Cumulus-oocyte Complexes Research Articles

Industrialization possibilities of purified pig sperm hyaluronidase.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

Decreased HAT1 expression in granulosa cells disturbs oocyte meiosis during mouse ovarian aging

BackgroundWith advanced maternal age, abnormalities during oocyte meiosis increase significantly. Aneuploidy is an important reason for the reduction in the quality of aged oocytes. However, the molecular mechanism of aneuploidy in aged oocytes is far from understood. Histone acetyltransferase 1 (HAT1) has been reported to be essential for mammalian development and genome stability, and involved in multiple organ aging. Whether HAT1 is involved in ovarian aging and the detailed mechanisms remain to be elucidated.MethodsThe level of HAT1 in aged mice ovaries was detected by immunohistochemical and immunoblotting. To explore the function of HAT1 in the process of mouse oocyte maturation, we used Anacardic Acid (AA) and small interfering RNAs (siRNA) to culture cumulus-oocyte complexes (COCs) from ICR female mice in vitro and gathered statistics of germinal vesicle breakdown (GVBD), the first polar body extrusion (PBE), meiotic defects, aneuploidy, 2-cell embryos formation, and blastocyst formation rate. Moreover, the human granulosa cell (GC)-like line KGN cells were used to investigate the mechanisms of HAT1 in this progress.ResultsHAT1 was highly expressed in ovarian granulosa cells (GCs) from young mice and the expression of HAT1 was significantly decreased in aged GCs. AA and siRNAs mediated inhibition of HAT1 in GCs decreased the PBE rate, and increased meiotic defects and aneuploidy in oocytes. Further studies showed that HAT1 could acetylate Forkhead box transcription factor O1 (FoxO1), leading to the translocation of FoxO1 into the nucleus. Resultantly, the translocation of acetylated FoxO1 increased the expression of amphiregulin (AREG) in GCs, which plays a significant role in oocyte meiosis.ConclusionThe present study suggests that decreased expression of HAT1 in GCs is a potential reason corresponding to oocyte age-related meiotic defects and provides a potential therapeutic target for clinical intervention to reduce aneuploid oocytes.

Conventional ovarian stimulation vs. delayed single dose corifollitropin alfa ovarian stimulation in oocyte donors: a prospective randomized study. Tail trial.

The present study compares two protocols for ovarian controlled stimulation in terms of number of cumulus-oocyte complexes and metaphase II oocytes. We employed a single injection of 150mcg of corifollitropin alfa after a 7-day oral contraceptive pill-free interval for TAIL group and a conventional administration of corifollitropin alfa after a 5-day OCP-free interval with additional rFSH from 8th of ovarian controlled stimulation. Prospective, randomized, comparative, non-inferiority, opened and controlled trial carried out in 180 oocyte donors 31 were excluded, 81 were randomized to the control group and 68 to the TAIL group. No differences were found in the number of follicles larger than 14 and 17 mm at triggering day. However, a lower number of cumulus-oocyte complexes and metaphase II oocytes were obtained in TAIL group compared to the control group, expressed as median (interquartile range): 10.5 (5.5-19) vs. 14 [11-21] and 9 (4-13) vs. 12 (9-17) respectively. Additionally, the incidence of failed retrieval or metaphase II oocytes = 0 was higher in TAIL group 7(10.3%) vs. 1(1.2%) p = 0.024. The use of a single injection of corifollitropin alfa after a 7-day oral contraceptive pill-free interval in oocyte donors resulted in a lower number of cumulus-oocyte complexes and metaphase II oocytes. No additional rFSH was administered in this group. Clinical Trial Registration: https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-001343-44/results.

Dynamic regulation of semaphorin 7A and adhesion receptors in ovarian follicle remodeling and ovulation

The ovarian follicle is a complex structure that protects and helps in the maturation of the oocyte, and then releases it through the controlled molecular and structural remodeling process of ovulation. The progesterone receptor (PGR) has been shown to be essential in regulating ovulation-related gene expression changes. In this study, we found disrupted expression of the cellular adhesion receptor gene Sema7A in the granulosa cells of PGR−/− mice during ovulation. We subsequently found that expression of Sema7A in preovulatory follicles is promoted by gonadotropins and hypoxia, establishing an asymmetrical pattern with the SEMA7A protein enriched at the apex of large antral follicles. Sema7A expression was downregulated through a PGR-dependent mechanism in the periovulatory period, the abundance of SEMA7A protein was reduced, and the asymmetric pattern became more homogeneous after an ovulatory stimulus. Receptors for Sema7A can either repel or promote intercellular adhesion. During ovulation, striking inverse regulation of repulsive Plxnc1 and adhesive Itga5/Itgb1 receptors likely contributes to dramatic tissue remodeling. The adhesive receptor Itga5 was significantly increased in periovulatory granulosa cells and cumulus–oocyte complexes (COCs), and functional assays showed that periovulatory granulosa cells and COCs acquire increased adhesive phenotypes, while Sema7A repels granulosa cell contact. These findings suggest that the regulation of Sema7A and its associated receptors, along with the modulation of integrin α5, may be critical in establishing the multilaminar ovarian follicle structure and facilitating the remodeling and apical release of the cumulus–oocyte complex during ovulation.

Improvement of oocyte competence and in vitro oocyte maturation with EGF and IGF-I in Guinea pig model

In vitro maturation (IVM) system is an alternative method to superovulation protocols to obtain mature oocytes. Epidermal Growth Factor (EGF) and Insulin-like Growth Factor I (IGF-I) have been widely used in IVM medium in different species. Although the guinea pig is a valuable animal model for reproductive studies, IVM is rarely used. We aimed to establish a suitable in vitro production system using EGF and/or IGF-I during IVM to improve oocyte competence. Firstly, immunolocalization of EGF and IGF-I receptors in the ovary was assessed. An IVM dose-response experiment was performed with cumulus-oocyte complexes (COCs) supplemented with: 1) EGF [0, 10, 50, 100 ng/mL or 10% fetal calf serum (FCS)]; 2) IGF-I [0, 50, 100, 200 ng/mL or 10% FCS]; or 3) the concentrations of EGF and IGF-I which showed the best IVM index in the previous experiments, with or without Fetal Calf Serum (FCS). Cortical granule and mitochondria distribution patterns were determined in in vivo and in vitro-matured oocytes for the first time in this species. Apoptotic rate after IVM and oocyte competence by in vitro embryo development were evaluated. Immunohistochemistry results showed positive immunostaining of EGF and IGF receptors in corpus luteum, oocytes, granulosa and theca cells in follicles in all stages of development. Supplementation of IVM medium with 50 ng/mL EGF or 100 ng/mL IGF-I or their combination with FCS successfully led to oocyte nuclear and cytoplasmic maturation and reduced the apoptotic rate. Both growth factors improved oocyte competence during IVM in this species since early embryos were in vitro developed, showing better results when FCS was used in the IVM medium.

Oocyte recovery after overnight ovary transport provides an alternative source of cumulus oocyte complexes that are competent to produce live piglets.

COVID-19 impacted abattoirs worldwide. The processing lines became a hotspot for the spread of COVID-19 resulting in plant restructuring and ultimately a critical loss of pig material for research. Commercial sources of pig oocytes are available but are costly and companies were already operating at a maximum capacity for supplying the oocyte needs around the United States. Here, we provide an alternative source of oocytes that are competent to produce live, healthy piglets.

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes.

In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

Deep machine learning applied to support clinical decision-making in the treatment of infertility using assisted reproductive technologies

Introduction. Machine learning (ML) applied to data analysis allows to more accurately and targetedly determine the most significant correctable and non-correctable predictors of onset of pregnancy in assisted reproductive technology (ART) programs in patients of different age groups. Analysis of data using various techniques and comparison of results obtained via two models will determine the most significant factors for onset of pregnancy in the ART program.Aim. To determine the most significant clinical and embryological predictors of onset of pregnancy using standard regression analysis and a decision tree algorithm to predict pregnancy in the ART program.Materials and methods. A total of 1,021 married couples were included in the retrospective study. The study analysed clinical and laboratory test findings and stimulated cycle parameters depending on the effectiveness of the ART program. A regression analysis was carried out and a decision tree algorithm was built using the Gini criterion to determine the most significant factors.Results. We identified “general” signs that require further validation on other models, including ML: the presence/absence of a history of pregnancies, stimulated cycle parameters (oocyte cumulus complex, number of metaphase II (MII) oocytes, number of zygotes), spermogram indicators on the day of puncture, number of high and good quality embryos, as well as the embryo grading.Conclusion. rFSH (follitropin-alpha, Gonal-f) gives a significant result in two of the five available age groups, follitropin-beta, corifollitropin alfa – in one of the five groups only. Building a model that includes not only the couple’s medical history data, but also molecular markers using machine learning methods will not only allow us to most accurately determine the most promising groups of patients for in vitro fertilization (IVF) programs, but also increase the efficiency of ART programs by selecting the highest quality embryo to be transferred.

Oocyte-Specific Deletion of Slc6a9 Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine. Glycine is transported during the mature egg and 1-cell to 4-cell embryo stages by a transporter identified as GLYT1, encoded by the Slc6a9 gene. Here, we have produced an oocyte-specific knockout of Slc6a9 by crossing mice that have a segment of the gene flanked by LoxP elements with transgenic mice expressing iCre driven by the oocyte-specific Gdf9 promoter. Slc6a9 null oocytes failed to develop glycine transport activity during meiotic maturation. However, females with these oocytes were fertile. When enclosed in their cumulus-oocyte complex, Slc6a9 null oocytes could accumulate glycine via GLYT1 transport in their coupled cumulus cells, which may support female fertility in vivo. In vitro, embryos derived from Slc6a9 null oocytes displayed a clear phenotype. While glycine rescued complete preimplantation development of wild type embryos from increased osmolarity, embryos derived from null oocytes failed to develop past the 2-cell stage even with glycine. Thus, Slc6a9 is required for glycine transport and protection against increased osmolarity in mouse eggs and early embryos.

Should the trigger to oocyte retrieval interval be different in progestin-primed ovarian stimulation cycles?

Research questionDoes the trigger to oocyte retrieval interval (TORI) affect oocyte maturation rates differently in progestin-primed ovarian stimulation (PPOS) and gonadotrophin-releasing hormone (GnRH) antagonist cycles? DesignThis was a retrospective cohort study. The interaction between the stimulation protocol and TORI was assessed in a linear mixed effects multivariable regression analysis with oocyte maturation rate as the dependent variable, and stimulation protocol (GnRH antagonist or PPOS), age (continuous), gonadotrophin type (FSH or human menopausal gonadotrophin), trigger (human chorionic gonadotrophin [HCG] or GnRH agonist), TORI (continuous) and days of stimulation (continuous) as the independent variables. Oocyte maturation rate was defined as number of metaphase II oocytes/number of cumulus–oocyte complexes retrieved. The maturation rate was calculated per cycle and treated as a continuous variable. ResultsA total of 473 GnRH antagonist and 205 PPOS cycles (121 conventional PPOS and 84 flexible PPOS) were analysed. The median (quartiles) female age was 36 (32–40) years. Of these cycles, 493 were triggered with HCG and 185 with a GnRH agonist. The TORI ranged between 33.6 and 39.1 h, with a median (quartiles) of 36.2 (36–36.4) hours. Maturation rates were similar between fixed PPOS, flexible PPOS and antagonist cycles (median 80%, 75% and 75%, respectively, P = 0.15). There was no significant interaction between the stimulation protocols and TORI for oocyte maturation. ConclusionsPPOS cycles do not seem to require a longer TORI than GnRH antagonist cycles.

Laparoscopic ovum-pick up and in vitro embryo production in gonadotropin-stimulated gilts: Preliminary results and envisioned applications

The present study was conducted to establish if laparoscopic ovum pick-up (LOPU) could be adapted to the swine species, and if the developmental competence of LOPU-sourced oocytes from peripubertal gilts could be improved by gonadotropin stimulation, by comparing with oocytes sourced from slaughtered gilts lacking hormonal stimulation. Estrus was synchronized in 34 gilts of ∼6–8 months of age by daily oral administration of 17.6 mg altrenogest for 13 days and 10 mg dinoprost IM on the last day of altrenogest. Follicular development was stimulated in all gilts with a single injection of 1250 IU eCG given 3 days before LOPU (together with the 12th dose of altrenogest). In about half of the gilts (Group eCG-hCG), 500 IU hCG were injected IM ∼72 h after eCG injection, or ∼16–18 h prior to LOPU, to initiate oocyte maturation in vivo, while the remaining animals only received eCG (Group eCG). Most gilts underwent LOPU twice alternating the gonadotropin protocol, thereby decreasing the impact of individual variation on results. Abattoir-sourced oocytes from prepubertal gilts served as Control. Following LOPU, oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage following standard procedures, while oocytes collected from Group eCG-hCG gilts were considered partly matured in vivo and were matured for ∼24 h instead of ∼44 h. Embryos reaching the blastocyst stage were fixed and stained to assess quality through cell numbers. There were no significant differences in the number of follicles aspirated and cumulus-oocyte complexes (COCs) recovered between Groups eCG-hCG and eCG (22.4 and 16.9 vs. 22.6 and 17.6, P > 0.05), as well as the recovery rate (76.6 vs. 78.1, P > 0.05). Cleavage rate was not different between Group eCG-hCG, Group eCG and Control (61.1 vs. 64.4 vs. 53.4 %, P > 0.05). However, the blastocyst rate over total oocytes (32.2 vs. 36.9 vs. 11.1 %, P < 0.05), blastocyst rate over cleaved oocytes (51.8 vs. 55.1 vs. 21.2 %, P < 0.01) and the average number of cells/blastocyst (89.6 vs. 87.5 vs. 62.2, P < 0.01) were unaffected by hCG treatment in LOPU-sourced oocytes, but both LOPU groups were significantly higher than abattoir-sourced oocytes, respectively. Our results suggest LOPU may become a powerful tool for sourcing swine oocytes with higher developmental competence than abattoir-sourced oocytes and known disease status for creating swine models for human biomedical applications, as well as for accelerated genetic gain in swine breeding programs.

MiR-155-5p improves oocyte maturation in porcine cumulus cells through connexin 43-mediated regulation of MPF activity

In this study, we aimed to investigate the effect of the expression of miR-155-5p and its target genes on oocyte maturation. We analyzed the expression of miR-155-5p and its target genes in cumulus cells and oocytes using quantitative real-time reverse-transcription polymerase chain reaction. Using carboxyfluorescein, porcine cumulus cells were transfected with mimics and inhibitors of ssc-miR-155-5p to induce in vitro maturation, and subsequently, cumulus expansion, oocyte maturation, and cleavage rate were measured. We found that miR-155-5p expression in cumulus cells at the metaphase II stage was significantly higher than that at the germinal vesicle (GV) stage, whereas Cx43 expression was significantly lower than that at the GV stage (P < 0.05). Compared with those in the negative control group, the cumulus diffusion area of cumulus oocyte complexes; oocyte maturation rate; cleavage rate; HAS2, PTGS2, CD44, PTX3, and TNFAIP6 expression in cumulus cells; and GDF9, BMP15, CyclinB1, and CDK1 expression in oocytes were significantly increased in the miR-155-5p mimics group (P < 0.05), whereas the mRNA and protein expression of CX43 were significantly decreased (P < 0.05). Compared with that in the negative control group, the protein expression of CyclinB1 and p-CDK1 (Thr14, Tyr15) in the miR-155-5p mimics group was significantly increased (P < 0.05). These results suggest that miR-155-5p regulates maturation promoting factor activity by targeting Cx43, which improves the in vitro maturation and cleavage rate of porcine oocytes.

Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan.

Reproductive aging is associated with ovulatory defects. Age-related ovarian fibrosis partially contributes to this phenotype as short-term treatment with anti-fibrotic compounds improves ovulation in reproductively old mice. However, age-dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time-lapse microscopy. The extracellular matrix integrity of expanded cumulus-oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA-associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age-related infertility and may serve as a target to extend reproductive longevity.

Putrescine alleviates the oxidative damage of cumulus-oocyte complex via improving fatty acid oxidation

BackgroundFatty acid oxidation of cumulus-oocyte complex (COC) provides sufficient energy for oocyte maturation. But, the relationship between fatty acid oxidation and oxidative stress in aging follicles, as well as the effect of putrescine, is still unclear. MethodsThe porcine COCs were randomly divided into four groups and cultured in in vitro maturation (IVM) medium with or without 1 mmol/L putrescine, with 50 μmol/L hydrogen peroxide (H2O2) or with 50 μmol/L H2O2 plus 1 mmol/L putrescine. Oocyte maturation was assessed by the first polar body extrusion. The expressions of genes involved in fatty acid oxidation were detected, and the mitochondrial function was analyzed by themembrane potential. ResultsThe maturation rate of oocyte was significantly lower in the H2O2 group when compared with the control group (P＜0.001), and putrescine significantly increased this rate in the H2O2 plus putrescine group when compared with the H2O2 group (P＜0.001). The expressions of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in cumulus cells (CCs) were significantly downregulated by H2O2 treatment, and partly rescued by putrescine addition (P＜0.05–0.001). However, the changes of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in oocytes were inapparent. The mitochondrial membrane potential of CCs in the H2O2 group was significantly lower than that in the control group, while putrescine addition significantly increased the mitochondrial membrane potential (P＜0.001). ConclusionThe decrease of oocyte maturation due to oxidative stress is related with the decreased fatty acid oxidation, and putrescine may alleviate the COCs damage via improving fatty acid oxidation.

The ERβ-cAMP signaling pathway regulates estradiol-induced ovine oocyte meiotic arrest

Although 17β-estradiol (E2) and its receptors (ERs) are reported to play important roles in regulating oocyte maturation, the specific mechanism remains unclear. First, we performed immunohistochemistry analyses to determine the expression of the ERα and ERβ proteins in ovine ovarian tissue. Second, E2 (0.5 ng/mL and 1 μg/mL) were added to pre-IVM medium for 0 h, 1 h and 2 h. The effects of E2 (0.5 ng/mL and 1 μg/mL) on cyclic adenosine monophosphate (cAMP) level in cumulus-oocyte complexes (COCs) and on oocyte meiotic progression were evaluated by ELISA and DAPI staining respectively. Third, the effects of E2 on the gene and protein expression of ERα and ERβ in COCs were investigated by Western blotting and real-time PCR. Afterward, ERβ and cAMP regulators were added to the 2-h pretreatment medium with or without E2 (0.5 ng/mL) to explore the possible interactions among E2, cAMP and ERβ. The results showed that both ERα and ERβ proteins were expressed in ovine cumulus layers and oocytes. E2 significantly increased intra-COC cAMP levels, maintained oocyte meiotic arrest, and promoted ERβ transcript and protein expression. E2 treatment increased the cAMP concentration, which was enhanced by ERβ agonist treatment and remarkably attenuated by ERβ inhibitor treatment. Forskolin plus IBMX treatment increased ERβ protein expression in COCs (P < 0.05), and this was attenuated by Rp-cAMP treatment. In conclusion, E2 (0.5 ng/mL) increased intra-COC cAMP levels by promoting ERβ expression, thereby maintaining oocyte meiotic arrest. cAMP in COCs has a positive feedback effect on ERβ expression, which provides a novel explanation for the positive role of E2 in regulating ovine follicle development and oocyte maturation.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Human TIMP1 Is a Growth Factor That Improves Oocyte Developmental Competence.

Oocyte developmental competence is the ability of a mature oocyte to be fertilized and subsequently support embryonic development. Such competence is gained during folliculogenesis and is facilitated by the bidirectional communication into a compacted cumulus-oocyte complex (COC). Human tissue inhibitor of metalloproteinases-1 (TIMP1) participates in biological processes, including cell growth, differentiation, and apoptosis. This study aimed to evaluate the influence of TIMP1 as a growth factor on the in vitro maturation (IVM) culture of bovine COCs to improve oocyte developmental competence. All TIMP1 treatments (50, 100, and 150 ng/mL) favored the COCs' compaction structure (p < 0.05). TIMP1 at 150 ng/mL produced more oocytes in metaphase II compared to the other treatments (p < 0.05). The 150 ng/mL TIMP1 generated oocytes with the most (p < 0.05) cortical granules below the plasma membrane (pattern I). In a parthenogenesis assay, oocyte IVM in 50 ng/mL of TIMP1 produced the most blastocyst compared to the other treatments (p < 0.05). The Principal Component Analysis (PCA) showed that 50 ng/mL of TIMP1 was the best condition to develop oocyte competence because it was associated with the COC compact and cortical granule pattern I. TIMP1 influences the development of oocyte competence when added to the IVM culture medium of COCs.

Experiences with transvaginal Ovum Pick-Up (OPU) in sows

Transvaginal ultrasound-guided Ovum Pick-Up (OPU) is an established technique in other species. Due to several challenges, there are few publications addressing the procedure in sows. An efficient OPU technique may allow for the collection of numerous oocytes from valuable sows for porcine in vitro embryo production, gene editing and cloning programmes, or cryopreservation. We aimed to improve transvaginal OPU and equipment for this technique in sows. In experiment 1, we conducted 13 OPU sessions on three Landrace x Large White hybrid sows under general anaesthesia, while the second experiment explored OPU in non-sedated animals (N = 6) physically restrained in a commercial claw trimming chute. The experiments resulted in 6.6 ± 5.6 (mean ± SD) and 7.7 ± 8.9 recovered cumulus-oocyte complexes per session, respectively. Post-mortem examination of the pelvic and abdominal cavities of the three sows subjected to repeated OPU sessions did not reveal major acute or chronic pathological lesions. The only sow which was inseminated after the experiment delivered 16 liveborn piglets at term. Salivary cortisol levels increased during the procedure in non-sedated and physically restrained sows but returned to baseline 1 h later (n = 5), indicating a short-term stress response. The described OPU technique and equipment have the potential to retrieve considerable numbers of oocytes by repeated procedures on valuable mature sows. Follow-up studies are needed to optimize the efficiency of the aspiration of high-quality oocytes and to describe the developmental competence of these OPU-derived oocytes. It is also essential to further investigate sow welfare during and after the procedure before recommending porcine transvaginal OPU as a sustainable and welfare-friendly procedure.

The birth of a healthy child in a married couple with a history of multiple unsuccessful attempts at ART by selecting sperm on oocyte-cumulus complexes. Case report

In male infertility, couples must use assisted reproductive technologies with the intracytoplasmic sperm injection (ICSI) technique. A significant disadvantage of ICSI is bypassing natural selection barriers since selective processes in the female reproductive tract are replaced by the choice of an embryologist based on sperm motility and morphological criteria. Various selection methods are being actively studied, bringing fertilization by ICSI closer to physiological, ensuring the selection of sperm with high DNA quality and good potential for fertilization. The article presents a clinical case of the birth of a healthy child in a married couple with severe male infertility and many ineffective attempts at assisted reproductive technologies using the method of sperm selection on oocyte-cumulus complexes. The child is physically healthy and develops according to age.

Assessment of developmental rate of mouse embryos yielded from in vitro fertilization of the oocyte with treatment of melatonin and vitamin C simultaneously

BackgroundIn recent decades, in vitro fertilization (IVF) has been widely used as a method of assisted reproductive technology (ART) to improve fertility in individuals. To be more successful in this laboratory method, we used the presence of two common types of antioxidants (melatonin and vitamin C) simultaneously and exclusively in IVF medium.MethodsThe cumulus-oocyte complexes (COCs) were obtained from Gonadotropin-releasing hormone (GnRH) and Human Chorionic Gonadotropin (HMG) -stimulated mice. Subsequently, metaphase II (MII) oocytes were fertilized in vitro. In the experiment, the IVF medium was randomly divided into two equal groups: The control group did not receive any antioxidants. In the treatment group, 100 µM melatonin and 5 mM vitamin C were added to the IVF medium. Finally, oocytes and putative embryos transferred into developmental medium and cultured 120 h after IVF to the blastocyst stage. After and before IVF, oocytes and putative embryos were stained with dichlorodihydrofluorescein diacetate (DCFDA) and the H2O2 level was measured with an inverted fluorescence microscope using ImageJ software. At the end of the fifth day after IVF, the expression of Bax and B cell lymphoma 2 (Bcl2) was evaluated using real-time PCR.ResultsThe levels of reactive oxygen species (ROS) in oocytes and putative embryos observed in the treatment group demonstrated a significant reduce compared to the control group (p ≤ 0.01. (.Furthermore, the number of embryos in the blastocycte stage(P < 0.05), the expression level of the Bcl2 (P < 0.05) gene, the Bax unlike gene, significantly increased compared with the control group.ConclusionWe conclude that the presence of melatonin and vitamin C antioxidants simultaneously and exclusively in the IVF medium leads to a reduction in ROS and ,as a result, improves the growth of the embryo up to the blastocyst stage.