Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts.

Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.

Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts.

PURPOSE: This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. METHODS: A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation. RESULTS: Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2- cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes. CONCLUSION: We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

Fetal Growth and Osteogenesis Dynamics during Early Development in the Ovine Species.

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops.

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Embryo biotechnologies in sheep: Achievements and new improvements.

To date, large-scale use of multiple ovulation and embryo transfer (MOET) programmes in ovine species is limited due to unpredictable results and high costs of hormonal stimulation and treatment. Therefore, even if considered reliable, they are not fully applicable in large-scale systems. More recently, the new prospects offered by in vitro embryo production (IVEP) through collection of oocytes post-mortem or by repeated ovum pick-up from live females suggested an alternative to MOET programmes and may be more extensively used, moving from the exclusive research in the laboratory to field application. The possibility to perform oocytes recovery from juvenile lambs to obtain embryos (JIVET) offers the great advantage to significantly reduce the generation interval, speeding the rate of genetic improvement. Although in the past decades several studies implemented novel protocols to enhance embryo production in sheep, the conditions of every single stage of IVEP can significantly affect embryo yield and successful transfer into the recipients. Moreover, the recent progresses on embryo production and freezing technologies might allow wider propagation of valuable genes in small ruminants populations and may be used for constitution of flocks without risks of disease. In addition, they can give a substantial contribution in preserving endangered breeds. The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.

Highly Photostable Carbon Dots from Citric Acid for Bioimaging.

Bioimaging supported by nanoparticles requires low cost, highly emissive and photostable systems with low cytotoxicity. Carbon dots (C-dots) offer a possible solution, even if controlling their properties is not always straightforward, not to mention their potentially simple synthesis and the fact that they do not exhibit long-term photostability in general. In the present work, we synthesized two C-dots starting from citric acid and tris (hydroxymethyl)-aminomethane (tris) or arginine methyl ester dihydrochloride. Cellular uptake and bioimaging were tested in vitro using murine neuroblastoma and ovine fibroblast cells. The C-dots are highly biocompatible, and after 24 h of incubation with the cells, 100% viability was still observed. Furthermore, the C-dots synthesized using tris have an average dimension of 2 nm, a quantum yield of 37%, high photostability and a zeta potential (ζ) around -12 mV. These properties favor cellular uptake without damaging cells and allow for very effective bioimaging.

Oocyte aging: looking beyond chromosome segregation errors.

The age-associated decline in female fertility is largely ascribable to a decrease in oocyte quality. This phenomenon is multifaceted and influenced by numerous interconnected maternal and environmental factors. An increase in the rate of meiotic errors is the major cause of the decline in oocyte developmental competence. However, abnormalities in the ooplasm accumulating with age - including altered metabolism, organelle dysfunction, and aberrant gene regulation - progressively undermine oocyte quality. Stockpiling of maternal macromolecules during folliculogenesis is crucial, as oocyte competence to achieve maturation, fertilization, and the earliest phases of embryo development occur in absence of transcription. At the same time, crucial remodeling of oocyte epigenetics during oogenesis is potentially exposed to interfering factors, such as assisted reproduction technologies (ARTs) or environmental changes, whose impact may be enhanced by reproductive aging. As the effects of maternal aging on molecular mechanisms governing the function of the human oocyte remain poorly understood, studies in animal models are essential to deepen current understanding, with translational implications for human ARTs. The present mini review aims at offering an updated and consistent view of cytoplasmic alterations occurring in oocytes during aging, focusing particularly on gene and epigenetic regulation. Appreciation of these mechanisms could inspire solutions to mitigate/control the phenomenon, and thus benefit modern ARTs.

Intrafollicular oocyte transfer (IFOT): Potential feasibility in the ovine species.

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 µL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

Molecular and Histological Evaluation of Sheep Ovarian Tissue Subjected to Lyophilization.

Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration.

3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors.

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus-oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

The subcortical maternal complex: emerging roles and novel perspectives.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

Use of Transparent Liquid Marble: Microbioreactor to Culture Cardiospheres.

Cells have a remarkable ability to self-organize and rearrange in functional organoids, this process was greatly boosted by the recent advances in 3D culture technologies and materials. Presently, this approach can be applied to model human organ development and function "in a dish" and can be used to predict drug response in a patient specific fashion.Here we describe a protocol that allows for the derivation of functional cardiac mini organoids consisting of cocultured cardiomyocytes and cardiac fibroblast. Cells are suspended in a drop of medium and encapsulated with hydrophobic fumed silica powder nanoparticles. These nanoparticles are treated with hydrophobic chemicals, hexamethyldisilazane (nHMDS), and result in the formation of microbioreactors. These microenvironments are defined as "liquid marbles," stimulating cell coalescence and 3D aggregation. Then nHMDS shell ensures optimal gas exchange between the interior liquid and the surrounding environment. This microbioreactor makes working in smaller volumes possible and is therefore amenable for higher throughput applications. Moreover, the properties of liquid marble microbioreactors makes it an excellent culture technique for cocultures. Here we demonstrate how cocultures of cardiac fibroblast and cardiomyocytes in a cardiosphere can be a valuable tool to model cardiac diseases in vitro and to assess cell interactions to decipher disease mechanisms.

Use of Virus-Mimicking Nanoparticles to Investigate Early Infection Events in Upper Airway 3D Models.

The current coronavirus disease-19 (COVID-19) pandemic, caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), underscores the threat posed by newly emerging viruses. The understanding of the mechanisms driving early infection events, that are crucial for the exponential spread of the disease, is mandatory and can be significantly implemented generating 3D in vitro models as experimental platforms to investigate the infection substrates and how the virus invades and ravages the tissues.We here describe a protocol for the creation of a synthetic hydrogel-based 3D culture system that mimics in vitro the complex architectures and mechanical cues distinctive of the upper airway epithelia. We then expose the in vitro generated 3D nasal and tracheal epithelia to gold nanoparticles (AuNPs) that display the typical shape and size distinctive of SARS-CoV-2 and of the majority of Coronaviridae presently known.The infection platform here described provides an efficient and highly physiological in vitro model that reproduces the host-pathogen early interactions, using virus-mimicking nanoparticles, and offers a flexible tool to study virus entry into the cell. At the same time, it reduces the risk of accidental infection/spillovers for researchers, which represents a crucial aspect when dealing with a virus that is highly contagious, virulent, and even deadly.

Liquid marble technology to create cost-effective 3D cardiospheres as a platform for in vitro drug testing and disease modelling.

Three-dimensional (3D) cell culturing has several advantages over 2D cultures. 3D cell cultures more accurately mimic the in vivo environment, which is vital to obtain reliable results in disease modelling and toxicity testing. With the introduction of the Yamanaka factors, reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) became available. This iPSC technology provides a scalable source of differentiated cells. iPSCs can be programmed to differentiate into any cell type of the body, including cardiomyocytes. These heart-specific muscle cells, can then serve as a model for therapeutic drug screening or assay development. Current methods to achieve multicellular spheroids by 3D cell cultures, such as hanging drop and spinner flasks are expensive, time-consuming and require specialized materials and training. Hydrophobic powders can be used to create a micro environment for cell cultures, which are termed liquid marbles (LM). In this procedure we describe the first use of the LM technology for 3D culturing in vitro derived human cardiomyocytes which results in the formation of cardiospheres within 24h. The cardiospheres could be used for several in depth and high-throughput analyses.

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window. In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues. The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

Distribution and size of lipid droplets in oocytes recovered from young lamb and adult ovine ovaries.

This study evaluated the distribution and size of lipid droplets (LDs) in oocytes recovered from young and adult ovine ovaries. Collected oocytes were categorised on the basis of their major diameter (small (SO), 70-90 µm; medium (MO), >90-110 µm; large (LO), >110-130µm) and were stained with Nile red to detect LDs. In adult and young oocytes, a diffuse pattern distribution of LDs was dominant in all classes except adult LO and young SO and LO. Larger LDs (i.e. >3µm) were mostly present in young SO and LO, whereas smaller LDs (1-3µm) were detected in the other adult and young oocyte categories.

Post-mortem recovery, in vitro maturation and fertilization of fallow deer (Dama dama, Linnaeus 1758) oocytes collected during reproductive and no reproductive season.

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.

DuoStim - a reproducible strategy to obtain more oocytes and competent embryos in a short time-frame aimed at fertility preservation and IVF purposes. A systematic review.

Recent evidence suggests that follicular development occurs in a wave-like model during the ovarian cycle, where up to three cohorts of follicles are recruited to complete folliculogenesis. This understanding overtakes the previous dogma stating that follicles grow only during the follicular phase of the menstrual cycle. Therefore, in in vitro fertilization (IVF), novel protocols regarding ovarian stimulation have been theorized based on the use of gonadotrophins to prompt the growth of antral follicles at any stage of the menstrual cycle. These unconventional protocols for ovarian stimulation aim at a more efficient management of poor-prognosis patients, otherwise exposed to conflicting outcomes after conventional approaches. DuoStim appears among these unconventional stimulation protocols as one of the most promising. It combines two consecutive stimulations in the follicular and luteal phases of the same ovarian cycle, aimed at increasing the number of oocytes retrieved and embryos produced in the short time-frame. This protocol has been suggested for the treatment of all conditions requiring a maximal and urgent exploitation of the ovarian reserve, such as oncological patients and poor responders at an advanced maternal age. At present, data from independent studies have outlined the consistency and reproducibility of this approach, which might also reduce the drop-out between consecutive failed IVF cycles in poor-prognosis patients. However, the protocol must be standardized, and more robust studies and cost-benefit analyses are needed to highlight the true clinical pros and cons deriving from DuoStim implementation in IVF.