Efficiency Of In Vitro Maturation Research Articles

Follicular fluid meiosis activating sterol supplementation enhances oocyte maturation and fertilization in a microfluidic system: A lab trial study

Background: In vitro maturation (IVM) is a promising technique in assisted reproductive technologies, offering benefits such as reducing the risk of ovarian hyperstimulation syndrome. Objective: This study aimed to evaluate the effects of timed follicular fluid meiosis-activating sterol (FF-MAS) supplementation on the IVM of germinal vesicle oocytes using a dynamic microfluidic system. Materials and Methods: In this lab trial study, 266 germinal vesicle oocytes were collected from the Infertility Center of Fatemieh hospital, Hamedan, Iran between June 2023 and January 2024. The oocytes were allocated into 3 groups for dynamic microfluidic culture. Each group received culture medium at a flow rate of 0.36 μL/min for 24 hr through inlet A and FF-MAS supplementation through inlet B for 1, 2, and 6 hr. The study evaluated maturation and fertilization rates, embryo development, and mitochondrial status, which was assessed using the JC-1 mitochondrial membrane potential assay. Results: Maturation rates were significantly higher in the medium-term FF-MAS exposure (MTG) and long-term FF-MAS exposure groups compared to the short-term FF-MAS group (STG) (p < 0.05). Fertilization rates were also higher in the MTG and long-term FF-MAS group compared to the STG (p < 0.05). Embryo formation rates and the proportion of good-quality embryos were higher in the MTG compared to the STG (100% vs. 75%; p = 0.03) and (83.3% vs. 33.3%; p = 0.01), respectively. Mitochondrial peripheral distribution was significantly higher in the MTG than in the STG (p = 0.04). Conclusion: Optimizing FF-MAS exposure duration enhances IVM efficiency, offering a promising strategy to increase oocyte utilization in in-vitro fertilization programs.

PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes.

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall invitro maturation efficiency and early embryonic stages.

Effect of Gentianella acuta (Michx.) Hulten against the arsenic-induced development hindrance of mouse oocytes.

The current study was designed to investigate the alleviative effect of Gentianella acuta (Michx.) Hulten (G. acuta) against the sodium arsenite (NaAsO2)-induced development hindrance of mouse oocytes. For this purpose, the in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs) was conducted in the presence of NaAsO2 and G. acuta, followed by the assessments of IVM efficiency including oocyte maturation, spindle organization, chromosome alignment, cytoskeleton assembly, cortical granule (CGs) dynamics, redox regulation, epigenetic modification, DNA damage, and apoptosis. Subsequently, the alleviative effect of G. acuta intervention on the fertilization impairments of NaAsO2-exposed oocytes was confirmed by the assessment of in vitro fertilization (IVF). The results showed that the G. acuta intervention effectively ameliorated the decreased maturation potentials and fertilization deficiency of NaAsO2-exposed oocytes but also significantly inhibited the DNA damages, apoptosis, and altered H3K27me3 expression level in the NaAsO2-exposed oocytes. The effective effects of G. acuta intervention against redox dysregulation including mitochondrial dysfunctions, accumulated reactive oxygen species (ROS) generation, glutathione (GSH) deficiency, and decreased adenosine triphosphate (ATP) further confirmed that the ameliorative effects of G. acuta intervention against the development hindrance of mouse oocytes were positively related to the antioxidant capacity of G. acuta. Evidenced by these abovementioned results, the present study provided fundamental bases for the ameliorative effect of G. acuta intervention against the meiotic defects caused by the NaAsO2 exposure, benefiting the future application potentials of G. acuta intervention in these nutritional and therapeutic research for attenuating the outcomes of arseniasis.

Follicular fluid C3a-peptide promotes oocyte maturation through F-actin aggregation

BackgroundImmature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides.ResultsIn current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC–MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with β-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes.ConclusionsOur results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

Regulation of hypoxia-inducible factor 1α by optimal oxygen concentration enhances oocyte maturation and early embryonic development in buffalo

Despite significant progress in vitro maturation (IVM) and in vitro culture (IVC) of oocytes and embryos, their developmental competence remains low. To address this issue, we used buffalo oocytes as a model system to investigate the effects and mechanisms of oxygen concentration on IVM and IVC. Our findings demonstrated that culturing buffalo oocytes with 5% oxygen significantly enhanced the efficiency of IVM and developmental competence of early embryos. Immunofluorescence results suggested that HIF1α played a critical role in these progresses. RT-qPCR results showed that maintaining a stable expression of HIF1α in cumulus cells with 5% oxygen concentration enhanced glycolysis, expansion, and proliferation abilities, up-regulated the expression of development-related genes, and suppressed apoptosis level. Consequently, it improved the maturation efficiency and quality of oocytes, leading to improve developmental capacity of buffalo early embryos. Similar outcomes were also observed when embryos were cultured with 5% oxygen. Collectively, our study provided insights into the role of oxygen regulation during oocytes maturation and early embryo development, and could potentially improve the efficiency of human assisted-reproduction technology.

Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation.

In vitro maturation (IVM) and cryopreservation of oocytes are two important parts of assisted reproductive technology (ART), but their efficacy is low. This study aimed to improve the quality of in vitro vitrified-warmed maturated oocytes using granulosa cell conditioned medium (GCCM). In the experimental study, fresh/non-vitrified and vitrified-warmed mouse germinal vesicle (GV) oocytes (as F and V) were In vitro maturated using basal medium (BM) and also BM supplemented with 50% GCCM as treated groups (GM), and categorized as FBM, FGM, VBM and VGM groups, respectively. The rate of successful IVM (MII oocyte formation), mitochondrial membrane potential and the viability of MII oocytes were determined using inverted microscopy, JC-1 and trypan blue staining. Then, the rate of In vitro fertilization (IVF) and subsequent two-cell embryo formation was calculated. Finally, the expression levels of Oct4, Sox2, Cdk-2, Gdf9, Integrin beta1 and Igf2 were analyzed using real-time polymerase chain reaction (PCR) in MII oocytes and two-cell embryos. These analyses showed that GCCM significantly increased the IVM rate, oocyte meiotic resumption and mitochondrial membrane potential (P<0.05). In addition, the rate of IVF and two-cell embryo formation was significantly higher in FGM and VGM compared to FBM and VBM (P<0.05). Interestingly, GCCM significantly affected the expression of the studied genes. Our findings suggest that GCCM might be useful for improving the efficiency of IVM and the subsequent IVF outcomes.

The Science of IVM

Oocyte in vitro maturation (IVM) makes use of oocytes from patients that have received minimal or no gonadotrophin stimulation. Whilst this brings many advantages to patients, this typically means oocytes are collected from small-medium sized (4-12 mm) antral follicles. These oocytes are under-developed, and yet removal of these from the follicle and culture in vitro leads to spontaneous oocyte meiotic maturation, without the benefit of the physiological signals that naturally induce oocyte maturation at ovulation. Using animals models, over the past decade seminal advances have been made in our understanding of the fundamental mechanisms regulating oocyte maturation in vivo. Most IVM systems, as practised clinically today, typically do not recapitulate these key cellular processes, possibly accounting for the lower efficiency of IVM compared to IVF. A key objective of modern approaches to IVM is to restore in vitro, as far as possible, the natural processes that occur during oocyte maturation in vivo. One strategy to achieve this in IVM is to: 1) prevent spontaneous meiotic resumption at oocyte collection using phosphodiesterase inhibitors, then 2) artificially maintain or elevate cumulus-oocyte complex (COC) cAMP levels, and finally to 3) induce oocyte meiotic resumption using EGF-like peptides. This typically requires the use of biphasic or 2-step IVM systems. One such biphasic IVM system is called capacitation-IVM (CAPA-IVM), as the oocyte is “capacitated” for development in vitro. CAPA-IVM uses the follicle’s natural oocyte meiotic inhibitor, c-type natriuretic peptide (CNP), in the pre-IVM phase, and amphiregulin as the oocyte meiotic inducer in the second IVM phase. Such biphasic-IVM systems typically lead to subsequent improvements in embryo yield compared to standard IVM, and indeed this is the case for CAPA-IVM. Several pilot RCTs and a large RCT of CAPA-IVM vs conventional IVF have been completed, and CAPA-IVM is now practiced in a few centres globally and is commencing in Sydney. The development and clinical application of modern IVM systems, built on decades of animal scientific research, is a bench-to-bedside success story of biomedical research. IVM is now a viable minimally invasive procedure for the treatment of infertility and for fertility preservation in female cancer patients.

NAT10 Maintains OGA mRNA Stability Through ac4C Modification in Regulating Oocyte Maturation.

In vitro maturation (IVM) refers to the process of developing immature oocytes into the mature in vitro under the microenvironment analogous to follicle fluid. It is an important technique for patients with polycystic ovary syndrome and, especially, those young patients with the need of fertility preservation. However, as the mechanisms of oocyte maturation have not been fully understood yet, the cultivation efficiency of IVM is not satisfactory. It was confirmed in our previous study that oocyte maturation was impaired after N-acetyltransferase 10 (NAT10) knockdown (KD). In the present study, we further explored the transcriptome alteration of NAT10-depleted oocytes and found that O-GlcNAcase(OGA) was an important target gene for NAT10-mediated ac4C modification in oocyte maturation. NAT10 might regulate OGA stability and expression by suppressing its degradation. To find out whether the influence of NAT10-mediated ac4C on oocyte maturation was mediated by OGA, we further explored the role of OGA in IVM. After knocking down OGA of oocytes, oocyte maturation was inhibited. In addition, as oocytes matured, OGA expression increased and, conversely, O-linked N-acetylglucosamine (O-GlcNAc) level decreased. On the basis of NAT10 KD transcriptome and OGA KD transcriptome data, NAT10-mediated ac4C modification of OGA might play a role through G protein–coupled receptors, molecular transduction, nucleosome DNA binding, and other mechanisms in oocyte maturation. Rsph6a, Gm7788, Gm41780, Trpc7, Gm29036, and Gm47144 were potential downstream genes. In conclusion, NAT10 maintained the stability of OGA transcript by ac4C modification on it, thus positively regulating IVM. Moreover, our study revealed the regulation mechanisms of oocytes maturation and provided reference for improving IVM outcomes. At the same time, the interaction between mRNA ac4C modification and protein O-GlcNAc modification was found for the first time, which enriched the regulation network of oocyte maturation.

The Improvement and Clinical Application of Human Oocyte In Vitro Maturation (IVM).

Oocyte in vitro maturation (IVM) is a technology with a long history that was established before IVF. Although it has been studied extensively, the efficiency of IVM has been poor for almost 30 years. In terms of the benefits of IVM, the efficiency and adoption of IVM are being improved by some notable improvements that have occurred in recent years. The establishment of biphasic IVM is the most important advancement in recent years. Biphasic IVM includes the pre-IVM culturing phase and IVM phase. The CNP-mediated pre-IVM culturing system is specifically tailored for non/minimally stimulated immature oocytes, and its efficiency has been shown. This is the most significant improvement made in recent decades in this area. In the clinic, IVM can be used for PCOS patients to avoid the occurrence of ovarian hyperstimulation syndrome (OHSS). Additionally, this method can solve the reproductive problems of some patients with special diseases (resistant ovary syndrome) that cannot be solved by IVF. In most fertility preservation procedures, oocytes in small antral follicles are lost. However, IVM has the ability to capture this kind of oocyte and save reproductive potential. IVM can be easily combined with fertility preservation strategies that have been applied in the clinic and improve the efficiency of fertility preservation. IVM is a useful and attractive technology and may be used widely worldwide in the near future.

Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation.

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p<.05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p<.05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p<.05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus-oocyte complexes (COCs). Our study indicated that TCCM (2days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.

Analysis of the activity of oncocalyxone A (Auxemma oncocalyx) and doxorubicin on the in vitro development of porcine oocytes

Most anticancer drugs like doxorubicin (DXR) have low specificity that results in undesirable effects especially when it comes to collateral effects on reproduction. Plants are excellent sources when searching for new drugs. Auxemma oncocalyx (A. oncocalyx) and its main component Oncocalyxone A (onco A) have anti-tumoral activity and are less toxic than DXR in reproductive parameters. However, there are no studies on the action of these drugs regarding the porcine in vitro oocyte competence and embryo development. The aim of this study was to evaluate the effect of A. oncocalyx and onco A exposure during in vitro maturation (IVM) of oocytes (Experiment 1) or in vitro embryo culture (IVC) (Experiment 2) on the oocyte developmental competence. For experiment 1, COCs were distributed in IVM medium alone (control) or supplemented with DXR (0.3 g/mL), A. oncocalyx (1.2 g/mL) and onco A (1 g/mL). Then, oocytes were submitted to in vitro fertilization (IVF) and in vitro embryo culture. For experiment 2, zygotes were cultured with DXR, A. oncocalyx and onco A for 7 days. Viability, maturation, fertilization and embryo developmental parameters were evaluated in both experiments. In experiment 1; DXR, A. oncocalyx and onco A reduced (P&lt;0.05) oocyte viability and IVM efficiency. Onco A increased (P&lt;0.05) the meiotic resumption. After IVF, all drugs reduced (P&lt;0.05) viability, IVF efficiency and percentage of cleaved embryos, nevertheless, only DXR decreased the percentage of blastocyst. In experiment 2; all drugs reduced (P&lt;0.05) the percentage of penetration, but only DXR and onco A decreased (P&lt;0.05) IVF efficiency. DXR and A. oncocalyx decreased (P&lt;0.05) the percentage of cleaved embryo, but had no effect on blastocyst formation. In conclusion, the addition of DXR during IVM or IVC negatively affected the IVF efficiency and cleavage rate. In addition, the exposure of COCs to DXR only during IVM was more detrimental to oocyte viability and blastocyst formation than A. oncocalyx and onco A.

Mogroside V improves porcine oocyte in vitro maturation and subsequent embryonic development

Oocyte in vitro maturation (IVM) plays a pivotal role in in vitro embryo production. However, the efficiency of IVM is still low and needs to be further improved. In the present study, we evaluated the beneficial effects of mogroside V, an extract derived from Siraitia grosvenorii, on oocyte IVM. Porcine cumulus–oocyte complexes were cultured in IVM medium supplemented or not supplemented with mogroside V for 40 h. We found that mogroside V supplementation increased the percentage of oocyte first polar body extrusion and improved subsequent blastocyst formation after parthenogenetic activation. Furthermore, mogroside V reduced the levels of reactive oxygen species (ROS) and increased the mRNA expression of oxidative stress-related genes (SOD, CAT and SIRT1). Moreover, mogroside V supplementation enhanced the mitochondrial content, mtDNA copy number, mitochondrial membrane potential (ΔΨm), ATP generation, and the relative mRNA expression of mitochondria-related genes (PGC-1α and TFAM). In summary, our findings demonstrate that mogroside V supplementation reduces intracellular ROS levels and enhances mitochondrial function to promote porcine oocyte IVM.

Effect of myostatin inhibitor (myostatin pro-peptide) microinjection on in vitro maturation and subsequent early developmental stages of buffalo embryo

Expression of myostatin (MSTN, also known as growth differentiation factor 8, GDF8) was recently detected in cumulus-oocytes complexes (COCs), however little is known about its role in in vitro maturation (IVM) and fertilization (IVF) in large animals. Therefore, this study was designed to investigate the effect of MSTN inhibition on IVM of buffalo oocytes through investigation of IVM efficiency and expression of some specific genes in COCs from IVM till subsequent developmental stages following IVF. To reach this goal, we prepared a construct of adeno-associated virus (AAV) carrying MSTN pro-peptides (AAV-MSTNP) to inhibit MSTN. Over-expression of MSTNP was verified by upregulated expression of MSTNP and downregulated expression of the TGFβ receptor ActRIIb, the TGFβ signal transducer SMAD2 in COCs using qPCR. Microinjection of AAV-MSTNP to oocytes before IVM yielded a significant decrease in maturation rate as revealed by less cumulus cells expansion, fewer oocytes reaching metaphase II, and downregulation of cumulus expansion-related genes pentraxin 3 (Ptx3) and prostaglandin-endoperoxide synthase 2 (Ptgs2) as compared to the control and vehicle groups. These changes were also accompanied by elevated intracellular reactive oxygen species (ROS), upregulated expression of the apoptotic Bax gene, reduced antioxidant enzymes (SOD, CAT, GPX) activities, and downregulated expression of the antioxidant gene nuclear factor erythroid 2 like 2 (Nrf2), and the anti-apoptotic gene Bcl2 in COCs after IVM. Overexpression of MSTN inhibitor, MSTNP, also inhibited GDF9 and BMP15 genes expression in COCs. Additionally, both the fertilization efficiency and cleavage and blastocyst rates were significantly lower in MSTNP group than in the control and vehicle groups. The obtained data suggest an important role for MSTN during IVM and the subsequent developmental stages probably through, at least in part, inhibition of ROS production and apoptosis and modulation of IVM-related gene expression in COCs.

177 Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes

In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P&amp;lt;0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P&amp;gt;0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P&amp;lt;0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate. Supported by RFBR (18-29-07089) and UAES (1343).

In vitro maturation (IVM) of oocytes in patients with resistant ovary syndrome and in patients with repeated deficient oocyte maturation.

To assess the efficiency of IVM in patients with repeated ART failure due to resistant ovary syndrome or due to deficient oocyte maturation. Clinical and laboratory data were obtained retrospectively from 28 patients who underwent 49cycles of IVM between 2010 and 2017; nine patients had resistant ovary syndrome and 19 patients had repeated deficient oocyte maturation. Nine patients with resistant ovary syndrome underwent 24 IVM cycles. In those, an average of 11.5 ± 10.4 cumulus-oocyte complexes (COC) was retrieved, and IVM resulted in 3.4 ± 3.1 mature oocytes. After ICSI and transfer of 23 cleavage-stage embryos, eight pregnancies were obtained, resulting in five healthy live births. The live birth rate was 16.7% per started cycle and 33.3% per patient. Nineteen patients with a history of deficient oocyte maturation underwent 25 IVM cycles. An average of 10.6 ± 9.2 COC was retrieved, and after IVM, 1.3 ± 2.1 oocytes were mature. No mature oocytes were obtained in 11cycles. In ten cycles with mature oocytes, none of them fertilized after ICSI. Out of four cycles with fertilized oocytes, only one good-quality embryo was obtained. No live births were obtained after IVM in patients with a history of deficient oocyte maturation. Based on our experience, IVM is a valuable approach in patients with resistant ovary syndrome, but should not be recommended for patients with deficient oocyte maturation.

A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach

The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.

Dose-dependent effects of frutalin on in vitro maturation and fertilization of pig oocytes

The aim of the present study was to evaluate the effect of frutalin (FTL) on in vitro maturation (IVM), and fertilization (IVF) of pig oocytes. In the Experiment 1, cumulus-oocyte complexes (COCs) were submitted to IVM in maturation medium alone or supplemented with different FTL concentration (0.6, 6 and 60 μg/mL), or 0.3 μg/mL doxorubicin (DXR). After IVM, some oocytes were evaluated for chromatin configuration, and the remaining oocytes were submitted to in vitro fertilization. In Experiment 2, matured oocytes were fertilized in IVF medium alone (control) or in presence of different FTL concentration (0.6, 6 and 60 μg/mL), or 0.3 μg/mL DXR. After 18 h post fertilization, the endpoints penetration rate, monospermy, spermatozoa per oocyte, and the IVF efficiency were evaluated in both experiments. In Experiment 1, 6 and 60 μg/mL FTL, as well as DXR increased (P < 0.05) the rate of oocytes with abnormal chromatin configuration when compared to oocyte matured in control medium alone or supplemented with 0.6 μg/mL FTL. The percentage of meiotic resumption in oocytes cultured with 60 μg/mL FTL or DXR was less (P < 0.05) than in the other treatments. Moreover, oocytes matured with 6 or 60 μg/mL FTL and DXR had a lesser IVM efficiency when compared to those matured with 0.6 μg/mL FTL or in control medium. Additionally, there was a greater (P < 0.05) with culture in a medium containing 6 μg/mL FTL for the rate of partenogenetically activated oocytes when compared with the other treatments. Culturing of COCs during IVM in a medium containing 6 or 60 FTL resulted in a lesser (P < 0.05) sperm penetration and spermatozoa/oocyte rates when compared to other treatments, and IVF efficiency was less (P < 0.05) than that in control medium alone or with a medium containing 0.6 μg/mL FTL. In Experiment 2, culturing in a medium containing 0.6 μg/mL FTL resulted in greater (P < 0.05) monospermy and IVF efficiency rates when compared to culturing in the control medium. In addition, culturing in a medium with 6 and 60 μg/mL FTL resulted in a lesser (P < 0.05) spermatozoa penetration, sperm/oocyte rates and IVF efficiency, although there were greater (P < 0.05) monospermy rates. In conclusion, culturing in a medium containing 0.6 μg/mL FTL resulted in lesser spermatozoa penetration rates and number of spermatozoa/oocyte increasing the IVF efficiency without harmful effects. Use of a greater concentration of FTL in the medium has toxic effects during oocyte maturation and results in a reduced IVF efficiency.

Effects of three different media on in vitro maturation and development, intracellular glutathione and reactive oxygen species levels, and maternal gene expression of abattoir-derived goat oocytes

This study was designed to compare the behavior of abattoir-derived goat oocytes submitted to different in vitro maturation (IVM) treatments. Cumulus-oocyte complexes collected from abattoir ovaries were subjected to IVM in three maturation media, which consisted of TCM199 supplemented either with: (1) 10ng/ml EGF and 100μM cysteamine (Defined medium); (2) 10% FCS, 5μg/ml FSH, 10μg/ml LH, 1μg/ml estradiol (FCS medium); or (3) combined components of both Defined and FCS media (Combined medium). After 22h of IVM, oocyte maturation rate was significantly higher in Combined medium compared to FCS medium (52.0±2.0% vs. 37.7±3.2%, P<0.05); however, no significant difference (P>0.05) was observed in maturation rates between Defined (43.4±4.7%) and FCS or Combined medium. There was no significant difference (P>0.05) in the rates of cleavage, blastocyst, or blastocyst cell number among different maturation conditions. The intracellular glutathione (GSH) level was observed to be significantly higher (P<0.05) in oocytes matured in Combined medium compared to either Defined or FCS medium. The level of intracellular reactive oxygen species (ROS) was lower (P<0.05) in Defined medium compared to both FCS and Combined media. Relative mRNA expression analysis of 39 developmentally associated genes revealed that four genes (MATER, PLAT, NANOG, and CCNB1) were differentially expressed between the groups. In summary, our results indicate that similar in vitro maturation rates and developmental ability can be obtained when Defined and Combined maturation media were used and the supplementation of FCS medium with EGF and Cysteamine improves IVM efficiency of goat oocytes.

Oocyte maturation and quality: role of cyclic nucleotides.

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities.

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.