Introduction

In vitro maturation (IVM) of mammalian oocytes is one of the oldest experimental successes in reproductive biology. In the 1930s, mouse oocytes were shown to resume meiosis and reach metaphase II (MII) after removal from their follicular environment (Pincus and Enzmann, 1935). This finding was later confirmed in livestock such as sheep, cattle, and goats, where oocytes collected ex vivo could undergo maturation, fertilization, and embryonic development (Moor and Trounson, 1977; Leibfried and First, 1979; Szöllösi et al., 1988). Clinical translation in humans followed in the 1990s, with improved culture systems allowing consistent embryo development and pregnancies (Chian et al., 2004).

In vivo, mammalian oocytes enter meiosis during fetal life and stop at prophase I, known as the germinal vesicle (GV) stage. After puberty, gonadotropin signaling induces meiotic resumption, first polar body extrusion, and arrest at MII until fertilization (Eppig and O’Brien, 1996; Conti and Franciosi, 2018). While nuclear maturation marks such as polar body release are critical, developmental competence requires cytoplasmic maturation: redistribution of mitochondria, maternal RNA regulation, cytoskeletal remodeling, and redox control (Combelles et al., 2002; Watson, 2007). These events are normally supported by cumulus and granulosa cells, cAMP/cGMP gradients, and kinase cascades (Ferreira et al., 2009; Gilchrist and Smitz, 2023; Martinez et al., 2023).

The motivation for IVM differs between livestock and human fields. In agriculture, IVM is indispensable for in vitro embryo production (IVP), enabling the maturation of oocytes from slaughterhouse ovaries or ovum pick-up (OPU) of genetically superior cows, followed by IVF and embryo transfer (Ealy et al., 2019; Lonergan, 2024). IVM oocytes also serve as essential cytoplasts for somatic cell nuclear transfer (SCNT) and as the entry point for genome editing by CRISPR/Cas9 in cattle, pigs, sheep, and goats (Campbell et al., 1996a; Campbell, 1999; Lin et al., 2022). In human assisted reproductive technology (ART), IVM lowers the risk of ovarian hyperstimulation syndrome (OHSS), benefits women with polycystic ovary syndrome (PCOS), and provides fertility preservation options for oncology patients (Archer and Chang, 2004; Siristatidis et al., 2018; Chatzimeletiou et al., 2025).

Climate change also highlights the importance of IVM. Heat stress reduces oocyte quality and embryo survival, especially in dairy cattle, where IVM can provide a stable in vitro environment less affected by seasonal variation (Hansen, 2009; Cavallari et al., 2019; Roth, 2021; Lee et al., 2023). Delayed childbearing and the need for fertility preservation further increase the demand for IVM in human medicine (Llonch et al., 2021; Telfer et al., 2023).

Despite progress, IVM-derived oocytes often show lower developmental competency, largely due to abnormal mitochondria and spindles, oxidative stress, and incomplete epigenetic remodeling (Dvoran et al., 2022; Jiang et al., 2023). To optimize media conditions, co-culture with cumulus cells, use of microfluidic systems, and identification of omics-based biomarkers have been employed (Trapphoff et al., 2016; Zhao et al., 2016; Iwasaki et al., 2018; Kassim et al., 2025).

In this review, we summarize recent progress and future perspectives related to IVM. First, we examine current methods for evaluating nuclear and cytoplasmic maturation, with emphasis on morphological, molecular, and functional indicators of oocyte competence. Second, we discuss improved strategies to enhance IVM efficiency, including optimized culture conditions, co-culture systems, and emerging biotechnologies such as microfluidics and omics-based biomarkers. Third, we explore the integration of IVM with oocyte cryopreservation in ART, highlighting its potential for fertility preservation and safer clinical applications. Finally, we review the use of IVM-derived oocytes in animal biotechnology, including IVP, SCNT, and genome editing.

Evaluation of In Vitro Oocyte Maturation

The evaluation of IVM requires an integrated consideration of nuclear and cytoplasmic events. Although extrusion of the first polar body is often regarded as the canonical endpoint of maturation, it does not fully reflect the competence of the oocyte. Reliable assessment therefore depends on combining morphological, cytoskeletal, molecular, and functional parameters (summarized in Table 1).

Nuclear maturation

The extrusion of the first polar body in the oocyte is the mostly widely used marker of meiosis I completion and entry into MII (Younis et al., 2009). More refined approaches quantify the size and morphology of the polar body, as excessively large polar bodies may indicate spindle positioning defects and compromised asymmetric division (Lee and Choi, 2023).

Assessment of spindle assembly provides additional resolution. Immunostaining of α- and β-tubulin is employed to evaluate spindle shape and integrity, while γ-tubulin localization highlights the organization of microtubule-organizing centers (Meng et al., 2004; Howe and FitzHarris, 2013).

The actin cytoskeleton also serves as a robust indicator. F-actin capping at the cortex overlying the spindle is required for asymmetric division and polar body extrusion. Fluorescent phalloidin staining combined with line-scan intensity profiling can be used to generate a capping index, reflecting the degree of actin polarization (Almonacid et al., 2014; Jo et al., 2015). Beyond morphology, protein–protein interactions can be probed by proximity ligation assays (PLA) to verify the functional association between spindle and polarity proteins. For example, PLA signals between SPECC1L and γ-tubulin have been shown to indicate their interaction during spindle positioning (Lee and Choi, 2023).

Oocyte ploidy analysis has progressively advanced since the introduction of chromosome-spreading techniques using MII oocytes, in which zona-free oocytes are spread on slides, stained, and scored for individual chromosome numbers (Stein and Schindler, 2011). This classic method remains a cornerstone for assessing aneuploidy rates in small-animal IVM models. Subsequently, fluorescence in situ hybridization (FISH) enabled targeted interrogation of specific chromosome sets. The development of array comparative genomic hybridization (array-CGH) provided genome-wide detection of full-chromosome copy-number changes in single oocytes or polar bodies (Colls et al., 2012). Most recently, next-generation sequencing (NGS) approaches have achieved single-cell resolution, offering high sensitivity and specificity for detecting aneuploidy and sub-chromosomal abnormalities in oocytes and blastocysts (Treff et al., 2016).

Cytoplasmic maturation

The early embryo largely relies on maternal transcripts and proteins accumulated during oogenesis. Maternal mRNA turnover, organelle redistribution, energy metabolism, and redox homeostasis are essential for successful development (Ferreira et al., 2009; Nikalayevich et al., 2025). In particular, maternal mRNA metabolism has emerged as a key determinant of oocyte quality and developmental competence. Recent studies show that translational control and selective degradation of stored transcripts are important for fertilization and the maternal-to-zygotic transition (Yang et al., 2024). For example, poly (A) tail remodeling, RNA-binding proteins, and small RNA-mediated pathways precisely regulate maternal transcript utilization in mammalian oocytes, and defects in these processes can lead to meiotic errors and compromised embryo development. Together, the remodeling of maternal mRNA stores is a central layer of cytoplasmic maturation, with direct implications for IVM protocols and their ability to generate competent gametes (Ermisch and Wood, 2024; Lee et al., 2024).

Mitochondrial quality can be accessed via morphology and the spatial organization. Distribution patterns, membrane potential (ΔΨm), and copy number are routinely assessed using fluorescent probes such as MitoTracker, JC-1, or TMRE, and qPCR-based mtDNA quantification (Wai et al., 2010; Feng et al., 2024; Wang et al., 2025). Aberrant clustering or peripheral localization correlates with reduced competence (Kirillova et al., 2021).

The oxidative state of the oocyte can be monitored using H₂DCFDA or MitoSOX probes to quantify ROS levels. High ROS and reduced glutathione (GSH) content are associated with poor developmental outcomes (Voros et al., 2025). Complementary to this, the lipid content and distribution of the ooplasm are emerging as quality markers, especially in bovine oocytes where high lipid accumulation predisposes embryos to heat and oxidative stress (De Andrade Melo-Sterza and Poehland, 2021). Fluorescent dyes such as Nile Red or BODIPY are used to quantify lipid droplets and map their spatial distribution (Genicot et al., 2005). The reduced GSH system is the most critical non-enzymatic defense mechanism in the ooplasm, and its concentration is a robust marker of competence. In oocytes with reduced developmental potential, such as prepubertal models, a decreased ability to synthesize GSH is observed. The ratio of reduced GSH to oxidized glutathione (GSSG) is the definitive measure of the cellular redox state, and a significantly lower GSH/GSSG ratio directly correlates with subsequent developmental failures (Jiao et al., 2013). Other structural indicators include endoplasmic reticulum (ER) reorganization and cortical granule migration. Immunostaining for ER markers (e.g., calreticulin) or lectin staining for cortical granules allows evaluation of cytoplasmic readiness for fertilization and polyspermy block (Mehlmann et al., 1995).

Metabolic activity provides an additional functional dimension for evaluating oocyte competence, as these assays are specifically designed to test whether energy utilization and redox regulation can predict developmental potential. The Brilliant Cresyl Blue (BCB) test remains one of the simplest approaches, assessing glucose-6-phosphate dehydrogenase (G6PD) activity that declines with oocyte growth; its purpose is to distinguish fully grown oocytes (BCB+) with higher competence from immature ones (BCB–) that are less likely to develop successfully (Wu et al., 2007; Wen et al., 2020).

The degree of cumulus expansion in the cumulus–oocyte complex (COC) is widely used as a morphological indicator during IVM, as more expanded cumulus layers are correlated with higher fertilization and embryo development rates. In addition, gene-expression profiling of cumulus cells has identified non-invasive biomarkers of oocyte competence, including elevated expression of HAS2, GREM1, and PTGS2, which is associated with improved oocyte quality and developmental outcomes (Uyar et al., 2013; Massoud et al., 2024).

Improved Strategies for Enhancing IVM Outcomes

Improving IVM outcomes has been a central focus across species, with efforts directed toward optimizing basal media, supplementing with functional additives, and introducing advanced culture systems (Tonai et al., 2023; Gotschel et al., 2024; Pham et al., 2024). Because oocytes from different species exhibit distinct metabolic requirements, the baseline composition of IVM media varies accordingly. In cattle, sheep, and goats, TCM-199 supplemented with gonadotropins (FSH/LH), estradiol, and growth factors remains the standard (Paramio and Izquierdo, 2014; Arias et al., 2022). In pigs, NCSU-23/37 media, enriched with hypotaurine and taurine, is frequently employed to reduce oxidative stress and improve cytoplasmic maturation (Kim et al., 2004). Mouse oocytes are typically matured in α-MEM or Waymouth medium with cAMP modulators and EGF (Richani et al., 2014). In humans, recent progress has been achieved with biphasic “CAPA-IVM” systems, where C-type natriuretic peptide (CNP) is used during a pre-IVM phase to maintain meiotic arrest, followed by conventional IVM for maturation (Gilchrist and Smitz, 2023). Together, these developments highlight the need for species-specific tailoring of basal media to reflect intrinsic follicular physiology (summarized in Table 2).

A major strategy for enhancing IVM has been the supplementation of culture systems with antioxidants and signaling molecules. Melatonin has been consistently shown to improve MII rates, reduce ROS levels, and increase blastocyst development in porcine oocytes through MT2 receptor-mediated mechanisms (Yang et al., 2020; Zhong et al., 2025). Similarly, resveratrol improves nuclear maturation and blastocyst quality under heat stress in pigs, acting via SIRT1 upregulation and redox balance restoration (Tatone et al., 2018; Meng et al., 2023). In cattle, antioxidant supplementation, including cysteamine and polyphenols, improves GSH levels and reduces ROS, thereby enhancing developmental competence, although concentration-dependent toxic effects have also been reported (Rakha et al., 2022). Manipulation of cyclic nucleotide signaling is another effective approach. In mice, pre-IVM with IBMX or dbcAMP stabilizes GV arrest, prevents premature meiotic resumption, and improves downstream competence (Wu et al., 2020).

Co-culture approaches represent another line of improvement. In humans, three-dimensional co-culture of COCs provides a more physiologic environment than simple monolayer systems, leading to better maturation and embryo outcomes (Combelles et al., 2005). In large domestic animals, granulosa or oviductal epithelial cells have been employed to provide paracrine factors that enhance cytoplasmic maturation, as demonstrated in equine (Zhu et al., 2022) and bovine models (Konishi et al., 1996; Zhu et al., 2022). Supplementation with follicular fluid or cumulus-conditioned medium improved maturation rates and embryo quality in human PCOS patients (Madkour et al., 2018).

Microfluidic culture platforms are emerging as promising tools to improve IVM by reproducing dynamic in vivo environments. In mice, microfluidic IVM systems improved fertilization and blastocyst development compared to static culture (Smith and Takayama, 2007; Mastrorocco et al., 2022; Alegretti et al., 2024). These approaches may become integral to standard IVM pipelines as scalability and reproducibility improve.

Finally, omics-based approaches have begun to uncover predictive biomarkers of oocyte competence, extending the evidence from cumulus cell gene expression to transcriptomic, proteomic, and metabolomic signatures that can more precisely capture the molecular state of the maturing oocyte. In cattle, proteomic analysis of cumulus cells revealed metabolic and stress-related pathways associated with oocyte competence (Uhde et al., 2018). Non-invasive metabolomic profiling of spent culture media and single-cell RNA-seq of oocytes are also being developed, providing real-time indicators of IVM quality (Kassim et al., 2025; Peserico et al., 2025). Overall, these developments show that improving IVM results requires a combination of better culture chemistry, improved physical culture conditions, and reliable biomarkers for selecting high-quality oocytes.

Integrated Protocols of IVM and Oocyte Cryopreservation in Reproductive Biotechnology

Oocyte cryopreservation helps solve practical problems such as distance between herds, transport of genetic material, and mismatched breeding seasons in farm animals. As a result, it improves the efficiency of IVP. The combination of IVM and oocyte freezing, mainly through ultra-rapid vitrification, has become widely used in modern reproductive biology. This approach provides safer and more flexible procedures in both human ART and animal breeding programs.

Conventional in vivo maturation requires high doses of hormone injections, which can increase the risk of OHSS, especially in patients with PCOS. IVM-cryopreservation procedures avoid heavy hormone treatment and therefore help prevent OHSS. They also reduce the cost of hormone drugs and limit the need for repeated hospital visits and monitoring (Son et al., 2019).

In animal science, IVM and cryopreservation are essential tools for large-scale germplasm management and the advancement of genetic technologies. Long-term conservation of female genetic resources via oocyte banking provides useful insurance for breeding programs and for preserving genetic diversity, especially in small or endangered populations (Ríos et al., 2025). The ability to reliably cryopreserve IVM-derived oocytes provides a steady, accessible supply of recipient cells necessary for advanced biotechnologies. These techniques include SCNT, cloning, and the production of genetically modified livestock via genome editing, which often requires a consistent, high-quality oocyte source (Park et al., 2015; Moawad et al., 2018).

Successful clinical use of oocyte freezing required replacing slow-freezing with rapid cooling methods, because rapid cooling reduces cold injury and damage to the fragile MII spindle. Studies also show that the timing of freezing is very important for preserving developmental potential (Vajta, 2000). Vitrification of MII oocytes after the completion of IVM (IVM → vitrification) is significantly more efficient than attempting IVM after warming GV-stage oocytes (vitrification → IVM). The lower success of the second method is mainly due to poor cytoplasmic maturation after warming. Even if GV-stage oocytes survive the freezing and warming steps, the cytoplasm often cannot carry out the metabolic and structural changes needed to support normal development. Therefore, completing cytoplasmic maturation before freezing is essential for maintaining oocyte competence (Fasano et al., 2012).

The disparity in success between human ART and livestock reproduction highlights critical species-specific vulnerabilities to cryopreservation, which is rooted in fundamental differences in oocyte physiology. A primary cryoinjury mechanism is chilling injury, which occurs when lipid-containing membranes and intracellular lipid droplets undergo irreversible phase transitions due to rapid cooling. This damage leads to major cellular dysfunction post-warming, and its severity is directly proportional to the cell’s lipid content. Cryo-stress consistently damages the meiotic machinery. Rapid changes in temperature and exposure to cryoprotectants can disrupt microtubules, leading to spindle breakdown and chromosomal displacement. These abnormalities are linked to increased aneuploidy and reduced embryo development (Hwang and Hochi, 2014; Moawad et al., 2018).

A major difficulty in livestock oocyte freezing arises from physical and biochemical features that differ from human and mouse oocytes. For example, pig oocytes are highly sensitive to cooling because they contain large amounts of cytoplasmic lipids. These lipid droplets slow heat movement within the oocyte, increasing the likelihood of cold damage. They also make the cytoplasm thicker, which slows the movement of water out of the cell and cryoprotectants into the cell during freezing. As a result, the inside of the oocyte cools more slowly, even when fast-cooling devices are used, and extra steps are needed to protect the cell (Hwang and Hochi, 2014). Livestock oocytes possess a significantly larger cytoplasmic volume compared with human counterparts. This increases the challenge of regulating osmotic stress, because a larger amount of water and cryoprotectant must be moved across the membrane. The resulting changes in volume place mechanical stress on the cytoskeleton and can further reduce developmental competence. Additionally, the high lipid content causes cytoplasmic darkening in domestic species, making crucial quality control assessments, such as non-invasive visualization of the meiotic spindle via polarized light microscopy, difficult or impossible. This requires the use of more invasive, fixative-dependent diagnostic methods (e.g., fluorescence microscopy), hindering real-time quality assurance and protocol refinement that is standard in human ART.

Applications of IVM-Derived Oocytes in Animal Biotechnology: IVP, SCNT, and Genome Editing

IVM-derived oocytes are now embedded across livestock biotechnology pipelines. In routine IVP, slaughterhouse ovaries or OPU from genetically elite donors supply immature COCs that are matured in vitro, fertilized by IVF or ICSI, and cultured to transferable blastocysts. This approach is standard in cattle and increasingly applied in small ruminants (Ferré et al., 2020).

For SCNT, IVM oocytes are indispensable as recipient cytoplasts. Enucleated MII-stage oocytes provide the reprogramming environment for donor nuclei, and virtually all large-animal SCNT protocols in cattle, pigs, goats, and sheep rely on IVM-derived oocytes. Species-specific refinements in culture media (e.g., SOF/mSOF derivatives) have improved cloning efficiency, but retrospective analyses consistently identify IVM oocyte quality as the primary determinant of developmental outcomes (Campbell et al., 1996a; 1996b; Campbell, 1999).

The most recent application is genome editing, in which IVM is widely used to obtain one-cell zygotes for genetic modification. In this approach, immature oocytes are matured in vitro, fertilized to form zygotes, and then edited by microinjection or electroporation of CRISPR–Cas reagents. In some systems, editing is carried out directly at the MII oocyte stage. This method shortens the generation interval, allows rapid introduction of desired traits, and supports precise allele replacement (Van Eenennaam, 2019; Zhang et al., 2023).

Species-specific applications further illustrate the centrality of IVM. In cattle, limited access to in vivo zygotes necessitates the use of IVM-IVF embryos for editing. Delivery of CRISPR editors by electroporation or microinjection into IVM-IVF zygotes has efficiently introduced targeted variants such as the PMEL coat-color allele, demonstrating on-target accuracy and manageable mosaicism (Wei et al., 2023). Given the long generation interval of cattle, IVM-IVF–based embryo editing remains the most time-efficient route to founder (Wei et al., 2023). In pigs, IVM-derived oocytes underpin both IVP and gene editing; landmark CRISPR/Cas9 studies demonstrated efficient editing in IVM-IVF zygotes that developed into blastocysts and liveborn animals (Gil et al., 2017; Su et al., 2019; Briski et al., 2024). In addition, single-step electroporation of IVM-IVF zygotes has yielded edited embryos at practical efficiencies (Piñeiro-Silva and Gadea, 2025). In small ruminant, gene-edited animals have been generated both by direct microinjection or electroporation into IVM-IVF embryos and by SCNT using edited donor cells (Mahdi et al., 2022; Liu et al., 2024). Taken together these results demonstrated the indispensable role of IVM oocytes as both cytoplast donors and zygote sources.

Conclusion and Future Perspectives

IVM has progressed from a supplementary technique to a widely applied method in both clinical ART and animal biotechnology. In livestock, IVM-derived oocytes are used for IVP, SCNT, and genome editing, allowing routine production of large numbers of embryos. However, differences in developmental competence between in vivo– and in vitro–matured oocytes still exist, mainly due to incomplete cytoplasmic maturation, cytoskeletal instability, and the naturally high lipid content found in many large-animal oocytes.

Future improvement is expected through omics-based biomarkers for oocyte selection, microfluidic and 3D follicle systems that better imitate in vivo environments, and optimized freezing methods for lipid-rich oocytes. Supplementation strategies—such as mitochondria-supporting metabolites, lipid-modifying additives, or cytoskeleton-stabilizing compounds—may further enhance maturation quality, particularly after cytoplasmic maturation. As genome editing becomes increasingly used in agriculture and biomedicine, IVM will continue to play a central role in scalable embryo production. Advances in culture systems and AI-based monitoring are likely to improve practical outcomes such as embryo survival, pregnancy rates, and healthy offspring.