Search Results (125)

Cacao genotypes propagation through plant tissue culture represents a strategic approach for establishing a core collection of elite plants to be used as a donor material source, necessary for increasing new planting areas of cacao. This study aimed to evaluate somatic embryo regeneration in ten native fine-aroma cacao genotypes (INDES-06, INDES-11, INDES-14, INDES-32, INDES-52, INDES-53, INDES-63, INDES-64, INDES-66, INDES-70) from the INDES-CES germplasm collection, under in vitro conditions using culture medium supplemented with different concentrations of Thidiazuron (0, 10, and 20 nM). Our results showed an average of 20 and 100% of callogenesis in all genotypes evaluated, but the callus development did not appear after early stages of its induction; however, primary somatic embryos were observed after 42 days after TDZ treatment in the INDES-52, INDES-53, INDES-64, INDES-66, INDES-70 genotypes. The INDES-52 genotype was more responsive to under 20 nM of TDZ, generating an average of 17 embryos per explant. This study contributes to the adaptation and establishment of a protocol for somatic embryo regeneration of fine-flavor cacao genotypes. Full article

The asynchrony of cytoplasmic and nuclear maturation in cumulus–oocyte complexes (COCs) due to prematurely declining concentrations of cyclic adenosine monophosphate (cAMP) has been shown to result in reduced oocyte developmental competence. The objective of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for different durations on the developmental potential of equine oocytes used for cloned embryo production. Collected COCs were transferred to cryovials filled with transport medium at 20–22 °C. Within the cryovials, the COCs were either untreated (Control) for 18 h or treated with 50 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine for the first 4 h (Pre-IVM 4 h) or the entire 18 h (Pre-IVM 18 h). Oocytes were then transferred to maturation medium and incubated for a further 22–24 h at 38.5 °C in 5% CO₂ in air. Somatic cell nuclear transfer embryos were then produced using the meiotically mature oocytes and donor cells from six different fibroblast cell lines. The rates of maturation and embryo development did not differ significantly between the groups, though blastocyst formation tended to be inferior in the Pre-IVM 4 h group compared with the Control group (p = 0.06). Of 67 blastocysts produced, 23 were transferred to recipient mares on Day 4 or 5 post-ovulation. Regarding the pregnancy outcomes, no significant differences were found between the groups, and four viable foals were born, each derived from a different donor cell line. The findings expand on those from previous evaluations of this biphasic IVM system, and indicate that the cAMP-modulating treatments exert limited effects under the pre-IVM conditions used here. Full article

The efficacy of in vitro fertilization (IVF) is significantly hindered by early embryonic developmental failure and oocyte maturation arrest. Recent findings in reproductive genetics have identified several oocyte-specific genes—TUBB8, KIF11, and CKAP5—as essential regulators of meiotic spindle formation and cytoskeletal dynamics. Mutations in these genes can lead to significant meiotic defects, fertilization failure, and embryo arrest. The links between genotype and phenotype, along with the underlying biological mechanisms, remain inadequately characterized despite the increasing number of identified variations. This systematic review was conducted in accordance with PRISMA 2020 guidelines. Relevant papers were retrieved from the PubMed and Embase databases using combinations of the keywords “TUBB8,” “KIF11,” “CKAP5,” “oocyte maturation arrest,” “embryonic arrest,” and “IVF failure.” Studies were included if they contained clinical, genomic, and functional data on TUBB8, KIF11, or CKAP5 mutations in women undergoing IVF. Molecular data, including gene variant classifications, inheritance models, in vitro tests (such as microtubule network analysis in HeLa cells), and assisted reproductive technology (ART) outcomes, were obtained. Eighteen trials including 35 women with primary infertility were included. Over fifty different variants were identified, the majority of which can be attributed to TUBB8 mutations. TUBB8 disrupted α/β-tubulin heterodimer assembly due to homozygous missense mutations, hence hindering meiotic spindle formation and leading to early embryo fragmentation or the creation of many pronuclei and cleavage failure. KIF11 mutations resulted in spindle disorganization and chromosomal misalignment via disrupting tubulin acetylation and microtubule transport. Mutations in CKAP5 impaired bipolar spindle assembly and microtubule stabilization. In vitro validation studies showed cytoskeletal disturbances, protein instability, and dominant negative effects in transfected animals. Donor egg IVF was the sole effective treatment; however, no viable pregnancies were documented in patients with pathogenic mutations of TUBB8 or KIF11. TUBB8, KIF11, and CKAP5 are essential for safeguarding oocyte meiotic competence and early embryonic development at the molecular level. Genetic differences in these genes disrupt microtubule dynamics and spindle assembly, resulting in various aspects of oocyte maturation and fertilization. Functional validation underscores the necessity of routine genetic screening for women experiencing unresolved IVF failure, as it substantiates their causal role in infertility. Future therapeutic avenues in ART may be enhanced by tailored counseling and innovative rescue methodologies like as gene therapy. Full article

Standard semen parameters remain the cornerstone of male infertility evaluation, though they often poorly reflect the likelihood of success in assisted reproductive technology (ART). This study evaluates sperm DNA fragmentation (SDF) as a diagnostic tool for male infertility and predictive biomarker for ART success. Semen samples were collected from 20 fertile donors and 40 infertile patients with abnormal semen parameters. A fraction of each sample was used for SDF assessment via TUNEL assay and flow cytometry, while the remaining portion was processed for conventional semen analysis and ART. Infertile patients exhibited higher SDF levels (32.77 ± 13.61%) compared to donors (22.19 ± 8.37%; p < 0.01), a difference that remained statistically significant across all subgroups stratified by semen parameters. Additionally, significant correlations were obtained between the percentage of SDF and sperm count (r = −0.4036), motility (r = −0.6377), and morphology (r = −0.2783). Regarding ART outcomes, patients with low-quality embryos exhibited higher SDF levels compared to those with high-quality embryos (30.02 ± 12.52% vs. 23.16 ± 8.41%; p = 0.0036). Receiver operating characteristic (ROC) curve analysis revealed an area under the curve (AUC) above 0.7 for the classification of male infertility as well as the assessment of embryo quality. Overall, our results support the utility of SDF as both a diagnostic biomarker for male infertility and a predictive indicator of embryo quality in ART, particularly in the presence of an oocyte-related female factor. Full article

A significant increase in life expectancy worldwide has resulted in a growing aging population, accompanied by a rise in aging-related diseases that pose substantial societal, economic, and medical challenges. This trend has prompted extensive efforts within many scientific and medical communities to develop and enhance therapies aimed at delaying aging processes, mitigating aging-related functional decline, and addressing aging-associated diseases to extend health span. Research in aging biology has focused on unraveling various biochemical and genetic pathways contributing to aging-related changes, including genomic instability, telomere shortening, and cellular senescence. The advent of induced pluripotent stem cells (iPSCs), derived through reprogramming human somatic cells, has revolutionized disease modeling and understanding in humans by addressing the limitations of conventional animal models and primary human cells. iPSCs offer significant advantages over other pluripotent stem cells, such as embryonic stem cells, as they can be obtained without the need for embryo destruction and are not restricted by the availability of healthy donors or patients. These attributes position iPSC technology as a promising avenue for modeling and deciphering mechanisms that underlie aging and associated diseases, as well as for studying drug effects. Moreover, iPSCs exhibit remarkable versatility in differentiating into diverse cell types, making them a promising tool for personalized regenerative therapies aimed at replacing aged or damaged cells with healthy, functional equivalents. This review explores the breadth of research in iPSC-based regenerative therapies and their potential applications in addressing a spectrum of aging-related conditions. Full article

Multiple System Atrophy (MSA) is a rare, rapidly progressive neurodegenerative disorder marked by autonomic dysfunction, parkinsonism, and cerebellar ataxia. While predominantly affecting individuals in their fifth or sixth decade, advancements in assisted reproductive technologies (ART) have created new clinical scenarios involving pregnancies in women within MSA’s typical onset range. Given the scarcity of documented MSA pregnancies, this review leverages insights from related Parkinson’s spectrum mechanisms to explore hypothetical scenarios for how pregnancy-induced physiological changes might influence MSA progression. Pregnancy-induced hormonal fluctuations, including elevated estrogen and progesterone levels, may modulate α-synuclein aggregation and neuroinflammatory pathways. Immune adaptations, such as fetal microchimerism and Th2-biased immune profiles, introduce additional complexities, particularly in donor embryo pregnancies involving complex microchimerism. Metabolic demands and oxidative stress further intersect with these mechanisms, potentially accelerating disease progression. We analyze existing literature and theoretical models, emphasizing the need for interdisciplinary research. Clinical implications are discussed to propose evidence-based strategies for optimizing maternal-fetal outcomes. This paper identifies critical knowledge gaps and proposes avenues for future investigation to optimize maternal-fetal outcomes in this unique and underexplored clinical intersection. Full article

For nearly three decades, interspecies somatic cell nuclear transfer (iSCNT) has been explored as a potential tool for cloning, regenerative medicine, and wildlife conservation. However, developmental inefficiencies remain a major challenge, largely due to persistent barriers in nucleocytoplasmic transport, mitonuclear communication, and epigenome crosstalk. This review synthesized peer-reviewed English articles from PubMed, Web of Science, and Scopus, spanning nearly three decades, using relevant keywords to explore the molecular mechanisms underlying iSCNT inefficiencies and potential improvement strategies. We highlight recent findings deepening the understanding of interspecies barriers in iSCNT, emphasizing their interconnected complexities, including the following: (1) nucleocytoplasmic incompatibility may disrupt nuclear pore complex (NPC) assembly and maturation, impairing the nuclear transport of essential transcription factors (TFs), embryonic genome activation (EGA), and nuclear reprogramming; (2) mitonuclear incompatibility could lead to nuclear and mitochondrial DNA (nDNA-mtDNA) mismatches, affecting electron transport chain (ETC) assembly, oxidative phosphorylation, and energy metabolism; (3) these interrelated incompatibilities can further influence epigenetic regulation, potentially leading to incomplete epigenetic reprogramming in iSCNT embryos. Addressing these challenges requires a multifaceted, species-specific approach that balances multiple incompatibilities rather than isolating a single factor. Gaining insight into the molecular interactions between the donor nucleus and recipient cytoplast, coupled with optimizing strategies tailored to specific pairings, could significantly enhance iSCNT efficiency, ultimately transforming experimental breakthroughs into real-world applications in reproductive biotechnology, regenerative medicine, and species conservation. Full article

Currently, the efficiency of somatic cell nuclear transfer (SCNT) technology is relatively low, primarily owing to reprogramming abnormalities in donor cells or reconstructed embryos. Using histone deacetylase inhibitor (HDACi) to artificially alter the epigenetic modifications of donor cells and improve the reprogramming ability of reconstructed embryos is effective in improving nuclear transfer efficiency. In this study, we used Albas cashmere goat cells as donor cells, treated them with Scriptaid, and constructed embryos using SCNT. The results suggest that donor cell treatment with Scriptaid significantly increased the cellular histone acetylation modification level, perturbed the expression of the pluripotency molecule NANOG, altered the reprogramming ability of embryos, and increased the developmental rate of SCNT-reconstructed embryos. Scriptaid inhibited donor cell proliferation, induced apoptosis, and blocked the G0/G1 phase of the cell cycle. These results provide a new research direction for improving SCNT efficiency and a new perspective in the fields of regenerative medicine, agriculture, and animal husbandry. Full article

Solid organ transplantation remains a life-saving treatment for patients worldwide. Unfortunately, the supply of donor organs cannot meet the current need, making the search for alternative sources even more essential. Xenotransplantation using sophisticated genetic engineering techniques to delete and overexpress specific genes in the donor animal has been investigated as a possible option. However, the use of exogenous tissue presents another host of obstacles, particularly regarding organ rejection. Given these limitations, interspecies blastocyst complementation in combination with precise gene knockouts presents a unique, promising pathway for the transplant organ shortage. In recent years, great advancements have been made in the field, with encouraging results in producing a donor-derived organ in a chimeric host. That said, one of the major barriers to successful interspecies chimerism is the mismatch in the developmental stages of the donor and the host cells in the chimeric embryo. Another major barrier to successful chimerism is the mismatch in the developmental speeds between the donor and host cells in the chimeric embryos. This review outlines 19 studies in which blastocyst complementation was used to generate solid organs. In particular, the genesis of the liver, lung, kidney, pancreas, heart, thyroid, thymus and parathyroids was investigated. Of the 19 studies, 7 included an interspecies model. Of the 7, one was completed using human donor cells in a pig host, and all others were rat–mouse chimeras. While very promising results have been demonstrated, with great advancements in the field, several challenges continue to persist. In particular, successful chimerism, organ generation and donor contribution, synchronized donor–host development, as well as ethical concerns regarding human–animal chimeras remain important aspects that will need to be addressed in future research. Full article

Background and Objectives: POSEIDON 4 (P4) patients face the most adverse outcomes among poor responders. Oocyte donation has overcome unsatisfactory live birth rates (LBRs) in P4 patients and has become an indispensable approach. However, many patients refuse oocyte donation despite poor live birth likelihood using autologous oocytes. This study aimed to determine clinical outcomes and live birth chances in P4 patients using autologous and donated oocytes. We also identified influencing factors of fertility outcome in P4 patients who underwent donor cycles. Materials and Methods: Retrospective data of 345 P4 patients who explored the first ovarian stimulation cycle (control group) were compared to 105 patients who failed to conceive and underwent repeated autologous ovarian stimulations with an increased starting gonadotropin dose and 100 unpregnant patients who received donated oocytes. Univariate analysis was used to identify prognostic factors of oocyte donation outcomes in P4 patients. Results: LBRs were significantly higher in the donor oocyte group. A higher number of retrieved and good-quality oocytes without differences in the blastocyst number and LBRs were found in the autologous patient group with adjusted gonadotropin dose compared to the control group. Univariate analysis showed that oocyte and embryo quality as well as blastocyst development had a positive impact on live birth in the donor patient group. Conclusions: Autologous in vitro fertilization (IVF) retreating and oocyte donation should be strongly advised for poor-prognosis P4 patients. Still, efforts in tailoring ovarian stimulation to obtain higher oocyte and embryo numbers in autologous cycles should be continued. Full article

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes. Full article

Background: Assisted reproductive technology (ART) nowadays plays a major role in the treatment of infertility, with the most frequently used techniques being in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The objective of this study is to analyze pregnancies achieved using these ART techniques and their correlations with the prematurity and congenital malformations rates. Methods: This is an observational retrospective longitudinal study that includes 814 newborns conceived through an ART, namely IVF or ICSI. Results: Using a multivariate logistic regression analysis mode, there is a higher prematurity rate in twin pregnancies OR 16 (95% CI 10.7, 23.8), donor conception OR 1.8 (95% CI 1.1, 3.3) and PIH pregnancy OR 2.6 (95% CI 1.5, 4.5). The odds of malformations in these ART pregnancies are increased by the stage of the embryo (day 3) OR 2.6 (95% CI 1.3, 5.2), fresh embryo transfer OR 2 (95% CI 1.2, 3.4) and donor conception OR 2.3 (95% CI 1.2, 4.4). The ART used (IVF/ICSI) does not influence the prematurity or birth defects rate. Conclusions: Donor conception is found to increase the odds of both prematurity and congenital malformations. The ART used (IVF/ICSI) does not influence the prematurity or birth defects rate. Full article

Background: T1D is a severe metabolic disorder due to selective autoimmune pancreatic islet β-cell killing, which results in complete abrogation of endogenous insulin secretion. The affected patients, once the disease is clinically overt, must immediately undertake insulin supplementation according to intensive therapy regimens to prevent the onset of acute and chronic complications, some of them potentially lethal. Replacement of the destroyed β-cells with fresh and vital pancreatic endocrine tissue, either of the whole organ or isolated islets transplantation, started a few decades ago with progressively encouraging results, although exogenous insulin withdrawal was obtained in a minor cohort of the treated patients. The restricted availability of donor organs coupled with general immunosuppression treatment of recipients to avoid graft immune rejection may, at least partially, explain the limited success achieved by these procedures. Results: The introduction of pluripotent stem cells (either of human embryonic origin or adult cells genetically induced to pluripotency) that can be differentiated toward insulin secretory β-like cells could provide an indefinite resource for insulin-producing cells (IPCs). Conclusions: Because the use of human embryos may encounter ethical problems, employment of adult multipotent mesenchymal stem cells (MSCs) extracted from several tissues may represent an alternative option. MSCs are associated with strong immunoregulatory properties that can alter early stages of β-cell-directed autoimmunity in T1D, other than holding the potential to differentiate themselves into β-like cells. Lights and shadows of these new strategies for the potential cure of T1D and their advancement state are reviewed. Full article

This study evaluates the utilization of in vitro embryo production (IVEP) technology for the conservation and breeding of the Auliekol cattle breed, a primary beef breed in Kazakhstan facing population decline due to the cessation of breeding programs and the incursion of transboundary diseases. We assessed the effect of consecutive ovum pick-up (OPU) procedures on oocyte yield and embryo production in Auliekol and Aberdeen Angus cows. A total of 2232 and 3659 oocytes were aspirated from Auliekol and Aberdeen Angus donors, respectively, with significantly higher yields and embryo production observed in Aberdeen Angus cows. The application of a meiotic block using Butyrolactone I (BLI) and subsequent in vitro fertilization (IVF) protocols was employed, with embryo development monitored up to the morula/blastocyst stage. Results indicated that Auliekol cows exhibited lower oocyte recovery, cleavage, and blastocyst rates compared to Aberdeen Angus cows, likely due to genetic characteristics. Despite the challenges, IVEP presents a valuable tool for the preservation and future propagation of the Auliekol breed, highlighting the need for further research to enhance reproductive outcomes and conservation strategies. Full article

Recombination-activating genes (RAGs) play a crucial role in the V(D)J recombination process and the development of immune cells. The development of the immune system and its mechanisms in pigs exhibit greater similarity to those of humans compared to other animals, thus rendering pigs a valuable tool for biomedical research. In this study, we utilized CRISPR/Cas9 gene editing and somatic cell nuclear transfer technology to generate RAG2 knockout (KO) pigs. Furthermore, we evaluated the impact of RAG2 KO on the immune organs and immune cell development through morphological observations, blood analysis and flow cytometry technology. RAG2 KO cell lines were used as donors for cloning. The reconstructed embryos were transplanted into 4 surrogate sows, and after 116 days of gestation, 2 sows gave birth to 12 live piglets, all of which were confirmed to be RAG2 KO. The thymus and spleen sizes of RAG2 KO pigs were significantly smaller than those of wild-type (WT) pigs. Hematoxylin-eosin staining results revealed that the thymus and spleen tissue structures of RAG2 KO pigs were disorganized and lacked the characteristic structures, indicating that RAG2 KO leads to dysplasia of the thymus and spleen. Hematological analysis demonstrated that the total number of white blood cells and lymphocytes in the circulation of RAG2 KO pigs was significantly lower, while the number of eosinophils was higher. Flow cytometry results indicated that the proportions of mature T and B lymphocytes were significantly reduced compared to WT pigs. These findings successfully verified the immunodeficiency phenotype of RAG2 KO pigs. This study may provide experimental animals for the development of tumor models and humanized animals. Full article