Search Results (74)

The preservation of porcine oocytes is critically important for advancing superior breeds and conserving genetic resources in pig production. Vitrification has gained traction as a preferred alternative to slow freezing for porcine oocytes because of its effectiveness in reducing ice crystal formation, yet it can still negatively affect oocyte quality, compromising their in vitro maturation (IVM) and later embryonic development. Long non-coding RNAs (lncRNAs) have proven to be key players in numerous biological processes, such as oocyte growth, maturation, and early embryogenesis. Despite this, the effects of vitrified porcine germinal vesicle (GV) oocytes, particularly regarding IVM and the dynamic expression patterns of lncRNAs during embryonic development, remain largely unclear. To address this gap, this study conducted lncRNA sequencing at the metaphase II (MII), parthenogenetic 4-cell embryo, and parthenogenetic blastocyst stages sourced from both fresh and vitrified GV oocytes. This method enabled us to ascertain the impact of vitrification on lncRNA expression throughout oocyte maturation and embryonic development. Results identified 773 differentially expressed lncRNAs (DELs) at the MII stage, 1973 at the parthenogenetic 4-cell, and 1192 at the parthenogenetic blastocyst. Enrichment analysis of forecasted target genes revealed their involvement in key regulatory pathways associated with the cell cycle, meiosis, stress response, and metabolic activity. Overall, this study provides a comprehensive overview of lncRNA expression during oocyte maturation and embryonic development following porcine GV oocyte vitrification, thereby shedding light on the molecular mechanisms behind vitrification-induced damage. Full article

Fertility-sparing treatments (FSTs) have gained importance for young female cancer patients, especially those with early-stage cervical, ovarian, and endometrial cancers. However, concerns about the long-term safety of these procedures, particularly in more advanced cancers, persist. A literature review was conducted using databases such as PubMed, Scopus, and Web of Science. The search terms included “fertility preservation” and “gynaecological cancer”. Articles published between 2014 and 2024 were considered, with 39 articles cited in the paper. The inclusion criteria were female patients undergoing FST. Studies were excluded if prior treatments impacted fertility or if oncological outcomes were inadequately reported. Radical trachelectomy, laparoscopic fertility-sparing surgeries, and cryopreservation techniques, such as ovarian tissue vitrification and oocyte cryopreservation, offer viable options for preserving fertility in early-stage gynecological cancer patients. Radical trachelectomy and cryopreservation showed positive reproductive outcomes, with pregnancy rates of 30–50% in early-stage cases. GnRH analogs during chemotherapy also demonstrated benefits in maintaining fertility. Despite these advances, recurrence in more advanced stages (FIGO IA2 and beyond) remains a concern. Minimally invasive surgeries like robotic-assisted procedures demonstrated comparable fertility outcomes to traditional methods but with fewer complications. FST is a promising option for women with early-stage cancer, offering favorable reproductive and survival outcomes. However, further research is needed to confirm long-term oncological safety in advanced stages. Multidisciplinary approaches and individualized treatment planning are essential for optimizing outcomes. Full article

The aim was to evaluate the effectiveness of semen cryopreservation and oocyte vitrification in roe deer as a potential method of gamete preservation for endangered deer species. Sperm were isolated from the cauda epididymis of fourteen bucks (n = 14). The motility measure (CASA) and morphology of fresh semen (FS) and frozen–thawed semen (TS) were compared. A hyaluronic binding assay was used to distinguish between mature FS spermatozoa expressing hyaluronan receptors and immature FS lacking these receptors, and the mitochondrial membrane potential (MMP) in TS was determined (flow cytometry). A Sperm–Hyaluronan Binding Assay (HBA) showed a viability rate of 61.9% in FS and 78.2% in TS. Oocytes received from eight does (n = 8) underwent a viability test and vitrification, and fresh oocytes from the other eight does (n = 8) were fertilized with TS and embryos were cultured until the blastocyst stage. The number of isolated oocytes, cumulus–oocyte complexes (COCs), cleaved embryos, and expanded blastocysts was evaluated. Higher percentages of morphological factors (acrosome, head, midpiece, and tail shape) were observed in FS compared to TS, whereas the motility and progressive movement were greater in TS (p ≤ 0.001). The viability was 50.5% and MMP was 40.6% in TS. A total of 311 oocytes were collected and from 150 COCs and 125 blastocysts developed. The viability of thawed oocytes after vitrification was 81%. The viability of vitrified oocytes and cryopreserved sperm confirmed the effectiveness of freezing protocols and highlights the potential for their implementation in other deer species. Full article

Background/Objectives: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autoimmune disorder caused by mutations in the AIRE gene. Approximately 60% of affected females develop premature ovarian insufficiency (POI) by age 30, often most commonly due to steroidogenic autoantibodies. Although APECED is typically diagnosed in childhood, its reproductive implications are underrecognized. This study reports a case of successful fertility preservation in an adult woman with APECED and reviews the relevant literature. Methods: We describe the clinical course of a 37-year-old woman with genetically confirmed APECED who underwent ovarian stimulation for fertility preservation. A comprehensive PubMed search was also conducted to identify English-language case reports on fertility preservation in APECED-associated POI. Results: The patient experienced menarche at age 13, adrenal insufficiency at 14, and menstrual irregularities from age 18. Genetic analysis confirmed an AIRE mutation (NM_000383: exon 11: c.1400+1G>A). Given her relatively high anti-Müllerian hormone level, she opted for fertility preservation and underwent six cycles of ovarian stimulation, resulting in the cryopreservation of 17 mature oocytes. During ovarian stimulation, multiple follicular developments were observed, but serum E2 levels remained low. The literature review identified fewer than 20 reported cases addressing fertility preservation in APECED, highlighting its rarity and the lack of standardized management. Conclusions: Although APECED frequently leads to early POI due to impaired steroidogenesis, residual ovarian function may persist. Early assessment of ovarian reserve and timely fertility counseling are crucial, even in asymptomatic patients or those diagnosed in childhood. Reproductive planning should be integrated into the long-term care of women with APECED. Full article

Background/Objectives: Cellular oxidative stress is crucial for GV stage oocyte vitrification quality. PI3K and the aquaporin family have been shown to facilitate various cellular processes related to redox homeostasis and energy balance; yet, the mechanisms underlying the involvement of aquaporin 7 (AQP7) in vitrified oocyte oxidative stress remain unclear. The purpose of the present investigation was to evaluate the role of AQP7 in vitrified oocytes and the mechanisms involved. Methods: AQP7 inhibitors were employed to investigate the effect of AQP7 on oxidative stress in mouse-vitrified oocytes, whereas PI3K activators were harnessed to ascertain whether AQP7 serves as a functional molecule involved in this process. Results: Our results indicate that AQP7 inhibition in vitrified oocytes results in a significant decrease in glutathione (GSH) levels associated with cellular oxidation and an elevation in H₂O₂ levels. This was accompanied by exacerbated mitochondrial dysfunction, weakened cytoskeletal proteins, accelerated early apoptosis. Consequently, both survival and maturation rates were markedly reduced. Interestingly, PI3K/AKT activation increased AQP7 expression, restored abnormal mitochondrial distribution, as well as calcium homeostasis, and rescued the oocyte survival/maturation rate. Conclusions: Our results provide new insights indicating that PI3K/AKT/AQP7 decreases oxidative stress by regulating mitochondrial morphology, function, and distribution, thereby rescuing oocyte maturation in vitrification. Full article

This study compared the DuoStim protocol with two conventional follicular phase stimulations for vitrified oocyte accumulation in poor-prognosis patients undergoing PGT-A. A retrospective analysis of 112 IVF cycles was conducted, with 66 cycles among patients undergoing DuoStim (DS-Group) and 46 among patients undergoing conventional follicular phase stimulations (DF-Group). The primary outcome was the time to live birth, while secondary outcomes included clinical pregnancy rate, miscarriage rate, live birth rate, and cumulative live birth rate. The final analysis included 66 patients in the DS-Group and 40 in the DF-Group, as 6 women (13%) in the DF-Group discontinued treatment after the first stimulation. Oocyte yield was similar between groups (8.4 ± 3.9 in DS-Group vs. 8.2 ± 4.0 in DF-Group, p = 0.80), as was the number of euploid blastocysts (0.9 ± 1.2 vs. 1.1 ± 1.1, p = 0.37). The cumulative live birth rate was 22.7% in the DS-Group and 25% in the DF-Group (multivariate odds ratio adjusted for maternal age and male factor: 1.05, p = 0.93). The time to live birth was significantly shorter in the DS-Group (81.5 ± 15.5 days) compared to the DF-Group (153.7 ± 78.2 days, p < 0.001). DuoStim showed similar efficacy but a shorter time to live birth. Full article

This study aimed to compare the morphometry, morphology, and maturation capacity of cattle oocytes subjected to vitrification using different vitrification and maturation media. In Experiment 1, a total of 900 oocytes were divided into three groups: (1) matured before vitrification, (2) non-vitrified, and (3) vitrified as immature oocytes using the straw vitrification method. Morphometric parameters, including oocyte diameter, ooplasm, zona pellucida width (ZPW), granulosa cell width (GRSW), and zona pellucida-granulosa cell width (ZP GRSW), were measured (µm) before and after cryopreservation. In Experiment 2, the maturation capacity of three in vitro maturation (IVM) media (VitroMat-Protect™, BO-IVM™, and TCM199) was evaluated based on cumulus–oocyte complex (COC) expansion and polar body (PB) extrusion. Morphological abnormalities such as fragmented polar bodies (FPBs), large vacuoles (LVs), degenerated oocytes (DOs), and cracked cytoplasm (CC) were recorded. While vitrification did not significantly affect the oocyte diameter, ooplasm, or ZPW, it significantly reduced the GRSW and ZP GRSW. BO-IVM™ supported the highest COC expansion rate, while TCM199 had the lowest. Among vitrified oocytes, the highest PB extrusion rates were observed in BO-IVM^TM (35.14 ± 5.01) and Vitromat-Protect^TM (24.60 ± 5.67) as compared to TCM199 (18.44 ± 8.00; p < 0.05). Oocytes with higher CC rates were observed in VitroMat-Protect™ (24.50 ± 10.53) and BO-IVM™ (31.42 ± 7.32) as compared to TCM199 (18.70 ± 7.04). In conclusion, the vitrification process affects the granulosa cells in both vitrified immature and mature oocytes. BO-IVM^TM and VitroMat-Protect^TM supported better oocyte maturation than TCM199, although vitrification increased FPB and CC rates. Full article

Donkeys and mules have historically played an important role in agriculture and are now gaining recognition for their contributions to animal conservation, milk production, tourism, and equid-assisted services. However, their distinctive reproductive challenges pose obstacles to breeding management. As a result, the application of assisted reproductive technologies (ARTs) could help address these challenges, enhancing their roles in both traditional and emerging industries. This review examines the current and emerging in vitro techniques for breeding donkeys and mules. Key methodologies such as sperm cryopreservation, innovative sperm preservation technologies, embryo transfer, ovum pick-up (OPU), oocyte maturation, and vitrification are discussed, emphasizing their importance in optimizing ARTs. Advances in in vitro embryo production technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer (SCNT) are reviewed, with particular attention to its success in producing the first donkey and mule blastocysts or foals. Despite significant progress in the last decade, standardization of protocols for gamete conservation and embryo transfer are still required in long-ear equids. Advancing these technologies in combination with established in vitro embryo production could significantly improve reproductive outcomes and enhance the genetic management of donkey and mule populations. Full article

Imperatorin (IMP) is a naturally occurring furanocoumarin with beneficial biological activities such as anticancer, antioxidant, and neuromodulatory properties. Currently, the protective effects and mechanisms of IMP on oxidative stress experienced by mouse oocytes after vitrification–thawing remain unclear. To investigate the influence of IMP on mouse oocyte development after vitrification–thawing, we added different concentrations of IMP to the vitrification and thawing media. Results indicated that the addition of 40 μM IMP enhanced post-thaw fertilization capacity, reduced intracellular reactive oxygen species (ROS) levels, and increased intracellular glutathione (GSH) levels. IMP also improved mitochondrial health by alleviating the decrease in mitochondrial membrane potential (MMP) and enhancing mitochondrial distribution. IMP reduced intracellular ROS levels by affecting the transcription of the antioxidant genes SOD2, NRF2, and HO-1 and enhancing SOD activity. It also elevated GSH levels via GPX1, improved mitochondrial function, and decreased early apoptosis through Bcl-2. In conclusion, IMP enhanced ovum health through the alleviation of oxidative stress. The present study provides useful information for further exploration of the molecular mechanisms of IMP in female reproductive cells and offers a novel approach for the improvement of vitrification technology. Full article

The cryopreservation of oocytes through vitrification is imperative for the conservation of livestock germplasm resources. However, as oocytes exhibit significant oxidative stress and organelle damage following vitrification freezing, it is crucial to optimise the vitrification conditions to mitigate the deleterious effects of freezing. In this study, we demonstrated that spermidine has been showed to enhance oocyte survival after vitrification freezing (92% ± 4% vs. 82% ± 3%, p < 0.01) and blastocyst formation after freezing for in vitro fertilisation (14.86% ± 7% vs. 6% ± 3, p < 0.05). Spermidine supplementation rescued 47.3% of dysregulated pathways, including ovarian steroidogenesis, and restored normal expression levels in 43.3% of aberrantly expressed genes. Subsequent studies elucidated that spermidine effectively rescued mitochondrial dysfunction after vitrification, alleviated oxidative stress damage, and regulated intracellular calcium homeostasis. Consequently, we concluded that the addition of spermidine during vitrification freezing is an effective method to protect oocytes from freezing damage. Full article

Cryopreservation is currently an essential technique in reproductive technologies that is used for the extended preservation of genetic material. Vitrification has become the industry’s standard cryopreservation technique for cattle oocytes and embryos. The current results of this technology, however, are still not good in terms of viability, fertilisation capacity, embryo development, or pregnancy. The oocytes’ susceptibility to freezing is associated with significant changes in the structures, functioning of the oocytes, and cryoinjury, which is harmful to the survival of cells and their subsequent growth. The effectiveness of producing embryos with in vitro techniques utilising vitrified cattle oocytes rarely exceeds 30–40%. A significant number of vitrified oocytes do not successfully develop into the embryo stage following in vitro fertilisation and culture. This review focuses on issues related to oocyte cryopreservation, ways to overcome them, and how to enhance the vitrified oocyte fertilisation process. Full article

Chlorogenic acid (CGA) has strong antioxidant properties. In order to improve the low maturation rate and poor vitrification freezing effect of sheep oocytes caused by oxidative stress. In this study, oocytes from 200 2–3-year-old Kazakh sheep were collected, and different concentrations of CGA were added to the maturation medium and vitrification freezing solution to study the effects of CGA on the maturation rate, cleavage rate, blastocyst rate, reactive oxygen species (ROS) and glutathione (GSH) levels, mitochondrial membrane potential, and the expression levels of oxidation and apoptosis-related genes in sheep oocytes. The results showed that adding 40 μmol/L CGA to the oocyte in vitro maturation solution significantly increased the maturation rate of oocytes, adding 50 μmol/L CGA to the vitrification cryopreservative solution significantly increased the cleavage and blastocyst rates of mature oocytes activated by parthenogenetic activation after freezing. During in vitro maturation and vitrification freezing in sheep oocytes, CGA significantly reduced the level of ROS and the expression of apoptosis-related genes (Caspase-3 and Bax/Bcl-2), and significantly increased the level of glutathione (GSH), mitochondrial membrane potential, and the expression of antioxidant and anti-apoptosis-related genes (SOD-2 and GPX-3). In addition, CGA significantly increased the expression of the anti-apoptotic gene (AKT) and anti-stress gene (FOXO) during vitrification freezing of sheep oocytes. In conclusion, 40 μmol/L CGA improves the maturation rate of sheep oocytes, and 50 μmol/L CGA improves the quality of parthenogenetic activation embryos after vitrification freezing of mature oocytes in sheep. These results provide a basis for the production of sheep in vitro embryos and the establishment of a germplasm resource bank. Full article

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further. Full article

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus–oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs. Full article

Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO₂ until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (BCL2, CAS9), oxidative stress (GPX4, CDX2), and development (GJB5) compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes. Full article