Introduction
Since the birth of the first baby conceived by in vitro fertilization (IVF, R. Edwards, 1978), a series of innovative events throughout the years have taken place in the field of Human Assisted Reproduction Technologies (ARTs), attempting to improve clinical outcomes. In particular, an alternative method to conventional IVF has been introduced by reproductive scientists, through which immature oocytes could mature in vitro, mimicking in vivo conditions, a technique known as in vitro maturation of oocytes (IVM). This method was first described in mammals, specifically using rabbit oocytes (Pincus and Enzmann, Reference Pincus and Enzmann1935), as a way to improve the efficiency of animal breeding in agriculturally important species. Moving towards the application of IVM in humans, the first successful fertilization of human in vitro matured oocytes was described in the late 1940s (Rock and Menkin, Reference Rock and Menkin1944; Menkin and Rock, Reference Menkin and Rock1948), while the remarkable work of Edwards defined the kinetics of oocyte nuclear maturation, as well as the ideal culture conditions for a successful IVM protocol (Edwards, Reference Edwards1962, Reference Edwards1965; Edwards et al., Reference Edwards, Bavister and Steptoe1969). Following the significant work made to optimize the technique, the first IVM birth was reported in 1991 by Cha and colleagues (Cha et al., Reference Cha, Koo, Ko, Choi, Han and Yoon1991), paving the way for a novel ART technique translated into clinical practice.
Application of IVM and clinical indications
IVM is based on the collection of immature cumulus-oocyte complexes (COCs) from antral follicles that are subsequently cultured in vitro until they reach the metaphase II (MII) stage (Edwards, Reference Edwards1965; Mikkelsen et al., Reference Mikkelsen, Smith and Lindenberg1999; De Vos et al., Reference De Vos, Grynberg, Ho, Yuan, Albertini and Gilchrist2021). Patients undergoing IVM receive no or minimal ovarian stimulation, instead of a conventional controlled ovarian stimulation IVF (COS-IVF) protocol. Once maturation in the laboratory is completed, IVM oocytes are normally fertilized and treated exactly as the oocytes retrieved after conventional IVF (Thompson and Gilchrist, Reference Thompson and Gilchrist2013).
As far as IVM protocols are concerned, there are four major protocols that are practiced in the laboratory. To start with, the standard IVM protocol represents the original one that was initially developed by Edwards (Edwards, Reference Edwards1965). The latter includes the collection of immature/GV-stage COCs, which undergo in vitro maturation in a single step until they reach the MII stage and are subsequently inseminated. It is of great importance that the cumulus cell-oocyte communication structure remains intact during in vitro culture from the GV to the metaphase II stage. Of note, follicle-stimulating hormone (FSH) might have been administered to patients, or not, prior to oocyte pick-up. Second, the so-called biphasic IVM protocol, which was the evolution of the standard IVM, involves a two-step procedure. The biphasic IVM protocol represents a variation of the standard IVM protocol, whose main difference relies on the additional pre-IVM step. More specifically, once collected, GV-stage COCs are cultured in a pre-IVM medium for approximately 24 h, where meiosis is inhibited at the GV stage, due to the presence of meiotic inhibitors in the culture medium. In the next step, oocytes finally mature from the GV to MII stage, through meiosis-inducing factors, such as epidermal growth factor (EGF-p). The meiotic induction step lasts 30–48 h in humans (Gilchrist, Reference Gilchrist2011; Richani and Gilchrist, Reference Richani and Gilchrist2022). Interestingly, both oocyte meiotic arrest and resumption are regulated by the C-type natriuretic peptide/ cyclic guanosine monophosphate (CNP/cGMP) signalling pathway, upstream of the intra-oocyte cyclic adenosine monophosphate (cAMP) (Gilchrist et al., Reference Gilchrist, Luciano, Richani, Zeng, Wang, De Vos and Thompson2016). Based on the above, the main principles of the biphasic IVM culture system include 1) the maintenance of the oocyte in a meiotically arrested- GV stage (in vitro), 2) the stable and not impaired communication between oocyte and cumulus cells, 3) the acquisition of the oocyte developmental competence during the pre-IVM step, and lastly 4) the resumption of meiosis under conditions mimicking the endogenous post-LH surge effect. Concerning the FSH priming during the implementation of the biphasic protocol, it remains optional. Finally, the pre-IVM procedure, also known as “capacitation-IVM” (CAPA-IVM) was tested for its safety and efficacy through pre-clinical trials, while its use in clinical practice is associated with healthy live birth rates, comparable to conventional IVF protocols (Gilchrist et al., Reference Gilchrist, Ho, De Vos, Sanchez, Romero, Ledger, Anckaert, Vuong and Smitz2024). Furthermore, the hCG-Primed IVM protocol represents an alternative protocol, where patients are triggered with human chorionic gonadotropin (hCG), in order to increase maturation success, while FSH priming still remains optional. The so-called “Truncated” IVM protocol, thus results in the presence of both immature (GV, MI) and mature (MII) stage oocytes, which are inseminated at different time points in the laboratory (Son et al., Reference Son, Chung, Chian, Herrero, Demirtas, Elizur, Gidoni, Sylvestre, Dean and Tan2008). In other words, oocytes are treated differently in the laboratory, with the MII oocytes necessitating fertilization on the same day of the oocyte retrieval, while the maturing and/or immature oocytes require IVM culture before the fertilization step, which might be a source of an additional laboratory burden. The hCG-primed IVM protocol thus excludes the use of any pre-IVM culture system. Finally, the “Rescue-IVM ” or Conventional IVF protocol, includes the in vitro maturation of immature oocytes (GV and/or MI stage), collected after a conventional IVF cycle, where FSH is normally being administrated and ovulation triggering is mostly followed after hCG priming. The oocytes collected by such protocols are commonly regarded as non-usable oocytes for medical practice and are normally discarded in the corresponding cycles. The “rescue-IVM” oocytes are usually denuded of their cumulus cells, after the oocyte retrieval and prior to intracytoplasmic sperm injection (ICSI) and as a result, these oocytes are invariably cultured in vitro in a denuded state, from the GV to the MII stage. Overall, due to their suboptimal quality and the presence of meiotic defects, the success of Rescue-IVM procedures remains questionable (De Vos et al., Reference De Vos, Smitz, Thompson and Gilchrist2016).
In terms of clinical application, IVM was basically designed as an alternative to standard ovarian stimulation protocols, in order to overcome the negative effects and risks associated with ovarian stimulation, such as ovarian hyperstimulation syndrome (OHSS) in high responders. In this regard, women with polycystic ovary syndrome (PCOS) represent the best candidates for IVM, as on the one hand, these women are expected to have a higher number of immature oocytes at oocyte pick-up, which is associated with better clinical outcomes when using IVM, while on the other hand, they risk being affected by OHSS (Cha et al., Reference Cha, Han, Chung, Choi, Lim, Lee, Ko and Yoon2000). More specifically, in women who are high responders and the final oocyte triggering is performed by GnRH agonist, IVM would be a useful approach. GnRH agonists have been successfully used to trigger final oocyte maturation in IVF cycles, occasionally leading to the collection of immature and/or reduced number of oocytes, as a preventive approach to OHSS (Casper, Reference Casper2015; Gonen et al., Reference Gonen, Balakier, Powell and Casper1990). Indeed, IVM represents a possible alternative to ovarian hyperstimulation, while priming in IVM cycles using GnRH agonists seems to be equally effective as hCG priming, although GnRH priming seems to be best-suited for fertility preservation in hormone-sensitive cancers and urgent fertility preservation cases (Hachem et al., Reference El Hachem, Sonigo, Benard, Presse, Sifer, Sermondade and Grynberg2018).
Furthermore, a good indication for IVM is also considered to be in cases of urgent fertility preservation, when conventional ovarian stimulation protocols cannot be applied and/ or are contraindicated. Such cases that cannot be treated with gonadotrophins include cancer patients who are scheduled to be exposed to gonadotoxic treatments and prepubertal girls (ESHRE Guideline Group on Female Fertility Preservation et al., Reference Anderson, Amant, Braat, D’Angelo, Chuva de Sousa Lopes, Demeestere, Dwek, Frith, Lambertini and Maslin2020). Lastly, IVM should ideally be applied in rare cases of patients with resistant ovary syndrome (ROS). ROS represents a rare endocrine disorder whose symptoms include hypergonadotropic anovulation and infertility, while patients experience primary or secondary amenorrhoea (Talbert et al., Reference Talbert, Raj, Hammond and Greer1984; Huhtaniemi and Alevizaki, Reference Huhtaniemi and Alevizaki2006). On the other hand, IVM may not be suitable for a certain group of patients. In fact, the use of IVM in normo-ovulatory patients (patients with regular cycles) might end up with a lower oocyte yield, compared to a conventional oocyte stimulation protocol, meaning fewer usable embryos, thus resulting in lower clinical pregnancy rates (Gilchrist and Smitz, Reference Gilchrist and Smitz2023). Accordingly, IVM is not indicated for poor responders with low ovarian reserve, as well as women of advanced reproductive age, since the success of IVM depends on the number of oocytes collected (the more the better), as already mentioned (Braga et al., Reference Braga, Figueira, Ferreira, Pasqualotto, Iaconelli and Borges2010). A higher number of oocytes collected after IVM might be able to compensate for the suboptimal clinical outcomes (De Vos et al., Reference De Vos, Grynberg, Ho, Yuan, Albertini and Gilchrist2021; Gilchrist and Smitz, Reference Gilchrist and Smitz2023). Finally, a small group of patients presenting oocyte meiotic defects, who obtain no mature oocytes after conventional oocyte stimulation procedures, are not suitable for IVM either. In fact, current IVM protocols are not able to “correct” oocyte meiotic abnormalities and thus do not result in encouraging clinical outcomes (Hourvitz et al., Reference Hourvitz, Maman, Brengauz, Machtinger and Dor2010; Galvão et al., Reference Galvão, Segers, Smitz, Tournaye and De Vos2018).
Taking into consideration the cases of patients that are suitable for an IVM procedure, IVM itself is not considered an experimental/add-on technique. On the contrary, this is not the case for the group of patients where IVM is contraindicated and therefore shouldn’t be applied, as recently declared by the ESHRE Add-ons working group (ESHRE Add-ons working group et al., Reference Lundin, Bentzen, Bozdag, Ebner, Harper, Le Clef, Moffett, Norcross, Polyzos and Rautakallio-Hokkanen2023). It is worth mentioning that the need for universal guidelines during the use of alternative IVF protocols, such as IVM, is urgent, as well as the expertise of reproductive scientists who need to discuss the clinical outcomes and protocol modifications, in the context of an inter-centre communication and exchange of knowledge and skills.
Effectiveness of IVM in clinical practice
Moving towards the efficacy of IVM on a clinical scale, reproductive scientists are questioning whether IVM increases success rates when applied to certain groups of patients (with an indication of PCOS, high responders and/or fertility preservation cases). Based on the recently published data, when comparing the outcomes of conventional IVF protocols to standard IVM in patients with a defined infertility cause (e.g. PCOS), IVM clinical pregnancy rates still remain lower (Vuong et al., Reference Vuong, Ho, Ho, Dang, Phung, Giang, Le, Pham, Wang, Smitz and Gilchrist2020; Gilchrist and Smitz, Reference Gilchrist and Smitz2023). Interestingly, several studies suggest that oocytes collected after an IVM procedure; once they reach the MII stage and therefore are inseminated, result in lower fertilization rates, as well as lower quality of embryos, contrasted to conventional IVF outcomes (Braga et al., Reference Braga, Figueira, Ferreira, Pasqualotto, Iaconelli and Borges2010). In fact, it is hypothesized that the significantly low clinical outcomes might be the result of a dysfunctional maturation process, an asynchronous nuclear-cytoplasmic maturation, owing to the in vitro culture conditions (De Vos et al., Reference De Vos, Van de Velde, Joris and Van Steirteghem1999; Bao et al., Reference Bao, Obata, Carroll, Domeki and Kono2000). Concerning the implantation capacity, a significant improvement in the success rates of IVM embryo transfers has been observed when choosing the freeze-all strategy and deferred transfer per cycle (De Vos et al., Reference De Vos, Ortega-Hrepich, Albuz, Guzman, Polyzos, Smitz and Devroey2011; Chang et al., Reference Chang, Song, Lee, Lee and Yoon2014; Vuong et al., Reference Vuong, Nguyen, Le, Pham, Ho, Le, Pham, Dang, Phung, Smitz and Ho2021). In particular, endometrial development seems to be compromised and insufficiently prepared for a fresh embryo transfer during an IVM cycle, compared to the natural and/or stimulated ones (De Vos et al., Reference De Vos, Ortega-Hrepich, Albuz, Guzman, Polyzos, Smitz and Devroey2011; Walls et al., Reference Walls, Hunter, Ryan, Keelan, Nathan and Hart2015; Ortega-Hrepich et al., Reference Ortega-Hrepich, Drakopoulos, Bourgain, Van Vaerenbergh, Guzman, Tournaye, Smitz and De Vos2019). Finally, IVM pregnancy rates are still controversial between centres, as observational studies demonstrate a live birth rate of 15,9% per retrieval (Child et al., Reference Child, Abdul-Jalil, Gulekli and Tan2001; Buckett et al., Reference Buckett, Chian and Tan2004), while others report pregnancy rates at approximately 22% (Söderström-Anttila et al., Reference Söderström-Anttila, Mäkinen, Tuuri and Suikkari2005). Based on the official report of the ESHRE Add-ons working group, ongoing pregnancy rates following IVM range from 36.8 to 31.9% in women aged from 20 to 39 years, while clinical pregnancies in women over 40 years are rarely detected (ESHRE Add-ons working group et al., Reference Lundin, Bentzen, Bozdag, Ebner, Harper, Le Clef, Moffett, Norcross, Polyzos and Rautakallio-Hokkanen2023). Of note, a positive correlation between IVM live birth rates and the number of oocytes collected at the time of egg retrieval has been observed, with a minimum of five oocytes needed to achieve a pregnancy (Al-Sunaidi et al., Reference Al-Sunaidi, Tulandi, Holzer, Sylvestre, Chian and Tan2007; Fadini et al., Reference Fadini, Comi, Mignini Renzini, Coticchio, Crippa, De Ponti and Dal Canto2011; Yang et al., Reference Yang, Patrizio, Yoon, Lim and Chian2012). Interestingly, a very recent article by Mostinckx and colleagues reported comparable reproductive outcomes between patients undergoing a conventional ovarian stimulation protocol and patients included in an IVM cycle, with serum anti-Müllerian hormone levels ≥10 ng/ml. Data from a large cohort of patients showed that ongoing pregnancy rates where not different in predicted hyper-responders undergoing ART after IVM compared with conventional IVF cycles (Mostinckx et al., Reference Mostinckx, Goyens, Mackens, Roelens, Boudry, Uvin, Segers, Schoemans, Drakopoulos, Blockeel and De Vos2024)
Safety issues and aspects of IVM
As expected, modified and/or newly performed ART protocols raise safety and ethical concerns about their potential adverse effects and the long-term safety of children conceived with these techniques, such as IVM. To overcome safety issues, scientists are investigating the impact of ART interventions performed on a clinical scale in human populations by using animal models, which represent an alternative approach to both understand the complexity of such reproductive treatments, as well as to collect useful data. Interestingly, the main advantage of studying ART treatments in animal models, compared to human clinical studies, is that animals selected for the studies normally do not present fertility complications, which could introduce a confounding factor of infertility in the population performing ART. Moreover, animal populations are characterized by a higher genetic homogeneity compared to human populations, which might also play an important role in detecting the variability in ART treatment effects.
As far as IVM is concerned, animal studies are primarily based on the bovine model, while clinical and laboratory protocols are slightly modified compared to human methodologies, during the hormonal priming and the in vitro maturation steps (Krisher, Reference Krisher2022). Overall, animal studies investigating the impact of IVM are focusing on several clinical outcomes, such as birth weight, length of gestation, cardiovascular (e.g. blood pressure) and metabolic (fasting glucose, insulin) parameters, behavioural traits and finally lifespan. First, results from a meta-analysis in bovine models showed a significant increase in the birthweight of the IVM group, when compared to the in vivo controls, while also a longer gestational length was found in the IVM group versus the controls (Beilby et al., Reference Beilby, Kneebone, Roseboom, van Marrewijk, Thompson, Norman, Robker, Mol and Wang2023). Studies focusing on the mouse model reported a significant increase in the systolic blood pressure in female mice conceived with IVM (Le et al., Reference Le, Lou, Wang, Wang, Wang, Li, Yang, Zhan, Lou and Jin2019), where metabolic outcomes from bovine studies (serum glucose and insulin levels after birth) were not found to be significantly different in the IVM group (Jacobsen et al., Reference Jacobsen, Schmidt, Hom, Sangild, Greve and Callesen2000; Sangild et al., Reference Sangild, Schmidt, Jacobsen, Fowden, Forhead, Avery and Greve2000; Bertolini et al., Reference Bertolini, Mason, Beam, Carneiro, Sween, Kominek, Moyer, Famula, Sainz and Anderson2002). Finally, no differences were reported in newborn behavioural traits, such as standing and suckling time, as well as respiratory distress, between IVM and in vivo conceived calves (Bertolini et al., Reference Bertolini, Mason, Beam, Carneiro, Sween, Kominek, Moyer, Famula, Sainz and Anderson2002), although the need for a breathing stimulus at birth was found to be significantly increased in calves conceived with IVM when compared to in vivo conceived animals (van Wagtendonk-de Leeuw et al., Reference Van Wagtendonk-de Leeuw, Mullaart, De Roos, Merton, den Daas, Kemp and De Ruigh2000). Lifespan data were not available for animals conceived with IVM (Beilby et al., Reference Beilby, Kneebone, Roseboom, van Marrewijk, Thompson, Norman, Robker, Mol and Wang2023).
On the other hand, in humans, currently available data do not support a globally negative impact of the use of IVM in clinical practice. To start with, very recent reports have evaluated the quality and ploidy status of embryos generated by an IVM procedure. These studies demonstrate that the ability of in vitro matured oocytes to be fertilized and form good quality embryos, as well as the production of euploid blastocysts, was similar to in vivo matured oocytes, whereas pregnancy and perinatal outcomes of these embryos were similar (Li et al., Reference Li, Chen, Sun, Zhang, Jiao, Chian, Li and Xu2021, Li et al., Reference Li, Jin, Tian, Yan, Li, Ren and Guo2024). Furthermore, concerns have also been expressed about the epigenetic abnormalities and imprinting errors in embryos resulting from an IVM procedure, as oocyte meiosis occurs in vitro. In fact, possible epigenetic modifications, such as methylation, as well as dysfunctional gene expression of imprinting genes, might occur, although published data do not report imprinting gene disorders in embryos/foetuses after IVM, suggesting that IVM does not compromise the epigenetic landscape and genomic imprinting establishment (Kuhtz et al., Reference Kuhtz, Romero, De Vos, Smitz, Haaf and Anckaert2014; Pliushch et al., Reference Pliushch, Schneider, Schneider, El Hajj, Rösner, Strowitzki and Haaf2015; Saenz-de-Juano et al., Reference Saenz-de-Juano, Ivanova, Romero, Lolicato, Sánchez, Van Ranst, Krueger, Segonds-Pichon, De Vos, Andrews and Smitz2019). However, while current data seem reassuring, increased methylation levels of the KvDMR1 locus were observed in arrested immature oocytes of unstimulated PCOS patients, compared to oocytes resulting from stimulated cycles, suggesting that stimulation may exert an impact over imprinting establishment (Khoueiry et al., Reference Khoueiry, Ibala-Rhomdane, Méry, Blachère, Guérin, Lornage and Lefèvre2008; Market-Velker et al., Reference Market-Velker, Zhang, Magri, Bonvissuto and Mann2010), making the subject a highly controversial one. Concerning neonatal health, as well as the development of children born after IVM, in the majority of cases, normal perinatal outcomes have been observed, when compared to babies born after a conventional IVF. In fact, factors such as miscarriage rate, preterm birth, birth weight, congenital anomalies, mental development and other pregnancy complications, seem to not be different from the ones described for IVF babies (Mostinckx et al., Reference Mostinckx, Segers, Belva, Buyl, Santos-Ribeiro, Blockeel, Smitz, Anckaert, Tournaye and De Vos2019; Belva et al., Reference Belva, Roelants, Vermaning, Desmyttere, De Schepper, Bonduelle, Tournaye, Hes and De Vos2020; Strowitzki et al., Reference Strowitzki, Bruckner and Roesner2021; Vuong et al., Reference Vuong, Nguyen, Nguyen, Ly, Tran, Nguyen, Hoang, Le, Pham, Smitz and Mol2022). While some reports do describe perinatal abnormalities (Cha et al., Reference Cha, Chung, Lee, Kwon, Chung, Park, Choi and Yoon2005; Söderström-Anttila et al., Reference Söderström-Anttila, Salokorpi, Pihlaja, Serenius-Sirve and Suikkari2006; Buckett et al., Reference Buckett, Chian, Holzer, Dean, Usher and Tan2007), clinical outcomes cannot be accurately assessed, as only a small number of babies are born after an IVM procedure, highlighting the importance of data availability. Indeed, the follow-up of children conceived from IVM is limited to ≤2 years, although current results from published studies have not identified differences between children born after IVM compared with those born after a conventional IVF cycle (Strowitzki et al., Reference Strowitzki, Bruckner and Roesner2021; Vuong et al., 2022; Reference Vuong, Pham, Ho and De Vos2023). Consequently, longitudinal data from prospective studies with longer term follow-up are needed, in order to draw solid conclusions about the safety of IVM (Vuong et al., Reference Vuong, Pham, Ho and De Vos2023).
IVM and mitochondrial function
While safety issues and complications after an IVM procedure have not yet been fully elucidated, concerns about critical components of oocyte competence are still a matter of discussion and need to be further investigated. For instance, mitochondria play a pivotal role during oocyte maturation and growth, as these procedures require a large amount of energy in the form of ATP. As a result, the correct functioning of mitochondria is crucial, otherwise these important steps will be compromised.
Mitochondria, also known as the power-house of the cell, are semi-autonomous organelles containing their own genetic information, called mitochondrial DNA (mtDNA). Their main functions include energy production for the cells, Ca2+ homeostasis, cell death regulation, iron metabolism and biosynthesis of several organic compounds (Spinelli and Haigis, Reference Spinelli and Haigis2018; Rossi et al., Reference Rossi, Pizzo and Filadi2019; Bock and Tait, Reference Bock and Tait2020; Boyman et al., Reference Boyman, Karbowski and Lederer2020; Lill and Freibert, Reference Lill and Freibert2020). During oogenesis and follicular growth, the number of mitochondria in oocytes increases exponentially, rising from approximately 10,000 to 20,0000 organelles (Jansen and de Boer, Reference Jansen and De Boer1998), while also the mtDNA copy number reaches up to 50,000 mtDNA copies in a mature oocyte (Reynier et al., Reference Reynier, May-Panloup, Chrétien, Morgan, Jean, Savagner, Barrière and Malthièry2001). This highlights the importance of an adequate number and good quality of mitochondria required to sustain oogenesis and the early stages of embryogenesis. However, before oocyte maturity completion, the energy needed to support the process must be provided by the surrounding granulosa and cumulus cells, as mitochondria from immature oocytes remain in a naïve state (Dumollard et al., Reference Dumollard, Campbell, Halet, Carroll and Swann2008). As oocytes lose progressively their connections with the cumulus cells, they need to activate their own mitochondria to complete the final stages of maturation. Any deviation from this well-defined mechanism may result in diminished ovarian reserve. In fact, in women with primary ovarian insufficiency, which is also known as premature ovarian failure, oocytes were found to contain less mtDNA copies, compared to women with a normal ovarian profile, suggesting that low values of mtDNA copy number are associated with an abnormal mitochondrial biogenesis (May-Panloup et al., Reference May-Panloup, Chrétien, Jacques, Vasseur, Malthièry and Reynier2005). Accordingly, mitochondrial distribution is also a crucial component during oocyte maturation, as it needs to be well-structured and dynamic (Takahashi et al., Reference Takahashi, Hashimoto, Yamochi, Goto, Yamanaka, Amo, Matsumoto, Inoue, Ito, Nakaoka and Suzuki2016). In fact, mitochondria preferentially migrate towards the perinuclear region and represent 80% of the cytoplasmic volume. After the germinal vesicle breakdown stage and until oocytes reach the MII stage, mitochondria are equally distributed and occupy almost the whole of cytoplasmic volume (Trebichalská et al., Reference Trebichalská, Kyjovská, Kloudová, Otevřel, Hampl and Holubcová2021).
However, during IVM, oocytes are exposed to different in vitro conditions, which may subsequently modify the well-defined pattern of mitochondrial function and localization. For instance, as mentioned before, upon maturation, mitochondria localize towards the perinuclear/central region of the oocyte, while their homogeneous distribution represents a sign of cytoplasmic maturity. On the contrary, the peripheral localization of mitochondria has been associated with meiotically incompetent oocytes (Sánchez et al., Reference Sánchez, Romero, De Vos, Verheyen and Smitz2015). Notably, it has been demonstrated that mitochondria from IVM oocytes tended to localize more abundantly in the peripheral region, instead of the inner cytoplasmic region, when compared to in vivo matured oocytes (Liu et al., Reference Liu, Li, Gao, Yan and Chen2010). To continue, while both mitochondrial number and ultrastructure were not found to be different between IVM and in vivo matured oocytes (Coticchio et al., Reference Coticchio, Dal Canto, Fadini, Mignini Renzini, Guglielmo, Miglietta, Palmerini, Macchiarelli and Nottola2016), data from mitochondrial DNA copy number assessment are scarce. Several studies based on the murine model have actually demonstrated that the mitochondrial DNA number of in vitro matured oocytes was significantly lower, compared to control oocytes, which was hypothesized to be a sign of a compromised mitochondrial biogenesis, as well as cytoplasmic immaturity (Ge et al., Reference Ge, Tollner, Hu, Da, Li, Guan, Shan, Lu, Huang and Dong2012; Tao et al., Reference Tao, Landis, Krisher, Duncan, Silva, Lonczak, Scott, Zhan, Chu, Scott and Treff2017). Furthermore, reports from mice showed that both mitochondrial membrane potential and the ATP amount were not found to be different between in vitro and in vivo oocytes (Ge et al., Reference Ge, Tollner, Hu, Da, Li, Guan, Shan, Lu, Huang and Dong2012). However, single-cell transcriptomic data from both in vitro and in vivo human oocytes demonstrated a significant number of alterations in gene pathways associated with mitochondrial function in the IVM group (Zhao et al., Reference Zhao, Li, Zhao, Tan, Liu, Liu, Chang, Huang, Li, Fan and Yu2019), making this subject a highly controversial one.
Whether in vitro conditions exert an impact over mitochondrial patterns still remains an open question. Nevertheless, the degree of the possible alterations previously mentioned, might depend on the specific cultured conditions and protocols used during an IVM procedure that might influence both nuclear and cytoplasmic maturity of the oocyte. In fact, it remains unclear whether IVM media influence the mitochondrial integrity of oocytes, as there is a large variety of protocols used in clinical practice. For instance, studies on the bovine model demonstrated that during an IVM procedure, relatively high oxygen concentrations (20%) resulted in a better embryonic yield, when compared to lower oxygen concentrations (5–7%) (Pinyopummintr and Bavister, Reference Pinyopummintr and Bavister1995; Whitty et al., Reference Whitty, Kind, Dunning and Thompson2021). However, attention should be paid to the oxygen concentrations applied, as excessive oxygen levels may result in increased production of reactive oxygen species (ROS), which subsequently compromise mitochondrial function. Although the impact of different oxygen concentrations, as a component of culture conditions, is not clear yet, it is likely that culture media composition might affect the redox state of the cells and ROS production (Cobley, Reference Cobley2020).
To overcome these issues, several studies propose the supplementation of IVM culture media with antioxidants, in order to enhance the mitochondrial function of in vitro matured oocytes, as well as to ameliorate the oocyte and embryonic competence upon IVM application. In fact, the supplementation of IVM media with the follicular fluid-derived melatonin has been shown to significantly increase the implantation rates in PCOS patients (Kim et al., Reference Kim, Park, Kim, Choi, Cho, Lee, Cha, Kim, Lee and Yoon2013), as well as the blastocyst formation rate in rescue-IVM oocytes (Hao et al., Reference Hao, Zhang, Han, Cao, Zhou, Wei, Lv and Chen2017; Zou et al., Reference Zou, Chen, Ding, Gao, Chen, Liu, Hao, Zou, Ji, Zhou and Wei2020). Other antioxidants that might improve IVM clinical outcomes include resveratrol, which resulted in improved spindle morphology and intact chromosomal localization in human rescue-IVM oocytes (Liu et al., Reference Liu, Sun, Zhao, Liu, Ma, Li, Huai, Zhao and Liu2018), quercetin, which was proven to improve the mitochondrial function in porcine, mice, goat and human IVM oocytes (Kang et al., Reference Kang, Kwon, Park, Kim, Moon, Koo, Jang and Lee2013; Silva et al., Reference Silva, Silva, Figueiredo, Gonçalves, Silva, Loiola, Bastos, Oliveira, Ribeiro, Barberino and Gouveia2018; Cao et al., Reference Cao, Zhao, Wang, Zhang, Bian, Liu, Zhang, Zhang and Zhao2020), leading to increased fertilization and blastocyst formation rates (Cao et al., Reference Cao, Zhao, Wang, Zhang, Bian, Liu, Zhang, Zhang and Zhao2020). Finally, addition of the antioxidant anethole in IVM culture media for bovine oocytes has resulted in higher cleavage, better embryonic development and higher cell number per blastocyst rates (Sá et al., Reference Sá, Vieira, Ferreira, Cadenas, Bruno, Maside, Sousa, Cibin, Alves, Rodrigues and Leal-Cardoso2019), while supplementation with the mitochondrial inner membrane coenzyme Q10 (CoQ10), is directly associated with increased mitochondrial function and better embryonic outcomes (Abdulhasan et al., Reference Abdulhasan, Li, Dai, Abu-Soud, Puscheck and Rappolee2017; Heydarnejad et al., Reference Heydarnejad, Ostadhosseini, Varnosfaderani, Jafarpour, Moghimi and Nasr-Esfahani2019). Surprisingly, addition of CoQ10 in the IVM culture medium of human oocytes resulted in a 20% increase of the maturation rate, as well as a significant decrease in aneuploidy rates in the first polar body of patients with an advanced maternal age (Ma et al., Reference Ma, Cai, Hu, Wang, Xie, Xing, Shen, Cui, Liu and Liu2020). Taken together, supplementation of the IVM media with antioxidants should be taken into consideration and be applied in clinical practice, by selecting the right combination and concentration of the agents, to avoid possible detrimental effects on the oocytes and embryos.
Conclusion
To summarize, IVM represents a procedure where minimal or absence of ovarian stimulation is required in patients with specific indications. As such, it is supposed to be an advantageous technique, as it requires less time, minimal medical monitoring and fewer to no hormone injections and blood monitoring. Cost-effectiveness studies also suggest that IVM is a less expensive choice, compared to a conventional ovarian stimulation protocol, while these characteristics are associated with a better mental and psychological status of patients undergoing such procedures (Braam et al., Reference Braam, Ho, Pham, Mol, Van Wely and Vuong2021; Practice Committees of the American Society for Reproductive Medicine, the Society of Reproductive Biologists and Technologists, and the Society for Assisted Reproductive Technology. Electronic address: jgoldstein@asrm.org, 2021).
If hormone-free protocols for both fertility preservation and in vitro fertilization represent the new era in ART, it still remains an open question. It is therefore crucial to highlight the importance of research and well-designed randomized controlled trials, in order to be able to resolve safety and effectiveness issues, as well as to improve protocols and culture conditions that are currently used during oocyte in vitro maturation. In conclusion, IVM requires specific expertise from both medical doctors and embryologists, while the follow-up of children born after IVM is urgently needed in order to ensure better clinical outcomes.
Competing interests
The authors declare no conflicts of interest.
Funding
Not applicable.
