In Vitro Maturation Of Oocytes Research Articles

Beneficial Effects of Catalpol Supplementation during In Vitro Maturation of Porcine Cumulus-Oocyte Complexes.

Oxidative stress degrades oocytes during in vitro maturation (IVM). Catalpol, a well-known iridoid glycoside, exhibits antioxidant, anti-inflammatory, and antihyperglycemic effects. In this study, catalpol supplementation was tested on porcine oocyte IVM and its mechanisms. Corticalgranule (GC) distribution, mitochondrial function, antioxidant capacity, DNA damage degree, and real-time quantitative polymerase chain reaction were used to confirm the effects of 10 μmol/L catalpol in the maturation medium during IVM. Catalpol treatment significantly increased the first-pole rate and cytoplasmic maturation in mature oocytes. It also increased oocyte glutathione (GSH), mitochondrial membrane potential and blastocyst cell number. However, DNA damage as well as reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Mitochondrial membrane potential and blastocyst cell number were also increased. Thus, the supplementation of 10 μmol/L catalpol in the IVM medium improves porcine oocyte maturation and embryonic development.

Prolonged maturation reduces cleavage and blastocyst rates after ICSI

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

Cumulus cell co-culture in media drops does not improve rescue in vitro maturation of vitrified-warmed immature oocytes

To assess whether co-culture with vitrified-warmed cumulus cells (CCs) in media drops improves rescue invitro maturation (IVM) of previously vitrified immature oocytes. Previous studies have shown improved rescue IVM of fresh immature oocytes when cocultured with CCs in a three-dimensional matrix. However, the scheduling and workload of embryologists would benefit from a simpler IVM approach, particularly in the setting of time-sensitive oncofertility oocyte cryopreservation (OC) cases. Although the yield of developmentally competent mature metaphase II (MII) oocytes is increased when rescue IVM is performed before cryopreservation, it is unknown whether maturation of previously vitrified immature oocytes is improved after coculture with CCs in a simple system not involving a three-dimensional matrix. Randomized controlled trial. Academic hospital. A total of 320 (160 germinal vesicles [GVs] and 160 metaphase I [MI]) immature oocytes and autologous CC clumps were vitrified from patients who were undergoing planned OC or intracytoplasmic sperm injection from July 2020 until September2021. On warming, the oocytes were randomized to culture in IVM media with CCs (+CC) or without CCs (-CC). Germinal vesicles and MI oocytes were cultured in 25 μL (SAGE IVM medium) for 32 hours and 20-22 hours, respectively. Oocytes with a polar body (MII) were randomized to confocal microscopy for analysis of spindle integrity and chromosomal alignment to assess nuclear maturity or to parthenogenetic activation to assess cytoplasmic maturity. Wilcoxon rank sum tests for continuous variables and the chi square or Fisher's exact test for categorical variables assessed statistical significance. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated. Patient demographic characteristics were similar for both the GV and MI groups after randomization to +CC vs. -CC. No statistically significant differences were observed between +CC vs. -CC groups regarding the percentage of MII from either GV (42.5% [34/80] vs. 52.5% [42/80]; RR 0.81; 95% CI: 0.57-1.15]) or MI (76.3% [61/80]; vs. 72.5% [58/80]; RR 1.05; 95% CI: 0.88-1.26]) oocytes. An increased percentage of GV-matured MIIs underwent parthenogenetic activation in the +CC group (92.3% [12/13] vs. 70.8% [17/24]), but the difference was not statistically significant (RR 1.30; 95% CI: 0.97-1.75), whereas the activation rate was identical for MI-matured oocytes (74.3% [26/35] vs. 75.0% [18/24], CC+ vs. CC-; RR 0.99; 95% CI: 0.74-1.32). No significant differences were observed between +CC vs. -CC groups for cleavage of parthenotes from GV-matured oocytes (91.7% [11/12] vs. 82.4% [14/17]) or blastulation (0 for both) or for MI-matured oocytes (cleavage: 80.8% [21/26] vs. 94.4% [17/18]; blastulation: 0 [0/26] vs. 16.7% [3/18]). Further, no significant differences were observed between +CC vs. -CC for GV-matured oocytes regarding incidence of bipolar spindles (38.9% [7/18] vs. 33.3% [5/15]) or aligned chromosomes (22.2% [4/18] vs. 0.0 [0/15]); or for MI-matured oocytes (bipolar spindle: 38.9% [7/18] vs. 42.9% [2/28]); aligned chromosomes (35.3% [6/17] vs. 24.1% [7/29]). Cumulus cell co-culture in this simple two-dimensional system does not improve rescue IVM of vitrified, warmed immature oocytes, at least by the markers assessed here. Further work is required to assess the efficacy of this system given its potential to provide flexibility in a busy, invitro fertilization clinic.

Modulation of Apoptosis and Autophagy by Melatonin in Juglone-Exposed Bovine Oocytes.

Melatonin, an antioxidant hormone secreted by the pineal gland, has been recognized as a regulator for numerous biological events. The deleterious effects of juglone, a polyphenolic extract of walnut trees, on embryo development has been previously reported. In the current study, we aimed to display the impact of melatonin administrated during in vitro oocyte maturation (IVM) on juglone-treated oocytes. Thus, in vitro matured oocytes were collected after 24 h post incubation with juglone in the presence or absence of melatonin. Reactive oxygen species (ROS), glutathione (GSH) content, mitochondrial distribution, and the relative abundance of mRNA transcription levels were assessed in oocytes, in addition, oocytes were in vitro fertilized to check the competency levels of oocytes to generate embryos. We found that administration of melatonin during the maturation of oocytes under juglone stress significantly improved the cleavage rate, 8-16 cell-stage embryos and day-8 blastocysts when compared to the sole juglone treatment. In addition, the fluorescence intensity of ROS increased, whereas the GSH decreased in juglone-treated oocytes compared to melatonin-juglone co-treated and untreated ones. Additionally, a significant increase in the mitochondrial aberrant pattern, the pattern that was normalized following melatonin supplementation, was observed following juglone administration. The mRNA analysis using RT-qPCR revealed a significant upregulation of autophagy and oxidative-stress-specific markers in the juglone-treated group compared to the co-treatment and control. In conclusion, the study reveals, for the first time, a protective effect of melatonin against the oxidative stress initiated following juglone treatment during the in vitro maturation of oocytes.

Combination of CNP, MT and FLI during IVM Significantly Improved the Quality and Development Abilities of Bovine Oocytes and IVF-Derived Embryos.

Oocyte maturation is a critical step in the completion of female gametogenesis in the ovary; thus, for subsequent fertilization and embryogenesis. Vitrification of embryo also has been shown to be closely associated with oocyte maturation. To improve the quality and developmental potential of bovine oocytes derived from in vitro maturation (IVM), Pre-IVM with C-type natriuretic peptide (CNP), melatonin (MT) and in combination, IGF1, FGF2, LIF (FLI) were supplemented in the IVM medium. In this current study, we cultured bovine oocytes in Pre-IVM with CNP for 6 h before transferring them to the IVM medium supplemented with MT and FLI. The developmental potential of bovine oocytes was then investigated by measuring the reactive oxygen species (ROS), the intracellular glutathione (GSH) and ATP levels, the transzonal projections (TZP), the mitochondrial membrane potential (ΔΨm), cacline-AM, and the expression of related genes (cumulus cells (CCs), oocytes, blastocysts). The results revealed that oocytes treated with a combination of CNP, MT, and FLI had dramatically improved the percentage of oocytes developed to blastocyst, ATP content, GSH levels, TZP intensity, the ΔΨm, cacline-AM fluorescence intensity, and considerably reduced ROS levels of oocytes. Furthermore, the survival rate and the hatched rate after vitrification of the CNP+MT+FLI group were significantly higher than those other groups. Thus, we speculated that CNP+MT+FLI increases the IVM of bovine oocytes. In conclusion, our findings deepen our understanding and provide new perspectives on targeting the combination of CNP, MT and FLI to enhance the quality and developmental potential of bovine oocytes.

Effect of Growth Factors and Hormones during In Vitro Growth Culture of Cumulus-Oocyte-Complexes Derived from Small Antral Follicles in Pigs

This study evaluated the effect of various growth factors and hormones in an in vitro growth (IVG) medium on the in vitro maturation (IVM) and developmental competence of oocytes derived from small antral follicles (SAFs) in pigs. Cumulus-oocyte complexes (COCs) derived from SAFs were either untreated or treated with epidermal growth factor (EGF), insulin-like factor-1 (IGF-1), insulin, or growth hormone (GH) for 2 days of IVG. Following IVG, COCs were cultured for maturation, and IVM oocytes were induced for parthenogenesis (PA). During IVG, the nuclear maturation of oocytes was significantly increased by the insulin treatment compared to other treatments. Moreover, the insulin treatment significantly increased blastocyst formation after PA relative to the No-IVG, control, EGF, and GH treatments. The cumulus expansion score after IVG-IVM was significantly higher in the insulin group than in the other groups. The glutathione (GSH) contents in IVM oocytes were increased through treatment with IGF, insulin, and GH compared to those of No-IVG oocytes. The level of reactive oxygen species (ROS) in IVM oocytes in all treatment groups was significantly lower after IVG culture than in the No-IVG group. The maturation-promoting factor (MPF) activity after IVM in the insulin-treated oocytes was significantly higher than that of the oocytes treated with EGF, IGF-1, and GH. In conclusion, this study demonstrates that insulin treatment during IVG culture improves the maturational and developmental competence of oocytes derived from SAFs in pigs through its effect on cumulus cell expansion and cytoplasmic microenvironments, such as GSH, ROS, and MPF activity.

The effect of antioxidants on increased oocyte competence in IVM: a review

In vitro maturation (IVM) is considered a potential assisted reproductive technology that is a safer and simpler alternative to conventional in vitro fertilization. It is primarily used in patients with impaired oocyte maturation and for the treatment of infertile women who are at risk of fertility loss. In addition, IVM is currently used mainly in polycystic ovarian syndrome patients with a high ovarian response and is still considered an experimental option in fertility preservation. Producing highly competent oocytes during IVM is considered a key step in the success of in vitro production (IVP) of embryos. Some factors, such as culture medium conditions and other supplements, have a significant impact on oocyte IVM performance. One of the known disruptors of oocyte developmental competence in IVP is oxidative stress (OS), which is caused by an imbalance between the production and neutralization of reactive oxygen species (ROS). In vitro conditions induce supraphysiological ROS levels due to exposure to an oxidative environment and the isolation of the oocyte from the follicle protective antioxidant milieu. Given the importance of OS in oocyte competence, the establishment of standardized antioxidant IVM systems is critical for improving the overall success of IVP. This review focuses on the main antioxidants tested to protect oocytes against OS in IVM.

The Effect of In Vitro Maturation (IVM) Protocol Changes on Measures of Oocyte/Embryo Competence

Purpose: In vitro maturation (IVM) continues its evolution as new ideas are introduced with the objective of making the IVM procedure easier and more effective. This study combines ideas believed likely to improve the IVM outcome or make the IVM oocyte identification process easier. Methods: A cohort of 45 women underwent an IVM cycle in which letrozole was used with the theoretical objective of improving the competence of small antral follicles, the oocyte aspiration technique was modified to minimize the time between oocyte aspiration and oocyte identification, and blastocysts were transferred during a subsequent cycle with controlled endometrial development. Results: Measures of oocyte competence used for these prospectively followed cycles were as follows: the maturation rate was 90.5%, the fertilization rate was 92.4%, the cleavage rate was 94.6%, the usable blastulation rate per zygote was 50.2%, and the implantation rate was 34.2%. Per transfer, the biochemical pregnancy rate was 63.2%, the clinical pregnancy rate was 55.3% and the ongoing/delivered pregnancy rate at the end of the first trimester was 47.4%. The miscarriage rate for clinical pregnancies in the first trimester was 14.3% and the ongoing twinning rate was 11.1%.

In vitro maturation of oocytes as a laboratory approach to polycystic ovarian syndrome (PCOS): From oocyte to embryo.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age, which in some case leads to infertility. This disorder is characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology. Infertile PCOS women that need in vitro fertilization (IVF) have greater risk of ovarian hyperstimulation syndrome (OHSS) if conventional ovarian stimulation is used. In vitro oocyte maturation (IVM) is an alternative technique that prevents OHSS in infertile PCOS women. In the last decade, IVM protocols have improved, particularly with the development of biphasic IVM culture accounting for better pregnancy and live birth rates. This technique has been extended to other treatments like, fertility preservation, when patients have no time, or a contra-indication for ovarian stimulation, and poor responders. In this review, we will discuss IVM as a viable option for PCOS infertile patients. This article is categorized under: Reproductive System Diseases > Molecular and Cellular Physiology Reproductive System Diseases > Environmental Factors Reproductive System Diseases > Genetics/Genomics/Epigenetics.

Isorhamnetin improves in vitro maturation of oxidative stress-exposed porcine oocytes and subsequent embryo development.

This study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro. Treatment with 2 μM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes (NFE2L2 and SOD2) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene (SHAS2) and improved chromosomal alignment. mRNA expression of maternal genes (CCNB1, MOS, BMP15 and GDF9) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes (SOX2, NANOG, and POU5F1) and anti-apoptotic genes (BCL2L1 and BIRC5) and decreased that of pro-apoptotic genes (CASP3 and FAS). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro. Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.

IVM of human immature oocytes for infertility treatment and fertility preservation.

Thousands of healthy babies are born from in vitro maturation (IVM) procedures, but the rate of efficiency differs with the source of immature oocytes obtained. Recently, there are different IVM protocols proposed for infertility treatment and fertility preservation. Based on the literature, the clinical application for IVM of immature oocytes was summarized. Immature oocytes may be retrieved from women after priming with or without the use of follicular stimulation hormone (FSH), human chorionic gonadotrophin (hCG) or a combination of both FSH and hCG. Successful pregnancy rates with IVM technology seem to be correlated with the number of immature oocytes obtained. With the source and culture course of immature oocytes, there are various IVM protocols. IVM of immature oocytes is profoundly affected by the culture conditions, but no breakthrough has been made by improving the IVM medium itself. Thus, the clinical application of IVM technology continues to evolve. IVM technology is a useful technique for infertile women and fertility preservation. Mild stimulation IVF combined with IVM of immature oocytes is a viable alternative to the conventional stimulation IVF cycle treatment as it may prove to be an optimal first-line treatment approach.

Antioxidant effect of ergothioneine on in vitro maturation of porcine oocytes.

Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.

The effects of cycloastragenol on bovine embryo development, implantation potential and telomerase activity.

Telomerase reverse transcriptase is a key factor responsible for structural and cellular alterations in aged oocytes and changes in the structure of the zona pellucida and mitochondria. Telomerase expression is reduced in aged cumulus oocyte complexes, and its activation or enhanced expression would be beneficial for in vitro oocyte maturation and in vitro embryo development. This study aimed to investigate telomerase activation by cycloastragenol and its effect on bovine oocyte in vitro maturation, fertilisation, and early embryo development. We used qPCR, Western blot, immunofluorescence, reactive oxygen species (ROS) assay,TUNEL assay, JC-1 assay, and invasion assay to analyse the affect of cycloastragenol (CAG) on bovine oocyte maturation, embryo development, embryo quality and implantation potential. Cycloastragenol treatment of oocytes in in vitro maturation (IVM) media significantly (P <0.05) improved oocyte IVM (90.87%), embryo cleavage (90.78%), blastocyst hatching (27.04%), and embryo implantation potential. Telomerase also interacts with mitochondria, and JC-1 staining results showed significantly (P <0.05) higher mitochondrial membrane potential (ΔΨ m) in the CAG-treated group. Furthermore, the inner cell mass (OCT4 and SOX2) and trophoblasts (CDX2) of the control and CAG groups were examined. Moreover, CAG treatment to primary cultured bovine cumulus cells substantially enhanced telomerase activity. Telomerase activation via cycloastragenol is beneficial for bovine oocyte IVM and for the production of high-quality bovine embryos. Cycloastragenol is a natural telomerase activator, and could be useful as a permanent component of oocyte maturation media.

Developmental competence of bovine oocytes in maturation media supplemented with follicular fluid exosomes

The present study evaluated the role offollicular fluid exosomes on the developmental competence of bovine oocytes. Ovaries from slaughtered crossbred cattle were collected, all visible surface follicles aspirated, and culture-grade oocytes were subjected to further study. Exosomes were isolated from bovine follicular fluid by differential ultracentrifugation. A total of 358 oocytes selected for the study were randomly divided into two groups. Group I constituted 111 oocytes, in which normal maturation was carried. Group II constituted 247 oocytes, in which in vitro maturation (IVM) medium was supplemented with follicular fluid exosomes at 1 μL/100 μL IVM medium. Maturation was assessed after 24h of culture in a CO2 incubator maintained at 38.5°C in 95 per cent humidified atmosphere of 5 per cent CO2. Following IVM of oocytes for 24 h, in vitro fertilisation (IVF)was carried out by co-incubating with capacitated spermatozoa for 18 h and embryo culture was carried out subsequently. In group II oocytes supplemented with exosomes, a significantly higher maturation rate (p ≤0.01) (95.80 ±1.67 vs76.10 ± 0.95), fertilisation rate (53.68 ± 3.02vs37.85 ± 7.01) and cleavage rate (p ≤0.01) (43.66 ± 2.13vs(32.47 ± 5.23) was noticed compared to oocytes in group I without any supplementation.The present study established that supplementation of follicular fluid exosomescould improve the developmental competence of bovine oocytes.

Effect of ovarian growth factors on ultra-structural maturation in frozen human immature oocytes after in vitro maturation: a comparative study

BackgroundIn artificial reproductive technique (ART), nearly 20% of human oocytes are immature in the germinal vesicle (GV) phase. Consequently, the best method for reserving them is cryopreserving GV oocytes, and in vitro maturation (IVM) is recommended. The aim of this study was to determine the ultrastructure characteristics of fresh and vitrified immature human oocytes after in vitro maturation in conditioned mediums.MethodsThis study was a comparative laboratory study carried out in 2018 at Afzalipur Infertility Center in Kerman. 170 fresh and 198 vitrified GV oocytes were cultured within three IVM mediums; α-mem as control medium, α-mem supplemented with human bone marrow mesenchymal stem cells (BM-MSCs) and α-mem supplemented with ovarian growth factors (O.F). After 48 h, the maturation rate and morphological feature of IVM oocytes [132 fresh IVM (fIVM) and 134 vitrified IVM (vIVM)] were evaluated. For the ultrastructure study, 10 IVM oocytes from each medium were compared with 10 fresh in vivo oocytes cancelled from ART.ResultsThe survival rate of vitrified GV oocyte after thawing was 88.88%. The oocyte maturation rate was reduced in vIVM compared to the fIVM group (76.33% vs. 77.95%); the highest oocyte maturation rate in the O.F fIVM and lowest in α-mem vIVM (82.35% vs. 71.42%). The lowest number of cortical granules was observed in α-mem vIVM, but the greatest presence of M-SER aggregates was in O.F fIVM. In vIVM oocytes, the oolemma contained irregular little microvillus organization.ConclusionsThe O.F mediums have shown the highest maturation which defends the oocyte ultra-structural conservation.

Rapamycin improves the quality and developmental competence of in vitro matured oocytes in aged mice and humans.

Women over age 35 suffer from the inadequate number and poor quality of oocytes during assisted reproductive treatment, and making full use of the oocytes by in vitro maturation (IVM) is crucial. Rapamycin could improve the developmental competences of the post-maturation oocytes during in vitro aging, yet its effects on the IVM process of oocytes from an aged population were not clear. In this study, the immature oocytes from aged mice or older women underwent IVM with or without 10 nM rapamycin, followed by parthenogenetic activation or insemination and embryo culture. The developmental competence and quality of IVM oocytes in both groups were compared. The results showed that in aged mice, the maturation rate, activation rate, and cleavage rate of IVM oocytes were significantly elevated in the rapamycin group. Additionally, oocytes cultured with rapamycin presented decreased ROS levels, reduced chromosome aberration, and attenuated levels of γ-H2AX. During IVM of oocytes from older women, the GVBD rate, 24 h maturation rate, and 48 h maturation rate were increased in the rapamycin group, compared with those in the control group, although without significant differences. After intracytoplasmic sperm injection (ICSI) and further culture of human oocytes, the high-quality embryo rate in the rapamycin group was significantly elevated. Overall, rapamycin improved IVM outcomes of oocytes from aged mice and older women. The specific mechanism of the positive effects of rapamycin on IVM outcomes might be reducing ROS levels, mitigating DNA damage, and promoting developmental potential.

In vivo and in vitro matured bovine oocytes present a distinct pattern of single-cell gene expression.

Oocyte gene expression is a well controlled event that promotes gamete competence to undergo maturation, fertilization, and to support early embryo development, directly affecting reproductive outcomes. Considering that in vivo controlled ovarian stimulation or in vitro maturation (IVM) for the acquisition of mature oocytes has distinct implications for gene expression, we sought to evaluate the effects of these procedures on the expression of competence-related genes in single-cell oocytes. Healthy Nelore cows of reproductive age were synchronized to harvest in vivo matured oocytes; ovaries from slaughtered animals were used to obtain cumulus-oocyte complexes that were in vitro matured. Single-cell gene expression was performed using TaqMan Low-Density Arrays and 42 genes were evaluated. In silico analysis of protein interactions and Gene Ontology (GO) analysis was performed. Reduced gene expression was observed for 24 targets in IVM oocytes when compared with those of in vivo matured oocytes (P < 0.05). Differences ranged from 1.5-fold to 4.8-fold higher in in vivo oocytes and the BMP15 (5.28), GDF9 (6.23), NOBOX (7.25), HSPA8 (7.85) and MSX1 (11.00) showed the greatest fold increases. The strongest score of functional interactions was observed between the CDC20 and CKS2, with the differentially expressed gene CDC20 being the main marker behind GO enrichment. IVM negatively affected the expression of important genes related to oocyte competency, and showed higher expression levels in in vivo matured oocytes. In vivo controlled ovarian stimulation may be a better strategy to achieve proper oocyte competence and increase the success of assisted reproductive technologies.

Supplementation of SDF1 during Pig Oocyte In Vitro Maturation Improves Subsequent Embryo Development.

The quality of in vitro matured oocytes is inferior to that of in vivo matured oocytes, which translates to low developmental capacity of embryos derived from in vitro matured oocytes. The developmental potential of in vitro matured oocytes is usually impaired due to oxidative stress. Stromal cell-derived factor-l (SDF1) can reduce oxidative stress and inhibit apoptosis. The aim of this study was to investigate the effects of SDF1 supplementation during pig oocyte in vitro maturation (IVM) on subsequent embryo development, and to explore the acting mechanisms of SDF1 in pig oocytes. We found that the IVM medium containing 20 ng/mL SDF1 improved the maturation rate of pig oocytes, as well as the cleavage rate and blastocyst rate of embryos generated by somatic cell nuclear transfer, in vitro fertilization, and parthenogenesis. Supplementation of 20 ng/mL SDF1 during IVM decreased the ROS level, increased the mitochondrial membrane potential, and altered the expression of apoptosis-related genes in the pig oocytes. The porcine oocyte transcriptomic data showed that SDF1 addition during IVM altered the expression of genes enriched in the purine metabolism and TNF signaling pathways. SDF1 supplementation during pig oocyte IVM also upregulated the mRNA and protein levels of YY1 and TET1, two critical factors for oocyte development. In conclusion, supplementation of SDF1 during pig oocyte IVM reduces oxidative stress, changes expression of genes involved in regulating apoptosis and oocyte growth, and enhances the ability of in vitro matured pig oocytes to support subsequent embryo development. Our findings provide a theoretical basis and a new method for improving the developmental potential of pig in vitro matured oocytes.

New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes

BackgroundEncapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes.Materials and MethodsEnzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes.ResultsA total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%).ConclusionHyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 μg/ml) or (ii) CD9 antibody (1, 5 and 25 μg/ml) or (iii) CD9 antibody (5μg/ml) + JUNO antibody (5μg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5μg/ml) + JUNO antibody (5μg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.