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REVIEW ARTICLE
Year : 2021 | Volume
: 5
| Issue : 3 | Page : 183-192
Research Progress of In vitro Oocyte Maturation
Yuan-Xue Jing1, Yi-Qing Wang2, Hong-Xing Li2, Feng Yue2, Shi-Long Xue2, Xue-Hong Zhang2
1 Reproductive Medicine Center, the First Hospital of Lanzhou University; Key Laboratory for Reproductive Medicine and Embryo of Gansu; The First School of Clinical Medicine, Lanzhou University, Lanzhou 730000, China
2 Reproductive Medicine Center, the First Hospital of Lanzhou University; Key Laboratory for Reproductive Medicine and Embryo of Gansu, Lanzhou 730000, China
Correspondence Address:
Xue-Hong Zhang
Reproductive Medicine Center, the First Hospital of Lanzhou University, No. 1, Donggangxi Road, Chengguan District, Lanzhou 730000
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2096-2924.325827
In vitro maturation (IVM) has been used in clinical settings for 30 years. The merits of IVM include that it needs a relatively small amount of hormones and short treatment period. However, because the effectiveness of IVM is lower than that of controlled ovarian hyperstimulation, there are few centers routinely use IVM, and it is only applicable to a few special populations. In this article, several oocyte sources related to IVM have been discussed and the effects of gonadotropin priming and triggering on IVM are described. Furthermore, we have reviewed the optimization of IVM culture conditions in recent years along with the effects of IVM on genes of oocytes and cumulus cells and the obstetric and neonatal outcomes. We aim to provide indications for future improvement of IVM technology so that the success rates of IVM technology in special populations can be improved. We hope that this mild and natural protocol can be applied to more populations, including individuals with normal ovulation.
Keywords: C-Type Natriuretic Peptide; Cyclic Adenosine Monophosphate; Epidermal Growth Factor-Like Peptides; In vitro Maturation; Melatonin; Oocyte
Oocyte in vitro maturation (IVM) was defined by Edwards in 1962 as the IVM of immature cumulus–oocyte complexes (COCs) collected from antral follicles.[1] The world's first baby successfully conceived using IVM was born in 1991,[2] and now, more than 5,000 babies have been born using IVM.[3] IVM provides patients with low-dose hormonal therapy, which reduces hormone-related side effects and the risk of ovarian hyperstimulation syndrome in patients. IVM is more suitable for patients with a polycystic ovary (PCO) or polycystic ovary syndrome (PCOS). In addition, IVM reduces treatment duration compared to routine controlled ovarian hyperstimulation (COH); therefore, it is more suitable for urgent fertility preservation. IVM also provides an additional way to facilitate pregnancy in patients with poor ovarian reaction and poor embryo quality for unknown reasons. In recent years, IVM has also made some progress in “rescue” of immature oocytes from COH.
The blastocyst formation rate and the live birth rate of oocytes derived from IVM are still significantly lower than those of mature oocytes obtained in vivo. IVM technology is not yet available in most reproductive centers. In the centers where it is available, it is not the first choice and should only be applied to the abovementioned special populations. In this paper, recent developments in IVM, corresponding obstetric outcomes, influence of oocyte genes, and existing problems of IVM are reviewed to lay a foundation for the further improvement of IVM technology.
Source of OocytesTo discuss the efficiency of oocyte maturation in vitro, the source of oocytes should first be clarified. Owing to differences in sources of immature oocytes, differences in the suitable culture protocol and the subsequent culture effect are noted.
The earliest IVM protocol did not use any gonadotropin (Gn), and the oocytes were retrieved when the follicle diameter reached approximately 8 mm. This process was not only suitable for patients with PCOS but also suitable for individuals with normal ovulation. Subsequently, owing to follicle-stimulating hormone (FSH) priming also yielded oocytes of the germinal vesicle (GV) stage that could reinitiate the first meiotic division in vitro and obtain the metaphase-II (MII) stage, the protocol of FSH priming began to use in human IVM. Generally, a small dose of FSH was added on the 3rd day of the cycle for 3 consecutive days. Most scientists and clinicians agreed that these two protocols met the basic definition of IVM.[4] If the patient received FSH for a short duration and was triggered by human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone agonist (GnRHa), this was called the “truncated IVF protocol.”[4] If it was triggered only by hCG or GnRHa, omitting FSH priming, this variation was called “truncated IVF without FSH.”[4] The oocytes obtained by truncated IVF and its variations were a mixture of immature, maturing, and mature oocytes. On the day of oocyte retrieval, intracytoplasmic sperm injection (ICSI) was performed on mature oocytes, and IVM culture was performed on immature and maturing oocytes. After 24–48 h of IVM culture, ICSI was performed on the mature oocytes obtained by IVM again. Hence, multiple rounds of ICSI in a single cycle were needed. This approach adds to the complexity of laboratory procedures, but it has significant meanings for an individual patient. We believed that the laboratory should not reject IVM combined with multiple rounds of ICSI because some special patients have no choice.
Immature oocytes retrieved from a COH cycle were different with the oocytes in the abovementioned IVM process; they were subjected to a large amount of exogenous Gn. Moreover, the oocytes were generally considered to have low developmental potential; IVM was of little significance. However, recent clinical trials using this “rescue protocol” have yielded healthy live births.[5] This changed our perception. It is clinically significant to improve the pregnancy outcomes of patients with low functional ovarian reserve (LFOR).Women with LFOR is characterized by abnormally elevated FSH and/or abnormally low age specific anti Müllerian hormone levels, and their oocyte quantity and quality following COH are always poor.[6] Every additional oocyte may offer potential additional benefits. The “rescue protocol” may alter pregnancy outcomes, reduce cycle cancellation rate, and increase cumulative pregnancy rate. It is necessary to actively explore the optimal culture scheme of immature oocytes produced by COH cycle.
In addition, immature oocytes could also be obtained directly from the removed ovarian tissue.[7],[8],[9],[10] IVM combined with cryopreservation of ovarian tissue can not only maximize the fertility preservation of cancer patients[11],[12],[13] but also maximize the fertility preservation of premature ovarian insufficiency patients caused by immune factors, genetic factors, iatrogenic factors, etc. For some patients with PCOS infertility, endoscopic transvaginal retrieval of immature oocytes to IVM before gynecological laparoscopic ovarian perforation surgery could enhance the scope of IVM for fertility preservation. The procedure is expected to increase the success rate of assisted pregnancy of the patients.[14]
In summary, this paper described several different sources of immature oocytes. We reviewed the progress in research on immature oocyte IVM, including the research related to various sources of immature oocytes that did not meet to the basic definition of IVM [Figure 1].
Follicle-Stimulating Hormone Priming and Human Chorionic Gonadotropin Trigger before Oocyte RetrievalFollicle-stimulating hormone priming
FSH is important for follicular development and oocyte maturation. Studies have shown that pretreatment with FSH before oocyte retrieval can increase oocyte yield and improve oocyte maturity and pregnancy rate.[15] This is quite different from the IVM practice in other mammals because the most common clinical practice of veterinary is not to use any Gn. A prospective multicenter cohort study of live birth rates per IVM cycles initiated showed that although the IVM cycles primed by recombinant FSH alone were less efficient than conventional IVF, they subsequently led to healthy live births.[16] Moreover, the oocyte retrieval in IVM may induce a change in the ovaries comparable to the result of laparoscopic ovarian drilling. The change in the ovary could have a beneficial effect on further IVF/ICSI treatment, leading to a higher pregnancy persistence rate in the first routine IVF/ICSI cycle after IVM failure.[16]
Human chorionic gonadotropin trigger
The effect of hCG on IVM outcomes remains controversial. Use of an hCG trigger before oocyte retrieval for IVM began in 1999.[17] Two patients with PCOS were injected with hCG 36 h before oocyte retrieval in a natural cycle, and clinical pregnancy was obtained by IVM of immature oocytes. The author suggested that hCG administration before the retrieval of immature oocytes could improve the maturational and developmental competence of the oocytes in patients with PCOS. Dahan et al.[18] believed that clinical IVM should include oocyte retrieval from small- and intermediate-sized follicles after hCG or GnRHa trigger. Gn combined with hCG was beneficial for patients with normal ovarian function and could improve the rate of oocyte retrieval and maturation in patients with PCOS.[19] Patients with PCOS who received IVM pretreated with hCG achieved acceptable live birth rates. High levels of anti-Müller hormone predicted a high number of oocytes and a high rate of oocyte maturation.[20] Benefits for patients with PCOS may be related to prematurely expressed and functionally active luteinizing hormone (LH)/chorionic Gn receptor in granulosa cells.[21]
However, hCG triggering reportedly had no positive effect on IVM in some studies. In a randomized controlled study of patients with cancer undergoing urgent fertility preservation who were primed with hCG or GnRHa before the IVM cycle, the total number of frozen oocytes in the untreated group, hCG group, and GnRHa group was 5.1 ± 3.8, 5.4 ± 3.8, and 6.0 ± 4.2, respectively. Mean differences between the three groups were not significant, i.e., the untreated group failed to show noninferiority compared with the hCG or GnRHa group.[22] A retrospective cohort study of unstimulated PCOS patients showed that hCG may not improve the clinical outcomes in the first IVM cycle.[23] In patients with PCOS, the oocyte maturation rate had a rising trend, but there was no statistically significant difference between the hCG group and the untreated group. In addition, there was no statistically significant difference in clinical pregnancy rates between the two groups; the rates of miscarriage and live birth were similar.[23] Another study also showed that there was no reliable evidence that hCG affected rates of live births or miscarriage in IVM.[24]
In summary, there was no consensus on the effectiveness of using hCG trigger in IVM protocols, and different centers have their own solutions and experience in implementing IVM. Care should be taken to distinguish the source of oocytes when comparing pregnancy outcomes because hCG triggers lead a mixture of in vivo maturation and IVM.
Estrogen supplement
Estrogen has a paracrine effect on oocytes and affects the maturation and fertilization of oocytes as well as the development and implantation of early embryos.[25] During the IVM cycle, estrogen supplementation may allow the endometrium to reach an appropriate thickness to compensate for the short IVM cycle.[26] However, estrogen will produce negative feedback to endogenous FSH secretion during the IVM cycle. How to properly supplement estrogen to improve pregnancy outcomes is still being explored.
Vitek et al.[27] found that in terms of laboratory and clinical results, the efficiency of estrogen supplements during the IVM cycle was similar to that of the natural cycle and low-stimulation IVM protocol. Moreover, the IVM protocol eliminated the need for FSH stimulation and cycle monitoring. Another study of the unstimulated IVM protocol showed that midfollicular exogenous estrogen endometrial priming was more effective than early endometrial priming.[28] The maturation rate, fertilization rate, and cleavage rate of the former were 60%, 75%, and 92%, respectively, and there was the delivery of a successful pregnancy.[28] Recent results in patients with PCOS who underwent FSH and hCG primed IVM have shown that early estrogen supplementation improved the quality of immature oocytes and had a beneficial effect on oocyte maturation compared with late estrogen supplementation.[29]
The difference in research results is related to the priming protocols adopted for IVM cycles and the baseline characteristics of the patients. Extensive studies are needed to determine the efficacy and optimal strategy of estrogen supplementation during the IVM cycle.
Additive Substances in In vitro Maturation Culture MediumThere are a few types of commercial IVM media available now. In practice, however, users often add substances to commercial media based on experience or use their own IVM medium completely. In this article, we introduce some commonly used additive substances and those with positive results.
Gonadotropins
Oocytes in IVM need an optimal environment similar to the natural milieu inside the body. Gn is the driving force of follicle development and oocyte maturation. Maturation media prepared in different types usually supplements with recombinant FSH and hCG.[30]
FSH promotes the growth and development of follicles through cyclic adenosine monophosphate (cAMP). Franciosi et al.[31] proved that FSH could improve the development ability of oocytes by regulating mRNA translation in oocytes. They also demonstrated that the effect of FSH was indirect, requiring epidermal growth factor (EGF) receptor signaling in the somatic compartments of the follicle. A low dose of FSH can upregulate the expression of natriuretic peptide precursor or its receptor through estradiol to prevent premature maturation of oocytes and facilitate the full growth of oocytes. Previous studies have shown that the addition of LH to the IVM culture medium can promote the maturation of immature oocytes and improve in vitro fertilization and blastocyst development rates. The FSH receptor appears earlier in human cumulus cells than in LH receptor. Recent evidence has shown that LH receptor expression in cumulus cells gradually increases with follicular volume.[32]
The addition of Gn to IVM medium is very important, and the FSH and LH receptors appear at different time. This means when immature oocytes are retrieved from small follicles or oocytes meet the classical definition of IVM, adding FSH to the culture medium should be initially considered in IVM. The IVM culture medium should be supplemented with LH or hCG in addition to FSH for immature oocytes from large follicles or those triggered by hCG or the common COH process.
Cyclic adenosine monophosphate
Oocyte maturation includes the maturation of the nucleus and cytoplasm. The two processes must closely synchronize to ensure oocyte viability. However, once oocyte retrieval from the antral follicle, spontaneous recovery meiosis may occur while the cytoplasm has not yet fully matured in vitro. The nucleus and cytoplasm of mature asynchronism affected subsequent development of the oocyte.
Oocytes maturated in vitro lacked the cAMP surge that occurs in vivo, and the developmental potential of oocytes is enhanced by artificially increasing cAMP in IVM COCs. Animal studies have shown that communication mediated by gap junction played a fundamental role in the regulation of chromatin remodeling and transcription of oocyte through a cAMP-dependent mechanism.[33] cAMP can induce cumulus expansion and improve oocyte development.[34] Postponement of nuclear precocity by cAMP modulatory agent was found to enhance the antioxidant defense of cumulus against oocytes and improve oocyte quality through gap junctions.[35] Further studies have shown that elevated cAMP altered adenine nucleotide metabolism and provided AMP for energy production through adenosine salvage pathways for the energy-demanding process of meiotic maturation.[36] Oocytes could generate ATP from AMP via the adenosine salvage pathway.[36]
Studies of immature human oocytes have shown similar results. Forskolin, an adenylate cyclase activator, postponed the spontaneous meiosis of immature human oocytes after continuous culture to obtain cytoplasmic maturation.[37] The development of mammalian follicles was influenced by both endocrine signals and paracrine factors. The cAMP and related pathways were required by endocrine stimulation to fine-tune paracrine oocyte signaling in follicular development. The importance of cAMP and related pathways has been demonstrated by studies on novel pathways for cAMP adenosine phosphate signaling in human granulosa cells.[38]
In summary, cAMP improved the quality of oocytes and embryo development potential by maintaining gap junction communication and regulating intracellular signaling, cumulus expansion, and steroidogenesis.[39] As well as enhancement of glutathione led to decreased reactive oxygen species[35] and enhanced glycolysis and oxidative metabolism. Ultimately, this enhancement led to the activation of EGF-like peptide cascades and other complex network pathways.[40] However, the current clinical IVM protocol cannot completely simulate the surge of cAMP in vitro, which may be the reason for the lower efficiency of IVM than that of clinical IVF.[40]
Growth hormone
Growth hormone (GH), a classical polypeptide hormone, is an important regulator of female reproduction in mammals and plays an important role in ovarian function, follicular growth, and steroid production. Addition of GH to IVM medium can promote oocyte maturation and embryo development in many animals.[41],[42],[43] Studies in ovine have shown that GH had a positive effect on nuclear maturation of oocytes. However, compared with GH or FSH alone, concomitant administration of both hormones had a negative effect on subsequent embryonic development. In contrast, concomitant administration of both hormones had no effect on subsequent embryonic development in the absence of serum. The results indicated that the presence of fetal bovine serum during oocyte maturation in vitro also affected the influence of GH on embryo development.[43]
A case report showed that naked GV oocytes derived from COH were matured by IVM containing GH, and a healthy girl was delivered after transfer of the frozen/thawed blastocyst.[44] The author suggested that GH allowed the rescue of naked GV oocytes, and GH action did not pass through the cumulus cells. However, this case report is unique in that the 15 oocytes obtained from COH were all immature oocytes, which was different than the usual 5%–10% immaturity rate of oocytes at that clinic. Li et al.[45] collected GV phase oocytes derived from patients with COH to study the effect of supplementing GH on oocytes maturation in vitro. GV-stage oocytes with a discernable GV were collected for this study after the corona-cumulus cells were removed by hyaluronidase. The results showed that the maturation, fertilization, and blastocyst rates were increased in the GH group compared with the control group. Further mechanism studies showed that GH could significantly enhance the expression of genes associated with meiotic progression and embryo development. The authors speculated that GH could promote oocyte maturation by accelerating the process of meiosis, balancing redox homeostasis of the cellular environment, and improving the developmental capacity of oocytes.
In summary, present studies show that GH can promote maturation in vitro of human immature oocytes derived from COH and improve subsequent embryonic development. However, whether GH can effectively play a positive role is also affected by other components in the culture medium, and the research related to GH promoting IVM requires further elucidation.
Epidermal growth factor-like peptides
The EGF pathway plays an important role in the fine regulation of the complex signal network of oocyte maturation and ovulation. It has been shown that EGF signaling is perturbed in COCs matured in vitro and inspired a new concept for optimizing IVM systems and enhance oocyte development.[46] Elucidating the fundamental molecular and cellular mechanisms of how the EGF network regulates oocyte maturation and ovulation is expected to expand new opportunities in assisted reproductive technology (ART).[46]
In animal models, adding tyrphostin Ag-1478 (a selective inhibitor of EGF receptor) to IVM culture medium can inhibit oocyte maturation, reduce cumulus expansion, and reduce blastocyst rate, which proves that EGFR activation plays an important role in IVM.[47] However, further studies showed that the addition of recombinant human EGF in IVM medium did not enhance outcome of IVM. The expression of mRNAs encoding EGF-like factors amphiregulin, epiregulin, and betacellulin in the cumulus cells showed that these factors produced by cumulus cells could be related to the maximum activation of EGF receptor during oocyte maturation and thereby precluded any further effects of exogenous recombinant human EGF.[47] This led to recognition of the importance of further understanding the activity of the entire pathway.[47] Studies on IVM in marmoset monkey oocytes have shown that the effect of EGF was highly dependent on the concentration of Gn in IVM culture medium. EGF negatively affected oocytes in low concentration of Gn but partially protected oocytes from the negative effects of high concentration of Gn.[48]
EGF-like peptides, such as amphiregulin and epiregulin, were better than FSH and EGF which were widely used IVM additives in promoting the development of mouse oocytes.[49] These two EGF-like peptides significantly increased blastocyst rates and improved blastocyst quality; therefore, amphiregulin and epiregulin could be preferable IVM additives compared to FSH or EGF.[49] Another study also showed that the addition of amphiregulin and epiregulin to culture medium significantly increased the maturation rate of human GV phase oocytes after Gn stimulation.[50] LH acted, at least in part, by stimulating preovulatory follicles to produce EGF family members, amphoteric regulators, and epidermal regulators as paracrine mediators for LH.
In summary, the addition of endogenous maturation mediators such as EGF-like peptides, which positively altered the COCs metabolic endpoints commonly associated with oocyte competence, was one strategy to improve IVM ability of oocytes.[51] The role of EGF-like peptides signaling pathway on oocytes matured in vitro deserves further studied.
C-type natriuretic peptide
Biphasic IVM is a two-phase IVM system that includes pre-IVM and IVM. Pre-IVM is a prematuration step that avoids the premature start of the meiosis process and maintains the transzonal projections between the cumulus cells and the oocyte membrane.[52] C-type natriuretic peptide (CNP) is one of the important additives in the pre-IVM stage. CNP, a physiological cAMP regulator in the ovary, can be used as a phosphodiesterase (PDE) inhibitor of biphasic IVM. CNP replaces other synthetic molecules, such as 3-isobutyl-1-methylxanthine, a non-specific PDE inhibitor, by preventing cAMP degradation to maintain meiosis block of oocyte.
Immature oocytes obtained directly from juvenile goat ovary sections were treated with this biphasic culture with CNP to maintain meiotic stagnation of oocytes for 6 h and to improve the developmental capacity of oocytes and embryos.[53] Similar results were obtained in immature cattle oocytes.[52] The pre-IVM containing CNP, estradiol, FSH, and insulin also improved the quality of oocytes matured in vitro in mildly stimulated 28-day-old mice.[54] The pre-IVM phase significantly influenced cumulus cell function, including prolonged cumulus–oocyte gap-junctional communication, and increased cell proliferation and cumulus expansion.[54]
In a prospective study of patients with PCO, the biphasic IVM culture containing CNP significantly increased oocyte maturity and the proportion of high-quality embryos at day 3. Moreover, oocytes from follicles <6 mm benefited more than oocytes from follicles ≥6 mm.[55] Other studies also confirmed that this IVM protocol improved the maturation rate of immature oocytes in small follicles (2–8 mm) and the utilization rate of embryo cleavage on day 3 in patients with PCOS, and high-quality blastocysts were obtained.[56] In addition, the clinical pregnancy rate was significantly increased, and a live birth after the biphasic culture was reported for the first time.[57] Compared with conventional ovarian stimulation (COS) embryos, blastocysts obtained by this culture showed similar methylation rates and gene expression at germline differentially methylated regions, as well as similar expression of major epigenetic regulators.[58]
In summary, the biphasic IVM system containing CNP has a good effect on the culture of immature oocytes from small follicle COCs, and its optimization effect is not limited to the laboratory, but it also feasible in the human population. This biphasic culture may be more suitable for oocytes directly obtained from removed ovarian tissue or oocytes obtained from small antral follicles.
Melatonin
Mature oocytes cultured in vitro lack the microenvironment required for normal development. The absence of an antioxidant system and additional exposure to exogenous reactive oxygen species are important factors for inefficiency of IVM culture of oocytes. Melatonin is a potent antioxidant that has been shown in animal studies enhanced the development of oocytes in IVM.[59],[60]
In immature oocytes derived from human COS regimen, appropriate concentrations of melatonin supplementation were beneficial to IVM.[61] It could significantly increase the rate of high-quality blastocysts, and the incidence of high-quality blastocyst aneuploidy was very low.[5] This clinical trial has obtained healthy live births.[5] In the same patients treated with a modified IVM/IVF protocol, there were no significant differences in fertilization rate, blastocyst formation rate, clinical pregnancy rate, implantation rate, and live birth/ongoing pregnancy rate between in vivo mature oocytes (IVF oocytes) and in vitro mature oocytes (IVM oocytes) obtained by the melatonin-added system.[62] This modified IVM/IVF regimen can be used effectively in patients with some indications with desirable clinical results.
A study on the mechanism by which melatonin improved the outcome of “rescue IVM” immature oocytes suggested that it may promote human oocyte maturation and early embryo development by enhancing clathrin-mediated endocytosis.[63] Another study showed that melatonin could reduce oxidative stress by ameliorating mitochondrial function and then improved the quality of human oocytes during IVM.[5],[64] The mechanism by which melatonin promoted IVM efficiency in animals was like that in humans. That is, by improving mitochondrial activity and ATP content, reducing ROS level, oocyte development ability, and blastocyst quality were improved, and melatonin had no effect on related gene expression.[59]
In summary, melatonin not only plays a positive role in the IVM of immature animal oocytes, but it also shows a good effect on the IVM of immature oocytes derived from human COS regimen. This has greatly encouraged clinical work and expanded the scope of application of IVM, ultimately benefiting patients [Supplementary Table 1].
COCs are multicellular structures characterized by a three-dimensional (3D) structure. Communication between oocytes and granulosa cells is bidirectional and metabolically interdependent, starting with the initial assembly of the primordial follicles and continuing throughout ovulation. Similarly, granulosa cells need to talk to each other, and granulosa cells also interact with theca cells. There is regulation between extracellular matrix and basal membrane, and there is material exchange between follicles and the surrounding environment.[65],[66],[67] Paracrine interactions between these cells control various processes of oocyte development. At present, most IVM-related studies have been carried out in the common 2D system. Such culture flattens the COCs, which cannot maintain the integrity of their original structure and function. This is an important factor restricting the improvement of IVM efficiency.
Ovarian follicle encapsulation in synthetic or natural matrixes based on biopolymers is a promising IVM process. It maintains 3D follicular tissue and maintains the functional relationship between oocytes and companion follicular cells by preventing them from flattening and subsequently destructing the gap junctions.[68] Compared to standard 2D culture, culturing COCs obtained from goat ovaries in 3D led to a significant increase in the percentage of oocytes reaching MII and a significant decrease in abnormal chromatin configuration of oocytes.[69] The cytoplasmic mitochondrial membrane potential and intracellular ROS levels of oocytes were significantly increased after 3D IVM. At the same time, mitochondrial/ROS co-localization, a healthy, nonoxidized, biomarker for assessing cytoplasmic status, did not change.[69] The CLSM-based multiparametric method used in this study correlated these data, and it was denoted that 3D culture system well balanced oocyte redox status.[69]
This new approach improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation, and it could be used for clinical purposes.[69] In the future, if some optimization schemes of culture medium can be combined with the new 3D culture mode, more ideal results should be obtained theoretically.
Effects of In vitro Maturation on the Oocyte and Cumulus CellsEffects on the oocyte
Compared to COS, IVM reduces the use of Gn and is a mild and friendly alternative for patient with PCOS. However, IVM extends the oocyte in vitro cultivation time, and the IVM process also introduces some unnecessary modification. It is still not clear whether the unnecessary modification can cause adverse effects on oocytes and embryo development. Study of the potential genetic and epigenetic modifications that may occur during the IVM process is urgently needed. Due to the extreme scarcity of human oocytes, there are few studies on the effects of IVM on oocyte genes.
Earlier studies have shown that IVM-MII oocytes were very similar to in vivo maturation-MII oocytes in terms of cellular pathways associated with nuclear maturity, but several pathways related to cytoplasmic functions were still expressed in an immature manner.[70] In addition, IVM-MII oocytes also differed in the expression of genes related to cellular storage and homeostasis. Differentially expressed genes/pathways provided clues for the optimization of IVM technology.[70] Another study showed that IVM strongly affected the gene expression profile of human oocytes, including several genes involved in transcriptional regulation, embryogenesis, epigenetics, development, and the cell cycle.[71] Optimizing the in vitro culture environment not only improves the maturation rate of oocytes, but it also improves the gene expression profile to the extent that they are more comparable to oocytes that naturally mature in the ovarian follicle.[71] Single-cell methylation analysis limited to a few carefully selected imprinted genes showed for the first time that the optimized human IVM program had no significant effect on the establishment of maternal DNA methylation patterns of LIT1, SNRPN, PEG3, and GTL2.[72]
A recent study using single-cell multiomic sequencing analysis compared transcriptomes, DNA methylations, and genomic copy number variations between mature in vivo and in vitro human oocytes.[73] Three conclusions were drawn from this study:[73] (1) The overall transcriptome profiles were similar among oocytes, but 507 genes were differentially expressed. The enrichment genes in vivo mature oocytes were related to the cell cycle process, while the enrichment genes in in vitro mature oocytes were related to the biogenesis of mitochondrial respiration; (2) there was no statistical difference in genome-wide CpG methylation level between in vivo and in vitro mature oocytes, but the non-CpG methylation level of in vivo mature oocytes was higher than that of in vitro oocytes, and there were significant differences between the two groups. Other studies have shown that abnormal methylation was independent of selected imprinted genes in human oocytes matured in vitro;[72] and (3) there was no significant difference in the proportion of aneuploidy between in vivo and in vitro mature, and the rate of aneuploidy was high in both groups, which was consistent with the results of previous studies.[74] However, the aneuploidy had no effect on transcription according to the correlation analysis. This finding is consistent with recent studies, showing that high rates of chromosomal abnormalities had minimal influence on chromatin accessibility.[75] The detrimental impact of aneuploidy on the embryo may be observed after fertilization. The authors suggested that some epigenetic differences between in vivo and in vitro mature oocytes were found in this study. Large and long-term cohort studies are needed to determine whether these epigenetic differences have any impact on embryonic development and offspring health. Further research should examine the duration of these epigenetic differences, as they may occur briefly during the IVM. If these differences are temporary, their effects may not be as harmful as initially predicted, and some oocytes derived from IVM develop into healthy embryos and babies after fertilization, suggesting that embryo development appears to be resistant to differentially expressed genes.
In general, there were no significant changes in the number of cytogenetic chromosomes of oocytes matured in vitro, and there were no significant differences in the major epigenetic gene changes between oocytes matured in vivo and in vitro. Oocytes matured in vitro are safe as the source of oocytes for ART therapy. However, it should not be ignored that, compared with in vivo mature oocytes, there are some differences in gene expression in vitro mature oocytes. The effects of these differences on subsequent development of oocytes and embryo development are not clear, and large-scale and long-term cohort studies are needed to confirm findings.
Effects on cumulus cells
In cumulus cells isolated after IVM compared with cumulus cells from in vivo matured COCs, genes related to cumulus expansion, TNFAIP6, PTGS2, and PTX3 were down-regulated. Moreover, genes associated with oocyte maturation, including several kinds of EGF-like growth factors (EREG, AREG, and BTC) were down-regulated, too while some genes were upregulated related to cell cycle, such as cyclins and CDKs.[76] This shows that the cumulus cells of IVM were still being proliferated, and the cumulus cells around mature oocytes (MII) were not yet fully matured.[76] Moreover, cumulus cells of oocytes matured in vitro were mainly enriched in genes related to DNA replication, recombination, and repair; while cumulus cells of mature in vivo oocytes were mainly enriched in genes related to lipid metabolism. Other studies have shown similar results that cumulus cells from IVM are rich in stress-related genes (HSPA5 and HSP90AB1).[77] Transcriptional abundance of key genes related to cumulus expansion (TNFAIP6) and regulation of oocyte maturation (INHBA and FST) from cumulus cells of oocytes matured in vivo are upregulated.[77]
These studies revealed differences in transcriptional abundance in cumulus cells surrounding mature oocytes from in vitro and in vivo.
Obstetric, Neonatal, and Long-Term OutcomesTo date, more than 5,000 IVM babies have been born worldwide.[78] With the clinical use of IVM gradually increasing, it is necessary to discuss the safety issues associated with IVM that may alter prenatal and postnatal normal development. Studies have shown that IVM does not pose any additional risks compared to IVF and ICSI.[79]
A retrospective cohort study showed that the obstetric and perinatal outcomes of deliveries following IVM cycles are comparable with those following conventional ICSI cycles, including the incidence of major and minor abnormalities of congenital malformations.[80] ICSI cycles were chosen as a control group in this study to exclude the possible effects of the insemination.[80] In a prospective controlled single-blind study, when compared with the counterparts after IVF and after ICSI, children (whether singleton pregnancies or not) conceived after IVM did not show differences during embryonic development, at birth, or in their neuropediatric development at the age of 2.[81] Furthermore, there was no significant difference between the pooled ART group and spontaneously conceived children.[81] However, the limitations of this prospective study lie in the small sample size and the need for a large multicenter cohort study on the health risks of children from IVM pregnancies.
Immature oocyte retrieval from antral follicles and subsequent IVM without ovarian stimulation (OS) is a mild procedure in the realm of ART. Patients with PCOS have many antral follicles on both sides, and most researchers consider patients with PCOS to be an ideal group for IVM. A paired retrospective case–control study involving patients with PCOS compared neonatal obstetric and long-term outcomes of IVM with conventional IVF.[82] There were no differences in the frequency of obstetric and neonatal outcomes between the two groups. No difference was found in birth weights between the groups, and the incidence of congenital anomalies was similar. There were no significant differences in the frequency and duration of hospitalization during childhood. Mean follow-up of 7.5 years showed that growth developmental status of both groups was within the normal range. Another study of IVM of immature oocytes from small antral follicles in patients with PCOS also showed that, compared to COS, IVM did not adversely affect the neonatal health of the offspring of patients with PCOS.[83] There were similar obstetric and neonatal outcomes between IVM and COS.
However, patients with PCOS are a heterogeneous population, and the phenotype of PCOS has a significant impact on obstetric and neonatal outcomes.[84] A single-center retrospective cohort study showed significant differences in cumulative live birth rates (CLBRs) among different PCOS phenotypes after IVM treatment, with the highest CLBR in patients with phenotypes A/Hop (hyperandrogenism + ovulatory disorder + polycystic ovary) and the lower CLBR in patients with phenotypes C/HP (hyperandrogenism + polycystic ovary) or D/OP (ovulation dysfunction + polycystic ovary).[85] This result is in sharp contrast to the lower CLBR in women with hyperandrogenism PCOS phenotype (A/Hop and C/Hp) after OS and ART. This study supports IVM as an effective treatment option, especially in the most severe PCOS phenotype (A/Hop). However, a retrospective study has shown that patients with PCOS phenotype A have increased risk of hypertensive disease pregnancy among patients treated with IVM compared to patients with COS.[83]
In summary, the current obstetric and neonatal outcomes demonstrate the safety of the IVM regimen for clinical use. However, large multicenter cohort studies are still needed to verify the safety of IVM. In view of the differences in pregnancy complications and pregnancy outcomes among different phenotypes of PCOS in patients, the application of IVM regimen in patients with PCOS is worthy of further study.
ConclusionAs IVM technology continues to improve, meaningful results have been achieved in pregnancy rates and implantation rates, with thousands of healthy children born. Particularly, when IVM is combined with other regimens, it expands the use of this technology and improves patient benefits. However, the efficiency of this technique is still lower than that of COS regimens, large-scale in-depth studies on obstetric and neonatal effects are still lacking, and long-term studies on IVM-related genetic changes in the oocytes need to be confirmed. The research depth of human oocyte is still weak compared with other mammals due to the scarcity and ethical principles of human oocytes. Only a better understanding of the entire molecular mechanism of oocyte maturation and adopting the most suitable culture methods for different stages of oocytes obtained by different ways can further improve the efficiency of IVM and make it applicable to a wider population.
Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.
Financial support and sponsorship
This study was supported by Gansu Province Natural Science Foundation (Grant No. 20JR10RA688) and Gansu Province Science Foundation for Distinguished Young Scholars (Grant No. 18JR3RA262).
Conflicts of interest
There are no conflicts of interest.
References
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