesicle (GV) rate and chromatin configuration were assessed. Then, oocytes were cultured in conventional IVM (c-IVM) or CAPA-IVM (pre-IVM + c-IVM) and EGF receptor (EGFR) protein expression, intra-oocyte ROS and blastocyst development were assessed. GV rate was higher in CNP groups than control (69% vs 28%, and 67% vs 31%, small and large follicles, respectively), but GV chromatin configuration was similar. In large follicles, EGFR expression was higher in oocytes and cumulus cells after CAPA-IVM, and ROS levels were lower. In small follicles these differences were not observed. c-IVM and CAPA-IVM produced similar blastocyst rates in small (3.7% vs 2.6%, respectively) and large follicles (8.3% vs 2.5%). Overall, CAPA-IVM enhanced EGFR expression for EGF peptide signalling and antioxidant capacity in oocytes from large follicles but oocytes from small follicles were too immature to benefit from it.

Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes Improved The Aged Mouse IVM Oocytes Quality.

During assisted reproductive technology (ART) treatment, the aged women, especially those over 35years old, have fewer mature oocytes and poorer quality of the oocytes comparing with the young women. In vitro maturation (IVM) technology facilitates the usage of immature oocytes, which is clinically important for the aged women. However, the maturation rate is low for the oocytes from the aged women. Human umbilical cord mesenchymal stem cells derived exosomes (HUCMSCs-exosomes), as important mediators of intercellular communication, have been widely used to restore ovarian function and improve female fertility. In this study, we isolated HUCMSCs-exosomes and collected the immature germinal vesicle oocytes from the naturally aged mouse model. And we added these HUCMSCs-exosomes to the conventional IVM culture system. The effects of HUCMSCs-exosomes on IVM oocytes were observed and analyzed from multiple aspects including maturation rate, spindle morphology, mitochondria function, and development potential. We found the quality of oocytes was improved by HUCMSCs-exosomes. Based on the results, we propose that HUCMSCs-exosomes may provide a novel and cell free strategy in the improvement of the IVM in elderly infertile women in the future.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus–oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

C-type natriuretic peptide treatment before in vitro maturation enhances maturation of yak oocytes and developmental competence of their crossbred embryos

ABSTRACT The effects of C-type natriuretic peptide (CNP) pre-treatment on the in vitro maturation (IVM) and developmental competence of yak (Bos grunniens) oocytes were evaluated. The results showed that 100 nM CNP could effectively maintain meiotic arrest of yak oocytes in vitro within 6 h. Yak oocytes matured in two-step IVM (pre-IVM with 100 nM CNP for 6 h followed by conventional IVM for 28 h) yielded a significantly (P < .05) increased cleavage rate, blastocyst rate after in vitro fertilization with cattle spermatozoa compared to the conventional one-step IVM method. The density of transzonal projections (TZPs) decreased after 6 h standard IVM (P < .05), whereas, COCs pre-IVM with 100 nM CNP for 6 h maintained the same density of TZPs compared to the fresh COCs. This pre-IVM resulted in lesser reactive oxygen species level (P < .05). Two-step IVM resulted in the increased glutathione levels, mRNA expression levels of BCL2, BMP15, and GDF9 (P < .05) in oocytes. In conclusion, these results indicate that CNP treatment (100 nM, 6 h) before IVM could enhance the maturation of yak oocytes and developmental competence of their crossbred embryos. This may have important applications in in vitro production of yak crossbred embryos.

Epigenetic variation in neonatal tissues in infants conceived using capacitation-in vitro maturation vs. in vitro fertilization

ObjectiveTo investigate alterations of the global DNA methylation profile in placenta, cord blood and neonatal buccal smears in infants conceived using in vitro maturation (IVM) with a prematuration step (capacitation-IVM [CAPA-IVM] versus in vitro fertilization (IVF). DesignAnalysis of data from the offspring of participants in a randomized controlled trial. SettingPrivate clinic. PatientsForty-six women with polycystic ovary syndrome and/or high antral follicle count and their offspring (58 newborns). Intervention(s)Women with polycystic ovary syndrome and/or a high antral follicle count participating in the clinical trial were randomized to undergo CAPA-IVM or conventional IVF. Main Outcome Measure(s)At delivery, biological samples including cord blood, placental tissue, and neonatal buccal smear were collected. Genome-wide DNA methylation was determined by Illumina Infinium MethylationEPIC BeadChip. Variability in methylation was also considered and mean variances for the two treatment categories were compared. ResultsIn neonatal buccal smears, there were no significant differences between the CAPA-IVM and conventional IVF groups based on CpG probe after linear regression analysis using a significant cut-off of false discovery rate <0.05 and |Δβ|≥0.05. In cord blood, only one CpG site showed a significant gain of methylation in the CAPA-IVM group. In placenta, CAPA-IVM was significantly associated with changes in methylation at five CpG sites. Significantly more variable DNA methylation was found in five probes in placenta, 54 in cord blood and two in buccal smears after IVM of oocytes. In cord blood samples, 20 CpG sites had more variable methylation in the conventional IVF versus IVM group. Isolated CpG sites showing differences in methylation in cord blood were not associated with changes in gene expression of the overlapping genes. Conclusion(s)CAPA-IVM appeared to be associated with only a small amount of epigenetic variation in cord blood, placental tissue and neonate buccal smears.

P-222 Differential regulation of active translation in mouse oocytes and zygotes upon maturation in vitro and in vivo

Abstract Study question What are the differences in the translation of specific transcripts between in vitro matured versus in vivo oocytes and IVF made versus in vivo zygotes Summary answer Our RNA-seq data show a selection of actively translated trancripts up and down regulated respective to total transcript among in vitro and in vivo conditions What is known already Our project wants to address whether there is room for an improvement towards the more physiological aspects of in vitro maturation technology. As the oocyte is transcriptionally silent during meiotic maturation, we want to approach this goal by addressing changes on global translational level. There is not much known about the employment of transcripts during meiotic maturation. Some get degraded, some silent and some recruited to be translated. To the current knowledge, nobody has so far focused on describing changes in active translation on the polysomal level between in vitro and in vivo culture conditions in mouse oocytes. Study design, size, duration Study performed on a mouse model. The following samples in quadruplicates have been analyzed and compared: in vitro meiotically matured denuded MII oocytes, in vitro meiotically matured cumulus-oocyte complexes supplemented with rFSH Gonal and hCG, in vivo meiotically matured MII oocytes, in vitro meiotically matured and IVF fertilized pronuclear zygotes, in vivo matured and fertilized pronuclear zygotes. Culture conditions were optimized according to the conventional clinical IVM technology. Participants/materials, setting, methods Mouse oocytes and zygotes were input material. Collected samples were subjected to a scarce sample profiling (polysomal fractionation on a sucrose gradient, total RNA isolation and qPCR). From respective quadruplicate fractionated samples, cDNA library was prepared and NGS deep RNA-sequencing performed. Raw data were analysed by Illumina DRAGEN software and differential gene expression was performed on Qiagen CLC Genomics. Candidate validation process is undergoing at the moment. Main results and the role of chance Results show a list of differentially regulated actively translated trancripts with implications for in vitro matured oocyte's ability to become fertilized. Similar pattern regarding developmental competence can be seen between in vitro and in vivo matured zygotes. Some candidate genes are in accordance with the latest publications regarding in vitro maturation. However due to ongoing validation and unpublished work we prefer not to disclose further and more concrete results. Limitations, reasons for caution First of all, it is a mouse model which is a kind of approximation regarding human oocyte and embryo development. RNA-seq results have to be cautiously interpreted, especially regarding the interpretation and translation of findings for further validation in clinical settings. Wider implications of the findings Place findings in context with the importance of correct translational regulation during oocyte meiotic maturation and present preliminary data for further validation in clinical assisted reproduction practice. Moreover we want to show key regulatory areas affected by in vitro maturation technology and suggest improvements for particular clinical applications. Trial registration number GA19-13491S

Sequential IVM by CNP preincubation and cooperating of PGE2 with AREG enhances developmental competence of SCNT reconstructs in goat

Developmental competence of in vitro matured cumulus oocyte complexes (COCs) in conventional IVM (C.IVM) is lower than in vivo maturated COCs and is related to unsynchronized nuclear and cytoplasmic maturation. To overcome this dearth, COCs can be exposed to granulosa secreted factors in a two-step system. Therefore, in the first experiment, 1000 nM of C-type natriuretic peptide for 8 h was determined (CAPA), as the best time and concentration to retain oocytes in germinal vesicle stage. This condition, also reduces lipid droplets and increases the expression of ATGL and PLIN2 involved in lipolysis and lipogenesis, respectively. In the second experiment, maturation was stimulated with prostaglandin E2 and amphiregulin for 18 h (CAPA-IVM), and their optimal concentrations based on blastocyst formation rates through in vitro fertilization (IVF) were determined as 1 and 600 nM, respectively. In the third experiment, the in vitro and in vivo developmental competency of SCNT embryos in CAPA-IVM group were determined. Despite similar blastocyst formation rates in IVF and SCNT between CAPA-IVM and C.IVM, the quality of blastocysts were quality was higher in CAPA-IVM, which reflected itself, as higher ICM/TE ratio and also expression of NANOG in SCNT blastocysts. Pregnancy rate, live births rate and SCNT efficiency were not significant between CAPA-IVM and C.IVM groups. Therefore, CAPA-IVM can improve the developmental competency of SCNT derived embryos.

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

O-231 In vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation with recombinant AMH in the maturation medium

Abstract Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged &amp;lt; 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

Assessment of bovine oocytes quality and their resistance to age-related changes after maturation in single-phase and two-phase culture systems

Existing approaches to in vitro maturation (IVM) of bovine oocytes do not take into account their specific demands during terminal phase of IVM including the need for increasing of their resistance to age-re-lated changes. In this work, we performed for the first time a comparative investigation of nuclear maturation and abnormal changes of MII chromosomes in bovine oocytes after their maturation in single-phase system and different two-phase systems and after the subsequent prolonged culture of the ova. When using the single-phase system, cumulus-oocyte complexes (COCs) were cultured for 24 h in the medium TCM 199 containing 10% fetal bovine serum (FBS), 10 μg/ml FSH, and 10 μg/ml luteinizing hormone (LH). In the two-phase system, oocytes matured in the same conditions for first 16 h and then in a new medium (TCM 199 containing 10% FBS (Control) or the same medium supplemented with 50 ng/ml progesterone) for the remaining 8 h of IVM. After maturation in the single-phase and the two-phase systems, a part of COCs were transferred to an aging medium (TCM 199 containing 10% FBS) and further cultured for 24 h. The state of the nuclear material (stage of meiosis and abnormal changes of MII chromosomes) in the matured and aged oocytes was assessed using cytogenetic analysis. The rate of oocytes being at the MII stage of meiosis after IVM in the single-phase and the two-phase systems was similar and amounted 82.7-86.3%. In addition, no effect of the culture system on the rate of MII oocytes with abnormal morphology of chromosomes was revealed. For the single-phase culture, this rate after the end of the IVM period was 32.2±0.5% and for the two-phase systems culture 38.5±4.0%. The prolonged culture of matured oocytes led to an increase the frequency of destructive changes in MII chromosomes to 56.9±2.9 and 68.4±3.0%, respectively. Progesterone in the IVM medium (during the last 8 hours) also did not effect on the rate of nuclear maturation, but reduced the rate of oocytes with abnormal changes in chromosomes: after IVM, as compared with the Control and the two-phase system; after 24-hours prolonged culture as compared to control (P &lt;0.05). The results of the study suggest that the two-phase maturation of bovine oocytes may be used as an alternative to the conventional IVM protocol and that progesterone during the second phase of IVM causes an increase in the quality of ova and their resistance to subsequent age-related transformations.

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery.

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte retrieval with routine gynecological surgery. OP-IVM is suitable for patients undergoing benign gynecological surgery who have the need for fertility preservation (FP) or infertility treatments such as in vitro fertilization and embryo transfer (IVF-ET). In the operating room, patients undergoing benign gynecological surgery are first anesthetized and receive ultrasound-guided immature follicle aspiration (IMFA) treatment. As the subsequent gynecological surgery is performed, the cumulus-oocyte complexes (COCs) are examined, and the immature COCs are transferred into the IVM medium and cultured for 28-32 hours in the IVF laboratory. After assessment, mature oocytes in the MII stage will be selected and cryopreserved in liquid nitrogen for FP or fertilized by intracytoplasmic sperm injection (ICSI) for IVF-ET. By combining IVM with gynecological surgery, immature oocytes that would have been discarded can be saved and used for assisted reproductive technology (ART). The procedure, significance and critical aspects of OP-IVM are described in this article.

85 The effects of heat exposure on the growth and developmental competence of oocytes derived from early antral follicles in dairy cows

Summer heat stress in dairy cows impairs the developmental competence of oocytes from antral follicles (2–8mm) which are used in conventional IVM and IVF systems. Moreover, summer heat stress is considered to impair the oocyte competence derived from smaller follicles; therefore, the impairment of oocyte competence possibly continues into the cooler autumn season. To investigate the thermosensitivity of early antral follicles (&amp;lt;1mm), we evaluated the effects of heat exposure on the growth and developmental competence of oocytes using invitro culture of oocyte–cumulus-granulosa complexes (OCGCs) derived from early antral follicles. OCGCs (n=315) were collected from early antral follicles (0.5–1mm) and cultured for 12 days. OCGCs in the heat shock group were cultured using a temperature cycle of 38.5°C for 5h, 39.5°C for 5h, 40.5°C for 5h, and 39.5°C for 9h, whereas those in the control group were cultured at a constant temperature of 38.5°C for 24h. The diameters of oocytes were measured before culture. Half of the culture medium was replaced every 4 days. Oestradiol-17β (E2) and progesterone (P4) production during the first, second, and third 4-day periods were measured by enzyme immunoassay; the viability of OCGCs was evaluated based on their morphology. Oocytes that survived after 12 days of culture (n=191) were subjected to IVM (38.5°C, 22h); their diameter and nuclear status were evaluated. Some oocytes (n=71) were subjected to IVF (38.5°C, 18h) and embryo culture (39.0°C, 150h). Cleavage and blastocyst rates were evaluated at 48h and 168h after IVF. Effects of treatment groups and culture periods on E2 and P4 production and diameters of oocytes were evaluated by two-way ANOVA followed by Tukey-Kramer or Student’s t-test. The viability of OCGCs, nuclear maturation, cleavage and blastocyst rates between two groups were compared by the chi-squared or Fisher’s exact test. E2 and P4 production and the viability of OCGCs were not different between the 2 groups. Although mean oocyte diameters before culture did not differ between the 2 groups, the mean diameters after IVM were significantly smaller in the heat shock group (108.0µm, n=56) than in the control group (111.7µm, n=61; P&amp;lt;0.05). The nuclear maturation rate in the heat shock group (36.4%, n=55) was significantly lower than in the control group (60.3%, n=58; P&amp;lt;0.05). Cleavage rates were similar between the control (54.5%, n=33) and heat shock groups (45.7%, n=35). However, no oocytes developed to blastocysts in the heat shock group (0%, n=35), whereas 30.3% (n=33) oocytes developed to blastocysts (cell number±s.d.; 92.4±28.4) in the control group (P&amp;lt;0.05). These findings suggest that summer heat stress in dairy cows impairs the growth, nuclear maturation, and developmental competence of oocytes derived from early antral follicles. This experimental model could be used to explore the mechanisms by which heat stress subsequently impairs oocyte competence during the cooler autumn season.

Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep.

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.

191 Effect of the time of oocyte collection through slicing method on meiosis resumption and invitro embryo production

Oocytes from juvenile goats are collected by slicing the ovary surface because the high percentage of small antral follicles limits follicular aspiration. The time of oocyte collection can impair oocyte developmental competence due to spontaneous resumption of meiosis. The aim of this study was to assess whether the time of slicing period affects oocyte meiosis and embryo development after invitro fertilization. Ovaries from juvenile goats (1-2 months old) were recovered at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were collected by slicing, selected, and kept in the slicing medium at 38.5°C in humidified air with 5% CO2 until analysis or culture. The slicing medium was HEPES-buffered (25mM) TCM-199 with 2.2mgmL−1 NaHCO3 and 50mgmL−1 gentamicin. Two slicing periods were tested: T1 (1 h) and T4 (4 h). After this time, a group of oocytes were stained with 1% orcein in 45% acetic acid solution for assessing meiotic arrest and observed as the rate of germinal vesicle (GV; 61-67 oocytes/group from 5 replicates). The remaining COCs were cultured in our conventional IVM medium (TCM-199 with FSH, LH, oestradiol, sodium pyruvate, glutamine, cysteamine, epidermal growth factor, and fetal bovine serum) at 38.5°C with 5% CO2. After 24h, a sample of oocytes were stained for assessing nuclear maturation (28-29 oocytes/group, 3 replicates), and the rest were invitro fertilized with 4×106 spermmL−1 in BO-IVF medium (IVF Bioscience) for 20h and embryo cultured in BO-IVC medium for 7 days (70-81 oocytes/group, 3 replicates). Blastocysts were stained with Hoechst 33258 for determining the number of cells. Data were analysed with two-way ANOVA with RStudio version 1.2.1335. The time of slicing was set as a fixed factor and the replicate as random variable. Data presented as percentage did not follow a normal distribution and were square root arcsine transformed before analysis. At the end of slicing periods T1 and T4, oocytes at GV were 100% and 84.7±5.0%, respectively (P&amp;lt;0.05). After 24h of IVM, the oocytes at MII were 77.0±7.1% and 88.6±7.3%, respectively, without statistical differences. However, oocytes from T1 produced a higher rate of cleaved oocytes (84.6±0.9%) and expanded blastocysts (11.03±5.2%) than T4 (49.8±7.9%, 0%, respectively; P&amp;lt;0.05). The total blastocyst rate for T1 and T4 was 25.4±5.8% and 9.4±4.9%, respectively (P=0.068). No differences were observed in blastocyst cell number (75.9±4.0 and 67.5±10.9, respectively). In conclusion, oocytes resume meiosis before IVM during a long slicing period, even though the slicing medium is not supplemented with hormones or growth factors. The longer slicing period does not affect nuclear maturation but impairs oocyte competence, observed as lower cleavage and blastocyst development. Further experiments are needed to determine whether the use of meiotic inhibitors in the slicing medium can prevent the negative effect of the long slicing period. This study was funded by the Spanish Ministry of Science, Innovation and Universities (AGL2017-85837-R).

33 Assessment of spindle morphology and reactive oxygen species production after vitrification of bovine oocytes following invitro maturation in the presence of glutathione ethyl ester

Oocyte cryopreservation by vitrification affects spindle integrity and causes oxidative stress induced by reactive oxygen species (ROS). Glutathione (GSH) ensures the correct assembly of microtubules, prevents ROS from attacking tubulin assembly, and ensures normal spindle function during meiosis. We hypothesised that addition of GSH ethyl ester (GSH-OEt), a cell-permeable GSH donor, to IVM medium before vitrification may improve oocyte cryotolerance by maintaining spindle morphology and preventing ROS production. For the IVM, viable cumulus-oocyte complexes were randomly distributed into two groups: 1) control: conventional IVM media with tissue culture medium-199 + 20% fetal calf serum + epidermal growth factor and 2) GSH-OEt: IVM medium supplemented with 5mM GSH-OEt at 38.5°C in a 5% CO2 humidified air atmosphere. After 22h of IVM, half of the oocytes from each group were vitrified/warmed using the Cryotop method (Kitazato Corp.). After 24h of IVM, oocytes were denuded, fixed, and microtubule and chromosome distribution were analysed by immunofluorescence. To measure ROS, oocytes were denuded and incubated in 5μM 2′,7′-dichlorodihydrofluorescein diacetate. Fluorescence was acquired, and resulting images were quantified with ImageJ software. A linear mixed effect followed by a pairwise comparison test (Tukey adjustment) were performed to analyse differences in meiotic spindle configuration and ROS production (P&amp;lt;0.05). No significant differences in percentages of MII or normal spindle configuration were observed among treatments. Treatment with GSH-OEt before vitrification resulted in similar percentages of abnormal spindle configuration to control oocytes, whereas vitrified oocytes showed significantly higher percentages of abnormal spindle configuration relative with control oocytes (Table 1). When the content of ROS was measured, vitrification resulted in significantly higher levels (15.1E+06±1.2E+06; P&amp;lt;0.05) when compared with other treatments. However, oocytes vitrified after IVM with GSH-OEt (11.8E+06±0.6E+06) showed similar ROS levels to control (10.7E+06±0.2E+06) and GSH-OEt (10.0E+06±0.1E+06) groups. In conclusion, addition of GSH-OEt to IVM before vitrification did not have detrimental effects on spindle morphology and was able to reduce ROS content. Further experiments are warranted to assess whether the addition of GSH-OEt to IVM medium can improve oocyte development competence after vitrification. Table 1.Assessment of spindle morphology after oocyte vitrification (VIT) following IVM in the presence of glutathione ethyl ester (GSH-OEt) Item n MII (%) Spindle configuration (%) Chromosome distribution (%) Normal Abnormal Absent Dispersed Decondensed Absent Control 127 79.4±92 74.1±4.4 16.2±5.6a 9.7±1.1 16.2±3.8 9.7±1.1 0 GSH-OEt 113 76.5±6.5 65.6±5.7 26.2±9.0ab 8.1±4.3 19.5±2.6 13.7±3.0 1.1±1.1 VIT 71 60.0±5.6 54.8±7.8 32.7±10.8b 12.4±1.4 34.4±8.7 10.7±6.4 0 GSH-OEt VIT 53 57.4±10.7 58.5±8.8 31.1±9.3ab 10.4±5.8 34.8±5.6 6.7±6.7 0 a,bDifferent superscripts indicate significant differences (P&amp;lt;0.05). *Percentage referred to the total number of oocytes at MII. This work was supported by MCIU, Spain (AGL2016-79802-P) and Generalitat de Catalunya (García-Martínez, 2017_FI_00451).

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes.

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats.

Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

171 PRE-IN VITRO MATURATION OF PORCINE OOCYTES USING PITUITARY ADENYLATE CYCLASE-ACTIVATING PEPTIDE: EFFECTS ON MEIOSIS PROGRESSION AND DEVELOPMENTAL COMPETENCE

Porcine immature oocytes derived from small follicle (SF, ≤3 mm in diameter) are able to resume meiosis, but few oocytes are capable to progress to the metaphase 2 stage. To improve capacitation of oocytes derived from SF and inhibit spontaneous maturation, a pre-in vitro maturation (IVM) system was applied to in vitro culturing of cumulus–oocyte complexes (COC). Pituitary adenylate cyclase-activating peptides (PACAP), increasing cellular cyclic adenosine 3′5′-monophosphate, have pleiotropous actions and multiple functions throughout the body as a neuropeptide. Recently, studies have described the role PACAP play in fertility and reproduction, including follicular development, antiapoptotic effects, and implantation. The purpose of this study is to improve the developmental competence of oocytes derived from SF by exogenous addition of PACAP on pre-IVM. In the conventional IVM group, COC obtained from follicles ≤3 mm in diameter (SF group) and 3 to 6 mm in diameter (medium follicles; MF group) were subjected to IVM for 42 h. In the pre-IVM group, COC obtained from SF were matured with nontreatment [pre-SF(-)PACAP group] and 1μM PACAP [pre-SF(+)PACAP group] for 18 h pre-IVM and were immediately subjected to IVM for 42 h. We examined nuclear stage assessment, intracellular reduced glutathione and reactive oxygen species levels, and embryo developmental competence by parthenogenesis and IVF. After IVM, the result of the nuclear stage assessment of groups showed that the pre-SF(+)PACAP group had the highest metaphase 2 rate in the groups (P &lt; 0.05). Reduced glutathione levels in MF and pre-SF(+)PACAP groups showed significantly higher levels than that of the other groups (P &lt; 0.05). After parthenogenesis, the cleavage rates were significantly (P &lt; 0.05) higher than the others in pre-SF(+)PACAP group. In the IVF experiment, the embryo cleavage rate was significantly higher in that of MF and pre-SF(+)PACAP groups compared with that of SF and pre-SF(-)PACAP groups (P &lt; 0.05). Moreover, no significant differences were found in the cleavage rate between MF and pre-SF(+)PACAP groups. In all groups derived from SF, the pre-SF(+)PACAP group rate of blastocyst formation and total cell number of blastocysts was significantly higher than that of the other groups (P &lt; 0.05). These results indicated that pre-IVM system using PACAP is able to improve meiotic maturation and developmental competence although the oocytes were derived from SF.

Effect of C-type natriuretic peptide pretreatment on in vitro bovine oocyte maturation.

C-type natriuretic peptide (CNP) has been considered as a physiological meiotic inhibitor that stimulates the cGMP production by cumulus cell natriuretic peptide receptor 2 (NPR2), which inhibits oocyte phosphodiesterase type 3 activity and increases cAMP. In this study, we explored the effect of CNP pretreatment on the in vitro maturation (IVM) of bovine oocytes by examining changes in cleavage rate, blastocyst formation, mitochondrial DNA (mtDNA) copy number, reactive oxygen species (ROS) level, glutathione (GSH) content, and redox state. Our results showed that 200nM CNP could effectively maintain meiotic arrest of bovine oocytes in vitro within 6h. The two-step IVM system in which oocytes were pretreated with 200nM CNP for 6h and then cultured IVM for 28h yielded a significantly (P<0.05) increased blastocyst rate and cell number after in vitro fertilization (IVF) while compared to the conventional one-step IVM method. In addition, in comparison with the conventional 24-h matured oocyte, oocytes pretreated with 200nM CNP for 6h followed by 28h IVM resulted in significantly (P<0.05) higher mtDNA copy number and ROS levels in oocytes, while GSH level significantly (P<0.05) decreased. Remarkably, regardless of treatment, no changes were observed in FAD++, NAD(P)H autofluorescence intensity, and redox ratio (FAD++/NAD(P)H) within the oocytes, maintaining a healthy metabolic equilibrium of redox throughout the two-step IVM. In conclusion, these results indicate that CNP pretreatment could dramatically improve the quality of bovine oocytes during in vitro maturation.

Zinc supplementation during in vitro maturation increases the production efficiency of cloned pigs.

Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.