**3. Definition of oocyte IVM**

*Embryology - Theory and Practice*

**2. Mechanism of oocyte maturation**

underlying the maturation of oocytes.

At present, with the improvement in the IVF efficiency and culture systems, a natural cycle or mild stimulation may be more suitable for women receiving IVF treatments. A previous study showed that natural cycle or mild stimulation IVF is more effective than conventional stimulation protocols in patients with a low functional ovarian reserve [3]. In contrast to the standard stimulation protocol, the mild stimulation protocol is a safer and more rational regimen that helps reduce the hormone dosage, lower treatment risks, and retrieve a small number of high-quality oocytes. Despite these theoretical advantages, the mild stimulation protocol has yet to become a mainstream treatment modality in the United States. With the development of IVM technology, a modified protocol able to increase the success rates of natural cycle or mild stimulation IVF has been established. In this protocol, in addition to the retrieval of mature oocytes in naturally or mildly stimulated cycles, immature oocytes from small follicles are also retrieved for IVM, thereby increasing the total number of retrieved oocytes in a single treatment cycle and the clinical pregnancy rate. Data from previous clinical studies has shown that the combined use of natural cycle or mild stimulation IVF with IVM can expand the applicable scope of IVM technology to the treatment of various types of female infertility and has resulted in satisfactory clinical pregnancy rates and live birth rates [4, 5].

Cyclic adenosine monophosphate (cAMP) plays an important role in regulating the maturation of oocytes. The mural granulosa cells (MGC) located on the follicular wall contain natriuretic peptide precursor C (NPPC), while the cumulus cells around oocytes express natriuretic peptide receptor 2 (NPR2). Oocyte-derived paracrine factors can promote the activation of NPR2 in cumulus cells, while the NPPC in mural granulosa cells can bind to NPR2 receptors in cumulus cells to produce cyclic guanosine monophosphate (cGMP), which then enters into oocytes through gap junctions to inhibit the activity of phosphodiesterase (PDE3A), thereby maintaining a high level of cAMP in oocytes and the arrest of oocytes in the meiosis cycle. The activation of PDE3A by luteinizing hormone (LH) downregulates the level of cAMP in oocytes and induces the maturation of oocytes, thereby relieving the immature oocytes in the germinal vesicle (GV) stage or first meiotic metaphase (MII) from cell cycle arrest, so that they can complete the first meiosis and enter the second MII to develop into mature oocytes [6]. Zhang et al. [7] reported that estradiol can promote and maintain the expression of NPR2 in cumulus cells and participate in NPPC-mediated meiotic arrest of oocytes in vitro. These studies have opened up a new field of molecular mechanistic research on resuming the meiosis of oocytes, providing a theoretical basis for revealing the molecular mechanisms

Studies have found that small molecule ribonucleotides (microRNAs) are also important for oocyte maturation. A certain number of dynamic and stable microRNAs were found in both mature oocytes and early-stage embryos, presumably contributing to the maturation of oocytes. Kim et al. [8, 9] reported that microRNAs may affect oocyte maturation by altering the gene expression and function of cumulus cells through cumulus cell interaction and paracrine secretion. Let-7 is one of the most abundant microRNAs in the ovary. Upregulation of Let-7c can increase the rate of oocyte maturation, suggesting that Let-7c may be involved in the information exchange between oocytes and surrounding mural granulosa cells. In addition, maturationpromoting factor (MPF), cytostatic factor (CSF), oocyte maturation inhibitor (OMI), and mitogen-activated protein kinase (MAPK) are involved in oocyte maturation and division [7]. The mechanisms underlying oocyte maturation awaits further studies.

**24**

The biological definition of oocyte IVM is to remove immature oocytes in the GV stage from antral follicles and culture them in a suitable culture system, so that these immature oocytes can mature to MII stage in vitro. However, the clinical definition of IVM technology for immature human oocytes is completely different from its biological definition. The differences include the different sources of immature oocytes, the different protocols used to induce ovulation, and the different time of oocyte retrieval. These factors may lead to the situation where the immature oocytes retrieved clinically are not in the GV stage. The use of human chorionic gonadotropin (hCG) to induce ovulation prior to clinical retrieval of oocytes may lead to the initiation of endogenous oocyte maturation, and hence some of the retrieved immature oocytes may have undergone germinal vesicle breakdown (GVBD) or entered the MI stage. Although immature oocytes in the MI stage have initiated the process of in vivo maturation, they still need to participate in the procedure of in vitro culture and maturation. Therefore, the definition of clinical IVM treatment should include the in vitro culture of immature oocytes in the GV and MI stages.

A recent point of view proposed to give the clinical definition of IVM of immature oocytes based on the diameter of follicles when the oocytes are retrieved [10]. However, this definition is not completely scientific, since the meiotic state of oocytes cannot be completely determined according to the size of follicles during the stimulation cycle [11, 12]. In addition, for immature oocytes collected from different clinical sources, their maturation rate and the rates to potentially develop into embryos and achieve live birth are different. Therefore, for clinical definition and research of IVM, attention should be paid to the effect of different sources of immature oocytes on the efficiency of IVM.
