Impact of Graphene Oxide Nanoparticles on In Vitro Development of a Mouse Preimplantation Embryo and Interaction With the Zona Pellucida.

The development of assisted reproductive technologies increases the likelihood of nanoparticles' (NPs) direct contact with gametes and embryos in in vitro conditions. Analyzing the influence of nanomaterials on the early mammalian embryo becomes increasingly relevant. This work is devoted to the effect of graphene oxide (GO) NPs on the in vitro development of mammalian embryos. Mouse 2-cell embryos were preincubated with GO NPs. The interaction of GO with the Zona Pellucida (ZP) of the embryo was investigated using fluorescence lifetime imaging with two-photon excitation (2p-FLIM). During embryo development, the NPs penetration into ZP (blastocyst stage) and perivitelline space (blastocyst hatching stage) was observed. Despite this, GO did not affect the embryo's ability to develop till late and hatching blastocysts. The mechanism of the NPs getting into the perivitelline space and the consequences of NP-embryo direct contact are discussed. The 2p-FLIM efficiency for studying NP interaction with mammalian embryos is evaluated.

Extracellular vesicles affecting embryo development in vitro: a potential culture medium supplement.

Extracellular vesicles (EVs) are nanometer-sized lipid bilayer vesicles released by cells, playing a crucial role in mediating cellular communication. This review evaluates the effect of EVs on early embryonic development in vitro by systematically searching the literature across three databases, Embase, PubMed, and Scopus, from inception (Embase, 1947; PubMed, 1996; and Scopus, 2004) to 30 June 2024. A total of 28 studies were considered relevant and included in this review. The EVs included in these investigations have been recovered from a range of sources, including oviduct fluid, follicular fluid, uterine fluid, seminal plasma, embryos, oviduct epithelial cells, endometrial epithelial cells, amniotic cells, and endometrial-derived mesenchymal stem cells collected primarily from mice, rabbits, cattle and pigs. This diversity in EV sources highlights the broad interest and potential applications of EVs in embryo culture systems. These studies have demonstrated that supplementation with EVs derived from physiologically normal biofluids and cells to the embryo culture medium system has positive effects on embryonic development. Conversely, EVs derived from cells under pathological conditions have shown a negative impact. This finding underscores the importance of the source and condition of EVs used in culture media. Further, the addition of EVs as a culture medium supplement holds significant therapeutic potential for optimizing in vitro embryo culture systems. In conclusion, this evaluation offers a thorough assessment of the available data on the role of EVs in embryo culture media and highlights the potential and challenges of using EVs in vitro embryo production.

Embryo Development in a Stochastic Universe.

Despite the elucidation of the many processes by which a single eukaryotic cell develops into a complex mature organism, it is still puzzling to some biologists how it is that an unvarying, interconnected set of processes becomes coordinated and insulated from a stochastic universe. This article suggests that electromagnetic processes deriving from the chemistry of an organism may provide such coordination. Specifically, the author develops the pacemaker concept, the periodic, autonomous electrical signal to the entire embryo, the result of which, after each pulse, is to alter or enlarge the transcriptome to produce the next level of complexity and maturity of the organism.

Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3ßi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

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 in vitro maturation efficiency and early embryonic stages.

Regulation of glucose metabolism: Effects on oocyte, preimplantation embryo, assisted reproductive technology and embryonic stem cell.

Glucose is a major energy substrate for cellular life activities, and its metabolic pathways include glycolysis, the pentose phosphate pathway, the hexosamine biosynthesis pathway, and the polyol pathway. Here, we review the glucose uptake pathways, metabolic characteristics, glucose transport, glucose metabolism-related enzymes, and biological importance in mammalian oocyte maturation, early embryo development, and embryonic stem cell proliferation and differentiation. Moreover, the interrelationships among glucose metabolism, female reproduction-related diseases and assisted reproductive technologies are focused. In addition, we review a number of analytical methodologies with the intention to integrate a multi-tiered strategy that encompasses cutting-edge metabolomics, artificial intelligence, epigenetics, and morphological assessments, setting the stage for a pivotal approach to cultivating high-caliber embryos in the future.

Advanced maternal age affects their frozen-thawed embryo susceptibility to high oxygen environment.

Preimplantation embryos can experience stress from laboratory interventions and a sub-optimal culture environment. Though research has demonstrated advanced maternal age impairs oocyte quality, the response of embryos derived from such oocytes to vitrification-thawing and culture in a high oxygen (O2) environment in the assisted reproductive technology laboratory is unknown. Therefore, in this study, embryos produced by in vitro fertilization (IVF) using oocytes from two- and eight-month-old Swiss albino mice were vitrified and thawed during their 6-8 cell stage. and cultured at low oxygen (5%) tension (LOT) and high oxygen (20%) tension (HOT). Embryo development, apoptosis, inner cell mass (ICM) outgrowth proliferation ability in vitro and pluripotency were assessed. Embryos from advanced maternal age cultured at HOT showed reduced fertilizing ability (p < 0.05), poor survival post-thawing (p < 0.05), and increased apoptosis (p < 0.01) in comparison to sibling embryos cultured at LOT. Importantly, the extended culture of vitrified-thawed embryos from advanced maternal age led to a significant (p < 0.001) reduction in complete ICM outgrowth formation at HOT in comparison to the LOT environment. The findings of this study suggest that HOT is detrimental to embryos from advanced maternal age, and importantly, vitrified-thawed embryos are more susceptible to stress, which could have negative implications, especially during the peri-implantation developmental period.

Paternal undernutrition and overnutrition modify semen composition and preimplantation embryo developmental kinetics in mice.

BACKGROUND: The importance of parental diet in relation to eventual offspring health is increasing in prominence due to the increased frequency of parents of reproductive age consuming poor diets. Whilst maternal health and offspring outcome have been studied in some detail, the paternal impacts are not as well understood. A father's poor nutritional status has been shown to have negative consequences on foetal growth and development and ultimately impact the long-term adult health of the offspring. In this study, we examined sperm- and seminal vesicle fluid-mediated mechanisms of preimplantation embryo development alterations in response to sub-optimal paternal diets. RESULTS: Male mice were fed a diet to model either under (low-protein diet (LPD)) or over (high-fat/sugar 'Western' diet (WD)) nutrition, LPD or WD supplemented with methyl donors or a control diet (CD) before mating with age-matched females. Male metabolic health was influenced by WD and MD-WD, with significant changes in multiple serum lipid classes and hepatic 1-carbon metabolites. Sperm RNA sequencing revealed significant changes to mRNA profiles in all groups when compared to CD (LPD: 32, MD-LPD: 17, WD: 53, MD-WD: 35 transcripts). Separate analysis of the seminal vesicle fluid proteome revealed a significant number of differentially expressed proteins in all groups (LPD: 13, MD-LPD: 27, WD: 24, MD-WD: 19) when compared to control. Following mating, in vitro time-lapse imaging of preimplantation embryos revealed a significant increase in the timing of development in all experimental groups when compared to CD embryos. Finally, qPCR analysis of uterine tissue at the time of implantation identified perturbed expression of Cd14 and Ptgs1 following mating with WD-fed males. CONCLUSIONS: Our current study shows that paternal nutritional status has the potential to influence male metabolic and reproductive health, impacting on embryonic development and the maternal reproductive tract. This study highlights potential direct (sperm-mediated) and indirect (seminal vesicle fluid-mediated) pathways in which a father's poor diet could shape the long-term health of his offspring.

NCOA3 knockdown delays human embryo development.

Embryonic development is a precisely controlled sequential process influenced by complex external and internal factors; therefore, this process holds paramount significance in the context of in vitro fertilization and embryo transfer (IVF-ET), with internal oocyte and embryo quality being pivotal in determining its success. Nuclear receptor coactivator 3 (NCOA3), a member of the p160 nuclear receptor coactivators family, has been extensively studied in tumorigenesis and reportedly plays a crucial role in maintaining pluripotency in mouse embryonic stem cells (ESCs). However, its functions in human embryo development remain largely unexplored. In this study, we collected human samples, including oocytes, zygotes, and embryos, from patients at the First Affiliated Hospital of Zhengzhou University to investigate whether NCOA3 regulates human embryonic development. To this end, we employed various assays, including immunofluorescence, quantitative real-time PCR (qPCR), microinjection, and RNA sequencing. Our findings suggested that NCOA3 expression level was low in inferior embryos (with >50 % fragmentation), and its presence is closely related to the expression of the pluripotency factor NANOG. Deletion of NCOA3 delays human embryonic development. Single-oocyte RNA sequencing revealed that NCOA3 primarily participates in metabolic alterations in oocytes. In sum, these findings elucidate the pivotal roles of NCOA3 in human embryonic development-NCOA3 deletion compromise the developmental potential of embryos. These mechanistic insights into the role of NCOA3 in human embryonic development not only advances our understanding of the intricate molecular mechanisms involved but also holds potential implications for improving assisted reproductive technologies (ART) and addressing developmental disorders in human embryos.

Sperm-carried IGF2: towards the discovery of a spark contributing to embryo growth and development.

Spermatozoa have been shown to carry key RNAs which, according to animal evidence, seem to play a role in early embryo development. In this context, a potential key growth regulator is insulin-like growth factor 2 (IGF2), a highly conserved paternally expressed imprinted gene involved in cell growth and proliferation which, recent observations indicate, is expressed in human spermatozoa. We herein hypothesized that sperm IGF2 gene expression and transmission at fertilization is required to support early embryo development. To test this hypothesis, we analyzed sperm IGF2 mRNA levels in the same semen aliquot used for homologous assisted reproductive technique (ART) in infertile couples and correlated these levels with embryo morphokinetics. To find a mechanistic explanation for the observed results, the transcriptomes of blastocysts obtained after injection of Igf2 mRNA in mouse parthenotes were analyzed. Sperm IGF2 mRNA negatively correlated with time of 2-cell stage (t2), t3, t4, t5, and time of expanded blastocyst (tEB), independently of maternal age, body mass index, anti-Müllerian hormone levels, and oocyte quality. An IGF2 mRNA index >4.9 predicted the ability of the embryos to reach the blastocyst stage on Day 5, with a sensitivity of 100% and a specificity of 71.6% (AUC 0.845; P < 0.001). In the animal study, transcriptome analysis demonstrated that 65 and 36 genes were, respectively, up- and down-regulated in the experimental group compared to the control group. These genes belong to pathways that regulate early embryo development, thus supporting the findings found in humans. This study has the potential to challenge the longstanding tenet that spermatozoa are simply vehicles carrying paternal DNA. Instead, it suggests that IGF2 mRNA in healthy spermatozoa provides critical support for early embryo development. Pre-ART sperm-carried IGF2 mRNA levels may be used as a marker to predict the chances of obtaining blastocysts to be transferred for infertile couples undergoing ART.

Extracellular vesicles: emerging paradigms in bovine embryo-maternal communication.

The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.

Comparative analysis of element and hormone content in zygotic embryos of Pinus elliottii and P. elliottii × P. caribaea.

Somatic embryogenesis is a crucial method for achieving clonal forestry in conifers. Understanding the development of zygotic embryos is essential not only for enhancing the efficiency and quality of somatic embryogenesis, but also for advancing forestry breeding programs. This study investigated dynamic changes of element and hormone contents during ZE development of Pinus elliottii and its hybrid P. elliottii × P. caribaea. Significant differences in embryo development speed among different clones were observed. Elemental analysis was conducted using inductively coupled plasma mass spectrometry (ICP-MS) and identified 68 elements, including major, minor, and beneficial elements. In both species, the contents of potassium (K), calcium (Ca), iron (Fe), boron (B) and five beneficial elements decreased during early ZE development, while phosphorus (P) and copper (Cu) increased. Significantly higher levels of K, Ca and Fe at the initial stage, and sulfur (S) and nickel (Ni) decreased at later stages were detected in P. elliottii than in the hybrid. For the other elements, except for very few significant differences at certain stages, most differences between the two species did not reach a significant level. The contents of endogenous hormones were determined and different accumulation patterns were detected in most hormones between the two species, except abscisic acid (ABA) which simultaneously decreased with developments by stage 8. Significant differences were found in indole-3-acetic acid (IAA) contents at most stages between species, while higher levels of total cytokinin (CK) at each stage were detected in the hybrid in comparison with those in P. elliottii. As a result, lower IAA to CK ratios in the hybrid than in P. elliottii. Methyl jasmonate (JA-me) and gibberellin A3 (GA3) contents showed a similar pattern and exhibited an M-shaped fluctuation in the hybrid. Furthermore, JA-me, GA3, gibberellin A4 (GA4) and brassinolide (BR) showed significantly higher levels in the hybrid than in P. elliottii. K-means clustering and correlation analyses were used to explore relationships between elements and hormones during embryo development, revealing complex interplay in both species. These data indicate different requirement in element and hormone contents for embryogenesis and suggest species-specific media composition for each step in somatic embryogenesis. The findings provide insights into their developmental processes and informing future research and applications in somatic embryogenesis and forestry breeding.

Does insulin-like growth factor-I level associate with pregnancy outcomes in primary and secondary infertile women undergoing in vitro fertilization? A prospective cohort study.

BACKGROUND: Infertility, which affects 8%-12% of couples worldwide and 21.9% of couples in Pakistan in particular, is a major reproductive health issue. In vitro fertilization (IVF) has emerged as a prevalent therapeutic intervention. Recent studies have identified insulin-like growth factor-I (IGF-I) as a promising biomarker for assessing embryo viability and predicting implantation outcomes in IVF procedures.

Arabidopsis TIC236 contributes to proplastid development and chloroplast biogenesis during embryogenesis.

Plastids are essential, semi-autonomous organelles in plants that carry out a multitude of functions during development. Plastids existing in different subtypes are derived from proplastids progenitors and interconvert in response to environmental and growth cues. Most efforts focus on the differentiation from proplastid to other forms. However, the studies of proplastid development are insufficient and whether proplastid biogenesis affects plant growth is yet to be determined. Arabidopsis TIC236, a translocon component at the inner membrane of the chloroplast envelope, is critical for importing chloroplast-targeted preproteins and chloroplast division. In this study, we uncovered the fundamental influence of proplastid biogenesis on embryo development by exploring the function of TIC236 during embryogenesis. Widespread and strong expression of TIC236 was observed in leaves and embryos. The null mutant tic236 had an embryo-lethal phenotype, with cell division in the mutant embryos delayed starting at the octant stage and arrested at the globular stage. Transmission electron microscopy revealed enlarged proplastids with an aberrant inner structure at the dermatogen and globular stages that ultimately did not differentiate into chloroplasts. Additionally, the fluorescence signal distribution patterns of tic236 embryos carrying the pDR5rev::3xVENUS-N7, pPIN1::PIN1-GFP, pWOX5::GFP, and pSCR::H2B-YFP reporter systems were altered. Together, we provide genetic evidence supporting proplastid biogenesis plays a vital role in embryo development and TIC236 is identified as an indispensable player, ensuring normal proplastid development.

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos.

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

The effect of resveratrol on the developmental competence of feline oocytes vitrified at the metaphase II stage.

The objective of this research was to assess the viability and developmental potential of feline oocytes following in vitro maturation (IVM), vitrification, and post-warming incubation with resveratrol. In the first experiment, warmed oocytes were incubated with 0.2 µM, 2 µM, or 20 µM resveratrol for 2 h. Oocytes treated with 0.2 µM resveratrol had the highest viability (68.89 %), as assessed by fluorescein diacetate and ethidium bromide staining, while higher concentrations were associated with diminished oocyte viability. In the second experiment, the warmed oocytes were inseminated following the 2-h incubation with the three concentrations of resveratrol. The presumptive zygotes were then maintained in culture and their development evaluated. The highest cleavage rate was observed when the oocytes had been incubated with 0.2 µM resveratrol (88.34 %), which was higher than for the control group (without resveratrol (75 %)). Moreover, this concentration of resveratrol also augmented the blastocyst formation rate. While the vitrification of oocytes often results in diminished developmental potential in the ensuing embryos, attributed to cryopreservation-induced injury, the utilization of low concentrations of resveratrol enhances the procedure's efficacy.

Single-cell RNA sequencing reveals the effects of high-fat diet on oocyte and early embryo development in female mice.

BACKGROUND: Obesity is a global health issue with detrimental effects on various human organs, including the reproductive system. Observational human data and several lines of animal experimental data suggest that maternal obesity impairs ovarian function and early embryo development, but the precise pathogenesis remains unclear. METHODS: We established a high-fat diet (HFD)-induced obese female mouse model to assess systemic metabolism, ovarian morphology, and oocyte function in mice. For the first time, this study employed single-cell RNA sequencing to explore the altered transcriptomic landscape of preimplantation embryos at different stages in HFD-induced obese mice. Differential gene expression analysis, enrichment analysis and protein-protein interactions network analysis were performed. RESULTS: HFD-induced obese female mice exhibited impaired glucolipid metabolism and insulin resistance. The ovaries of HFD mice had a reduced total follicle number, an increased proportion of atretic follicles, and irregular granulosa cell arrangement. Furthermore, the maturation rate of embryonic development by in vitro fertilization of oocytes was significantly decreased in HFD mice. Additionally, the transcriptional landscapes of preimplantation embryos at different stages in mice induced by different diets were significantly distinguished. The maternal-to-zygotic transition was also affected by the failure to remove maternal RNAs and to turn off zygotic genome expression. CONCLUSIONS: HFD-induced obesity impaired ovarian morphology and oocyte function in female mice and further led to alterations in the transcriptional landscape of preimplantation embryos at different stages of HFD mice.

MicroRNAomic Analysis of Spent Media from Slow- and Fast-Growing Bovine Embryos Reveal Distinct Differences.

In bovine embryos, the microRNA (miRNA) expression has been profiled at each stage of early development in vitro. The miRNAomic analysis of spent media has the potential to reveal characteristics of embryo health; however, applications are limited without categorizing miRNA profiles by embryo quality. Time-lapse imaging has shown the timing of embryo development in vitro may be indicative of their developmental potential. The study aimed to characterize miRNAs in the spent media of bovine embryos with different growth rates during the pre-implantation phase. Bovine cumulus-oocyte complexes were aspirated from ovaries, fertilized, and cultured to blastocyst stage of development. At the 2-cell, 8-cell, and blastocyst stage, each microdrop of 30 presumptive zygotes were classified as slow- or fast-growing based on the percentage of embryos that had reached the desired morphological stage. A comparative analysis was performed on the spent media of slow- and fast-growing embryos using the results of a GeneChip miRNA 4.0 array hybridization. In total, 34 differentially expressed miRNAs were identified between the comparison groups: 14 miRNAs were found in the 2-cell samples, 7 in the 8-cell samples, and 12 in the blastocyst samples. The results demonstrate distinct miRNAs populations can be identified between slow- and fast-growing embryos, highlighting the novel biomarkers of developmental potential at each stage of pre-implantation development.

Transcriptional insights from the human embryo identify laminin-511 as a suitable matrix for human trophoblast stem cell culture.

The establishment of culture conditions to propagate self-renewing human trophoblast stem cells in long-term culture provides a paradigm for in vitro modelling of trophoblast. The extracellular matrix (ECM) is a critical determinant of cell identity and behaviour. Therefore, models aiming to reproduce cells in vitro should recapitulate the native cell-ECM microenvironment. Here, we mine human embryo transcriptional datasets to identify ECM components and cognate receptors expressed in the trophectoderm. Following, we identify laminin-511-E8 protein fragment as a physiologically relevant ECM capable of maintaining hTSCs in the stem cell state and retaining differentiation ability.

Bioengineered 3D ovarian model for long-term multiple development of preantral follicle: bridging the gap for poly(ε-caprolactone) (PCL)-based scaffold reproductive applications.

BACKGROUND: Assisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development. METHODS: PA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level. RESULTS: The ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence. CONCLUSIONS: The study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.