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Year : 2018  |  Volume : 2  |  Issue : 4  |  Page : 237-248

Strategies of infertility treatment with human immature oocytes

Center for Reproductive Medicine, The 10thPeople's Hospital of Tongji University, Shanghai 200072, China

Date of Submission28-Mar-2018
Date of Web Publication11-Jan-2019

Correspondence Address:
Ri-Cheng Chian
Center for Reproductive Medicine, The 10th People's Hospital of Tongji University, 301 Yanchang Road, Shanghai 200072
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.249885

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Human immature oocytes can be matured in vitro following culture. In vitro maturation (IVM) refers to maturation in culture of immature oocytes at different stages that may or may not have been exposed to short courses of gonadotropins. The source of immature oocytes is an important feature for the subsequent embryonic development and pregnancy, as well healthy live births. IVM is an efficient treatment that has already achieved significant outcomes in terms of acceptable pregnancies and implantation rates and resulted in the births of several thousands of healthy babies. As the development of IVM treatment continues, an attractive possibility for improving the already successful outcome is to combine a natural cycle in vitro fertilization (IVF) treatment with an immature oocyte retrieval followed by IVM of those immature oocytes. If the treatment processes can be simplified with immature oocyte retrieval, different types of infertile women may be able to take advantage of these treatments. Although IVM treatment is still considered experimental by the society, it is time to reconsider the IVM technological evolution. Mild stimulation IVF combined with IVM treatment represents a viable alternative to the standard treatment, and as data accumulate over time, it may prove to be an optimal first-line treatment approach.

Keywords: Immature Oocytes; In vitro Maturation; Infertility Treatment

How to cite this article:
Chian RC, Wang L, Yang ZY. Strategies of infertility treatment with human immature oocytes. Reprod Dev Med 2018;2:237-48

How to cite this URL:
Chian RC, Wang L, Yang ZY. Strategies of infertility treatment with human immature oocytes. Reprod Dev Med [serial online] 2018 [cited 2021 Aug 1];2:237-48. Available from: https://www.repdevmed.org/text.asp?2018/2/4/237/249885

  Introduction Top

Initial attempts in human in vitro fertilization (IVF) were used in vitro- matured (IVM) oocytes in the 1930s[1],[2],[3],[4] because it was impossible to obtain human IVM oocytes. The landmark work on IVM of human immature oocytes was carried out in the 1960s,[5],[6] and the human IVF techniques were also established with IVM oocytes.[7],[8],[9],[10] Therefore, one can assume that the current advanced assisted reproductive technologies (ARTs) for infertility treatment are based on the evolution of IVM.

In the 1970s, laparoscopy was introduced to collect human mature oocytes from preovulatory follicles,[11] resulting in the possibility to retrieve IVM oocytes for IVF.[12] Although the first human live birth resulting from IVF was produced by natural cycle IVF,[13] this procedure was gradually replaced by controlled ovarian hyperstimulation combined with IVF treatment because the number of oocytes retrieved determined the embryos available for transfer which, in turn, directly affected the chance of successful pregnancy.[14],[15],[16]

Initially, clomiphene citrate (CC) was used as a single ovarian stimulation agent.[17],[18],[19] Subsequently, in combination with human menopausal gonadotropin (hMG), it was utilized to generate multiple follicle developments and to increase the yield of more than one oocyte.[20],[21],[22] However, the current standard protocols use gonadotropins (recombinant or hMG) combined with luteinizing hormone (LH)-releasing hormone agonists to prevent the problem of premature ovulation with the aim of obtaining an average of 10–15 mature oocytes per retrieval from each woman. Although high-dose gonadotropin treatments may obtain more oocytes, this approach is associated with a number of adverse short- and long-term side effects, including a greater risk of ovarian hyperstimulation syndrome (OHSS).[23] Thus, natural cycle and mild stimulation IVF as well as IVM treatments have become appealing options for infertile couples.

Currently, given the efficiency of IVF and improvements in the culture system, natural cycle IVF or mild stimulation may be more suitable for women undergoing IVF treatment. Several studies have shown that natural cycle IVF treatment has advantages over standard stimulation IVF treatment, particularly in the management of women with low ovarian reserve.[24],[25] In contrast to stimulation IVF treatment, the aim of mild stimulation is to develop safer and patient-friendlier protocols where the risks of the treatment are minimized. Interestingly, despite theoretical advantages, mild IVF treatment has not yet become a mainstream approach in the United States. A recent large retrospective study found a significant decrease in live birth rates associated with increased follicle-stimulating hormone (FSH) dose, regardless of the number of oocytes retrieved,[26] and cautioned against high doses of FSH in IVF treatment cycles, albeit falling short of recommending mild IVF treatment. There is also evidence that mild stimulation or modified natural cycle protocols may have equal or even improved success rates compared with conventional IVF in women with a history of poor ovarian response.[27]

Recovery of immature oocytes followed by IVM of these oocytes is a potentially useful treatment for infertile women. This method seems particularly effective for women with polycystic ovaries (PCO) or PCO syndrome (PCOS)-related infertility, because there are numerous antral follicles within the ovaries of these patients.[28],[29],[30] To date, IVM treatment has been mainly applied to women with PCOS and is not considered applicable to all types of infertility with acceptable outcomes.[31] Clearly, it is impossible for IVM treatment to replace the current stream of IVF treatment procedures. In 2013, the Practice Committee of the American Society for Reproductive Medicine and the Society for ART indicated that IVM should only be performed as an experimental procedure evaluating both efficacy and safety in carefully selected patients.[32]

As we accumulate more experience and outcome data, natural cycle IVF, mild stimulation IVF, and IVM treatment may prove to be not just alternatives to standard stimulation treatments, but potentially first-line treatment choices.[33] As the development of IVM treatment, one very attractive possibility for enhancing the successful outcome is to combine natural cycle IVF treatment with immature egg retrieval followed by IVM of those immature oocytes.[34] It has been proven that the use of IVM technology can thus be broadened to treat women suffering from all types of infertility with acceptable pregnancy and live birth rates.[35],[36],[37],[38] Recently, we have published a review paper invited by fertility and sterility;[39] however, the number of words was limited to 3,000 in which was not what we wrote initially. Because we have written the review with approximately 8,000 words, we would like to publish what we wrote initially with the space of this journal. Maybe, some parts repeated with the previous review. However, this review would be more comprehensive and valuable for the readers. This review aims to share our views of the strategies of infertility treatment with immature oocytes.

  Definition of IVM Top

The oocyte is a unique cell in a woman's body, because of its special structure, function, and the fact that it undergoes meiosis. Meiotic progression in the oocyte is defined as the oocyte maturation from reinitiation of the first meiotic division to metaphase-II (M-II) stage accompanied by cytoplasmic maturation to successfully prepare the oocyte subsequent fertilization and early embryonic development.[40] In vivo meiotic resumption of oocyte is initiated in response to the preovulatory surge of LH. LH surge triggers oocyte maturation from germinal vesicle (GV) stage to M-II stage. For infertility treatment with IVF technology, the patients are given human chorionic gonadotropin (hCG) to induce the completion of oocyte meiosis in the follicles to retrieve mature M-II oocytes after 36 h of hCG injection. Without hCG injection in IVF treatments, most of the retrieved oocytes will remain at an immature GV stage.

Human immature GV-stage oocytes can be matured spontaneously to M-II stage in vitro when they are removed from the antral follicles and cultured in the proper culture media. This is the biological definition of oocyte IVM. With the development of IVM technique, IVM of human immature oocytes as a clinical procedure has been practiced a few decades after the first live birth of IVM oocytes.[41] However, the techniques used for IVM of human immature oocytes differ from protocols, and the clinical definition of IVM treatment also differs with biological definition of oocyte IVM. Such differences include the source of immature oocytes that may not be at the GV stage due to patient selection and different stimulation protocols. In some cases, the human immature oocytes were retrieved from the follicles that have been stimulated for a few days with gonadotropin to support moderate follicles growth and have been triggered with hCG before oocyte retrieval. Recently, there is a proposed clinical definition of IVM based on the size of follicles during the immature retrieval,[42] but this proposed definition may be cumbersome and complicate. Clearly, it has been criticized that the definition based on the size of follicles is not a scientific rationale, and such definition can lead to false results in interpreting the follow-up of children conceived with IVM technique.[43],[44]

It is interesting to mention here that the first reports of pregnancies from IVM oocytes were from stimulated cycles where the immature oocytes retrieved were followed by IVM and IVF,[45],[46] in which it was not clearly defined for IVM procedure of immature oocytes based on the size of follicles. It seems difficult to clearly define the definition of the clinical meiotic status of oocytes because there are different situations with patient selection and the sources. Therefore, the definition of clinical IVM should be defined with the origin of immature oocytes to clarify the outcomes derived from different sources of immature oocytes for follow-up. Nevertheless, we believe that the clinical IVM treatment should be defined as IVM of any immature oocytes, regardless of stage from GV and M-I to M-II stage for clinical application, since it involves the procedure of IVM for immature oocytes. It is important to point out for scientists, especially for basic scientists, that they should distinctly understand that the situation of clinical procedures is quite different with the laboratory materials. It is not possible to evaluate the clinical procedures as evaluation of laboratory procedures for IVM of oocytes. The accumulation of our combined knowledge is to better understand the purpose of performing clinical IVM of oocytes and use this knowledge for our health care.

  Source of Immature Oocytes for IVM Top

As mentioned above, today's human IVF technology was developed with in vitro-matured oocytes derived from surgical materials.[8] Thus far, there is no unique IVM technology applied in the world.[30] Essentially, clinical IVM technology may be divided into the differences based on the source of immature oocyte retrieval: (1) from C-section, (2) from patients with PCO and PCOS, and (3) from patients with normal ovaries [Figure 1]. For patients with PCO or PCOS and normal ovaries, IVM can be further subdivided into the categories of priming with FSH alone, FSH + hCG, and hCG alone. However, the details about the origin of oocytes and their associated outcomes have previously been discussed.[30]
Figure 1: Immature oocytes from different sources. (a) GV-stage oocyte from woman with C-section; (b) GV-stage oocyte from woman with PCOS after priming with FSH alone; (c) M-I stage oocyte from woman with regular ovaries after priming with FSH and hCG. Bar indicates 60 μm. Arrows indicate GV in the cytoplasm. GV: Germinal vesicle; M-I: Metaphase-I; FSH: Follicle-stimulating hormone; PCOS: Polycystic ovaries syndrome; hCG: Human chorionic gonadotropin.

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From women with cesarean section

In 1991, a successful live birth from IVM was reported using immature oocytes that had been collected during a cesarean delivery.[41] A pregnancy resulting from the fertilization of immature oocytes derived at the time of a cesarean delivery was confirmed by another report.[47] However, the oocyte maturation rate seemed relatively low when these immature oocytes were cultured in vitro.[48] It has been reported that immature oocytes collected from the small follicles at cesarean delivery followed by IVM and IVF can develop to blastocyst stage when the embryos are cocultured with human ampullary epithelial cells.[49] There is few research reported using the immature oocytes obtained from cesarean section.[50] Immature oocytes derived from cesarean delivery followed by IVM may be an available source for a donor oocyte program.[51] More research is required to clarify the efficiency and safety issues with the use of immature oocytes derived from cesarean delivery.

Apart from cesarean section, it was shown that immature oocytes could be retrieved from the ovaries obtained surgically regardless of follicular or luteal phase,[52] and that the number of immature oocytes collected was related mainly to the age of the women, pathology, day of the menstrual cycle, and cyclic versus noncyclic ovaries. Interestingly, it has been mentioned that immature oocytes from the luteal phase have a significantly higher maturation rate compared with those collected from the follicular phase. It seems that the number of immature oocytes collected from the ovary decreases as women's age increases, but there is no statistically significant difference in oocyte maturation and cleavage rates in the different age groups.[53]

It has been reported that immature oocyte retrieval followed by IVM and vitrification is the possibility of retrieving oocytes regardless of the phase of the menstrual cycle for fertility preservation,[54] indicating that it is possible to perform immature oocyte retrieval sequentially in either the follicular or luteal phases of the menstrual cycle in breast cancer patients without affecting the quantity or maturation rate of the oocytes. Thus, in cancer patients under severe time constraints, immature oocyte retrieval in the luteal phase followed by IVM and vitrification can be considered before chemotherapy to maximize the possibility of fertility preservation, as has been suggested.[55],[56] There were no statistically significant differences found in the number of retrieved oocytes, maturation rate, fertilization rate, or total number of cryopreserved oocytes and embryos when immature oocyte retrieval followed by IVM of oocytes for fertility preservation was performed during the follicular phase compared with the luteal phase of the cycle.[57]

From patients with polycystic ovarian and polycystic ovarian syndrome

Since the first report of a pregnancy in a woman with anovulatory PCOS after undergoing IVM and IVF,[58] several groups have made efforts to develop this treatment for infertile women with PCO or PCOS, because a large number of antral follicles in the ovaries are seen in infertile women with PCO or PCOS. This group of patients is sensitive to ovarian stimulation with gonadotropins and has an increased risk of OHSS compared with women who have normal ovaries. The technique involves modified IVM treatment, with priming with FSH or hCG before immature oocyte retrieval. It seems that the successful pregnancy rates for IVM treatment correlated with the number of immature oocytes retrieved.[59]

Ovarian stimulation with FSH before immature oocyte retrieval has been applied, indicating that FSH pretreatment promotes efficient recovery of immature oocytes and maturation in vitro.[60] Suikkari et al.[61] reported that although using low-dose FSH priming started from luteal phase improves the efficiency of immature oocyte recovery, maturation and fertilization rates, the average number of immature oocytes collected, the rates of oocyte maturation and fertilization are not different between women with regular menstrual cycles and women with irregular cycles of PCOS. Priming with r-FSH during follicular phase before harvesting of immature oocytes from the patients with PCOS improves the maturational potential of the oocytes and the implantation rate of the cleaved embryos, indicating that significantly higher pregnancy (29%, 7/24) and implantation (21%, 8/37) rates were obtained when priming with FSH before immature oocyte retrieval.[62] Recently, it has been reported that FSH priming is beneficial for promotion of the maturation and quality of oocytes, leading to a higher embryo cleavage rate and lower rate of pregnancy loss.[63] Theoretically, the use of FSH priming at the beginning of follicular or luteal phases may enhance more follicular development and the maturational competence of immature oocytes in vivo. It has been found that oocyte maturation rates were not different between the immature oocytes priming with and without hCG, but the time course of oocyte maturation was different.[40] It appears that the oocytes retrieved from follicles in respond to hCG that may promote the initiation of oocyte maturation in vivo, because it has been reported there were LH/hCG receptors in the granulosa and cumulus cells from the antral follicles as small as 3 mm in diameter, suggesting a benefit to most follicles when using hCG priming.[64] It has been demonstrated that the time course of oocyte maturation in vitro is hastened and the rate of oocyte maturation is increased by priming with 10,000 IU hCG 36 h before retrieval of immature oocytes from women with PCOS.[65],[66] Therefore, it is possible that pregnancy rate may potentially be improved by priming with hCG before immature oocyte retrieval.[67] This notion was confirmed by other reports.[68],[69],[70],[71]

It has been reported that women with PCOS, the time course, and maturation rate are different when the GV-stage oocytes divide into different groups based on the pattern of cumulus cells after hCG priming.[72] It seems that, with hCG priming, it not only does promotes some oocytes initiated maturation process but also enhances some GV-stage oocytes from the small follicles to acquire maturational and developmental competence in vivo. Nevertheless, a recent review indicated that there is no conclusive evidence that hCG priming had an effect on live birth, pregnancy, or miscarriage rates in IVM treatment and that there was low-quality evidence that suggested that hCG priming may reduce clinical pregnancy rates. However, these findings were limited by the small number of data included, as no data were available on adverse events (other than miscarriage) or on drug reactions.[73] Therefore, the authors could not adequately assess the safety of hCG priming, suggesting that further evidence from well-designed randomized controlled trials is needed before making definitive conclusions about the role of hCG priming, as well as the optimal dose and timing.

Interestingly, it has been reported that FSH priming with 75 IU/day for 6 days in combination with hCG priming 36 h before immature oocyte retrieval has no additional benefit on women with PCOS.[69] There were hardly seen leading or dominant follicles in the ovaries during IVM treatment cycles from women with PCOS, but it is possible to retrieve M-I oocytes from the small follicles after hCG priming. Therefore, it is important to mention that the situation and results will be quite different with normal ovaries and PCOS after hCG priming.

From patients with normal ovaries and regular menstrual cycling women

With the advantages of IVM treatment, it has been applied to some infertile women with normal ovaries and regular menstrual cycles who prefer IVM treatment. Gonadotropins (FSH or hCG or FSH) combined with hCG have been used to obtain a greater number of immature oocytes in this group of patients. In such cases, if there are leading or dominant follicles produced and hCG primed before egg retrieval, the mature oocyte collection should be the first choice, and the second consideration should be given to retrieve the immature oocytes from the small follicles. Priming with a fixed dose of FSH (150 IU/day) for 3 days from day 3 of the menstrual cycle without priming with hCG for IVM treatment did not increase the number of oocytes obtained per aspiration and does not improve oocyte maturation, cleavage rates, or embryonic development.[74] It seems that oocyte maturation, fertilization, cleavage, or implantation rates were not different between “coasting” for 2–3 days before immature oocyte retrieval when women with normal menstrual cycles were given 150 IU FSH/day for 3 days started from day 3.[75] Interestingly, it has been shown that using low-dose FSH priming starting from the luteal phase improves the efficiency of immature oocyte recovery and maturation as well as the rates of fertilization with IVM treatment.[62]

A study was designed to determine whether the efficiency of IVM in women with normal ovaries can be improved by gonadotropin administration,[76] indicating that a more favorable result can be obtained with a combination of FSH plus hCG priming; FSH priming or hCG priming alone had no significant effect on the clinical outcome. In women with normal ovaries and a regular menstrual cycle, the significant advantage of priming with hCG alone before egg retrieval is to collect the mature oocytes from leading or dominant follicles other than immature oocyte retrieval. In these women, consideration should be given to natural cycle IVF and administration of hCG when the leading or dominant follicles reach a certain size. In such cases, one or two mature oocytes together with several immature oocytes can be retrieved at the time of oocyte retrieval 36 h after hCG priming.[33] A protocol has been developed for infertile women with normal ovaries and regular menstrual cycles, namely natural cycle IVF combined with IVM,[35],[36] which will be described in detail later.

  Methods of Immature Oocyte Retrieval Top

Transvaginal ultrasound-guided follicular aspiration has now become the preferred procedure for immature oocyte retrieval in IVM treatment cycles. The same principles applied to IVF oocyte retrieval are also valid for IVM procedure. However, a smaller gauge needle (19G or 20G) is preferable. There are several designed aspiration needles for immature oocyte retrieval, one of them called “Steiner-Tan” needle. However, the use of the Steiner-Tan needle did not increase the number of oocytes retrieved.[77] It also did not increase the time required for retrieval, but flushing of antral follicles significantly decreased clot formation in fluid aspirates. The use of the Steiner-Tan needle also significantly decreased the number of vaginal needle punctures during each case. Therefore, the authors concluded that the primary benefit of the Steiner-Tan needle was on the embryological aspects of IVM and that decreased blood and blood clots in the aspirates made IVM retrieval comparable to conventional IVF for the embryologist. This causes less pain and less damage to the smaller follicles, thereby allowing greater numbers of immature oocytes to be collected. Although IVM ovum pick-up (OPU) requires more punctures per ovary, it is well tolerated and does not seem to be associated with a higher complication compared to IVF OPU.[78] It is important to note that no report indicates increased bleeding or complications during immature oocyte retrieval compared to regular mature oocyte aspiration. This may be explained by the use of thinner needles and lower aspiration pressure for immature oocyte retrieval. Anesthesia is recommended for immature oocyte collection due to the length of retrieval time. Types of anesthesia used for transvaginal follicle aspiration for IVM include conscious sedation and local injection.

Because the intrafollicular pressure is already higher in small follicles, the aspiration vacuum pressure is reduced to 85 mmHg, which is approximately half of the conventional IVF aspiration pressure. A higher aspiration pressure provokes an increase in the number of denuded oocytes. It has been reported that immature oocytes were aspirated with 20G needles with a vacuum of 180 or 300 mmHg, and the authors indicated that the percentages of cumulus cell-enclosed oocytes and transferable embryos per retrieved oocytes at 180 mmHg (69.7% and 23.8%, respectively) were significantly higher (P < 0.01) than those at 300 mmHg (46.2% and 12.8%, respectively).[79] The ongoing pregnancy rate per retrieval cycle at 180 mmHg (30%) was higher (P < 0.01) than that in 300 mmHg (4.3%). The data indicate that lower pressure of vacuum aspiration with a 20G needle improves the developmental competence of immature oocytes following IVM, IVF, and embryo transfer. However, the pressure of 180 mmHg is still too high for immature oocyte retrieval.

Good ultrasonographic visualization is a vital element of successful immature oocyte retrieval. Follicular sizes vary, and certain follicles may be difficult to aspirate or, even if they are aspirated, no oocytes may be recovered, especially from the very small-sized follicles (<4 mm in diameter). Aspirates are collected in 10 mL culture tubes containing approximately 2 mL of heparinized warming medium (usually containing 2 units/mL of heparin). It is possible to use 0.9% saline containing 2 units/mL of heparin. It has been described that there are two ways to search and collect immature oocytes from follicular aspirates in the literature: (1) dish search – the follicular aspirates are poured directly into a  Petri dish More Details and examined for immature oocytes under a stereomicroscope and (2) cell strainer – the follicular aspirates are filled through a cell strainer (70 μm nylon). After filtering, the collected aspirates can be rinsed with a prewarmed flushing medium and transferred to a petri dish to search for immature oocytes under a stereomicroscope.

How to inseminate the in vitro-matured oocytes, especially for the oocytes already denuded from cumulus cells? Is intracytoplasmic sperm injection (ICSI) the essential procedure for in vitro-matured oocytes? We have found that ICSI is not an essential procedure for in vitro-matured oocytes after denuding if the parameters of sperm are normal (Chian et al., 2000, unpublished data with 16th Annual Meeting of ESHRE, O-061 presentation). Interestingly, recent data showed that the normal fertilization rate was similar in both methods of the conventional IVF insemination and ICSI for IVM oocytes derived from GV and MI stages,[80] indicating that although fertilization methods resulted in a similar abnormal fertilization rate in GV-derived oocytes, the multipronuclear (multi-PN) formation rate was significantly lower with ICSI than with conventional IVF insemination. The authors concluded that both fertilization methods could be applied to GV-derived oocytes, but ICSI is favored in M-I-derived oocytes because ICSI could prevent multi-PN formation.

  IVM Culture Systems Top

Human oocytes acquire a series of competencies during follicular development (oocyte growth and maturation) that play critical roles in fertilization and subsequent early embryonic development. Even if high rates of IVM of immature oocytes are obtained, the developmental competence of the in vitro-matured oocyte is still be suboptimal as indicated by the relatively minimal development up to blastocyst stage and the poor viability to term after transfer. Oocyte maturation in vitro is profoundly affected by culture conditions.[81] Although numerous data have been accumulated from studies, the current rationale for choosing a specific medium for IVM of human immature oocytes appears to stem largely from adapting methods developed for culturing other cell types. Complex culture media, tissue culture medium-199, Ham's F10 medium, and Chang's medium buffered with bicarbonate and/or N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) supplemented with various sera, gonadotropins (FSH and LH), growth factors, and steroids, have been most widely used in research and the clinical application of oocyte IVM.[46],[58]

All existing media for oocyte maturation in vitro have a base of complex culture media supplemented with different substances. There are commercially available IVM media, indicating that there was no difference in maturation rate or fertilization rate of oocytes matured in vitro in both media.[82] There is also no difference in the formation of good-quality blastocysts in the two groups. Therefore, in terms of human infertility treatment with IVM technology, it seems that no breakthrough has yet been made by improving the IVM medium itself.

It has been proposed that the nuclear maturation and cytoplasmic maturation were not synchronized during IVM of immature oocytes,[83] and ways to synchronize the nuclear and cytoplasmic maturations have been explored.[84] In animal models of IVM of immature oocytes, some of these approaches resulted in high blastocyst formation and fetal production,[85] and there is no report indicating that they can be applied safely to human infertility treatment. While the use of cell cycle-modulating agents or spindle formation inhibitors during oocyte IVM may be acceptable within the animal industry, the safety issues associated with the use of these agents for the clinical application of in vitro human oocyte maturation should be carefully investigated before adoption of the methodology.

The physical requirements of IVM system and role of culture platforms or devices both play a role in influencing immature maturation and subsequent embryonic development. Recently, several new systems have been introduced for culturing animal gates and embryos as well other cells,[86],[87],[88] but the systems have not yet been used in the field of clinical ART. Application of microfluidic technologies has been proposed in human ART, indicating that such technologies may be influential in the field of human gametes and preimplantation embryo biology.[89] Interestingly, a microfluidic system supports murine ovarian follicles to mimic the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single-, dual-, and multiunit microfluidic platforms.[90] These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary,  Fallopian tube More Details, uterus, cervix, and liver, with a sustained circulating flow between all tissues. However, the practical application and the mechanical and biochemical characteristics of microfluidics still need to be studied properly before clinical implementation, especially for IVM of human immature oocytes.

In addition, an encapsulated three-dimensional (3-D) culture system was used for culturing mouse follicular growth.[91] The encapsulated 3-D culture system utilizes biomaterials to maintain cell-to-cell communication and supports follicle development to produce a mature oocyte that has been reported in nonhuman primate preantral follicles.[92] Although many reports indicated that the encapsulated 3-D culture system was efficient for follicle growth in vitro,[93],[94],[95],[96] it seems that there is still no report for human oocytes matured in vitro with encapsulated 3-D culture system. Interestingly, it has been reported that liquid marbles (LMs) consist of a drop of liquid encapsulated by hydrophobic powder particles, in which 3-D bioreactor could form to support the growth of different type of cells.[97],[98] However, a recent report showed that the similar percentages of oocyte maturation were observed in LM and regular culture medium droplets (control) and that there were no differences in embryonic development (blastocyst formation rates) after IVF following IVM of sheep immature oocytes, indicating that LM microbioreactor may be a viable technique that provides a suitable microenvironment to induce oocyte IVM.[99] Nevertheless, the efficiency and the clinical outcomes of IVM with human immature oocytes by the encapsulated 3-D culture system must be further confirmed before clinical application.

  Clinical Outcomes and Safety of IVM Treatment Top

As mentioned above, immature oocytes priming with FSH or hCG before immature oocyte retrieval improve oocyte maturation rate and embryo quality as well as pregnancy rates when retrieved from infertile women with PCOS, normal ovaries, and regular menstrual cycling women. The source of follicles may be important for the subsequent embryonic development, but the developmental competence of immature oocytes derived from the small antral follicles seems to not be adversely affected by the presence of a dominant follicle. Hence, a novel strategy emerged, known as natural cycle IVF combined with IVM, since IVM oocytes and immature oocytes are retrieved at the same time and in the same cycle. So far, it has been estimated that there are more than a few thousand healthy infants born following immature oocyte retrieval and IVM from women with PCOS.[30],[100] In general, the clinical pregnancy and implantation rates per embryo transfer have reached approximately 35%–40% and 15%–20%, respectively, in infertile women with PCOS following IVM of immature oocytes based on different reports.[30] If those reports included the aforementioned natural cycle IVF combined with IVM, the positive clinical outcome rates would be higher.

Since the introduction of IVF and other ARTs for infertility treatment, the health of infants born from these techniques, including IVM technology, has been of major concern, especially for the extra period of in vitro culture of immature oocytes. In fact, IVM of the immature oocytes from GV via M-I to M-II stage normally requires 36–48 h of culture in vitro.[6],[40] For clinical IVM of the immature oocytes from M-I to M-II stage, only 6–24 h of culture in vitro is necessary.[101] Interestingly, it has been reported that there was no significance in morphokinetics of embryos developed from oocytes matured in vivo and in vitro in IVM cycles, indicating that the embryonic developmental potential may not different between the oocyte matured in vivo and in vitro.[102] There were insignificant differences for zona pellucida birefringence and meiotic spindle between the in vivo- and in vitro-matured oocytes.[103] Furthermore, it has been indicated that the in vivo- and in vitro-matured oocytes showed normal ooplasm with uniform distribution of organelles.[104] Mitochondrial morphology appeared similar between the maturation conditions. Cortical granules were found typically stratified in a single, mostly continuous row just beneath the ooplasm in the in vivo- and in vitro-matured oocytes.

One study showed that optimized human IVM procedures have no significant effects on the establishment of maternal DNA methylation patterns at LIT1, SNRPN, PEG3, and GTL2.[105] There were no differences in terms of oxygen consumption between embryos derived from in vitro and IVM oocytes, indicating that there was also no imprinting gene disorder found in IVM babies.[106] No statistically significant impact was found from IVM on chorionic villi and cord-blood DNA methylation at developmentally important genes and interspersed repeats, suggesting that IVM-induced epigenetic changes in offspring, if any, will be relatively minor in magnitude and/or infrequent.[107] For human IVM, no definitive conclusions on imprinting establishment can be drawn as well-designed studies are currently not available. Therefore, epigenetic analysis should be performed in children born from pregnancies after IVM to draw definitive conclusions on the epigenetic safety of human IVM.[108]

The number of live births from IVM oocytes has been increasing over the past three decades. It has been estimated that more than 5,000 IVM infants have been born worldwide.[30] There are many concerns regarding obstetric and perinatal outcomes of IVM infants, as well as their long-term development.[109] Several studies have reported that the mean birth weight and the incidence of congenital anomalies seem to be comparable with spontaneous conceptions or conceptions of infertile women undergoing IVF treatment.[110],[111],[112] In 196 infants conceived after IVM of immature oocytes, an increased risk of adverse obstetric or perinatal outcomes was not shown compared with children conceived by IVM oocytes or children conceived by conventional ovarian-stimulated ICSI cycles.[113]

Recently, it has been reported that 1421 IVM babies were born from 1187 pregnancies in 31 IVF clinics located in 22 countries, and the data were collected at the time of birth, including stillbirths but not pregnancy terminations.[31] Of the 1421 IVM infants born, there were 18 major congenital abnormalities [Table 1]. However, this proportion is comparable with the prevalence of major birth defects per birth with spontaneous conception according to the International Clearinghouse for Birth Defects Surveillance and Research (2011).[114] Accordingly, IVM may not actually be associated with a significantly increased risk of poor obstetric outcomes or congenital abnormalities compared to the risk level deemed acceptable for IVF or other ARTs.
Table 1: Obstetric outcomes and congenital abnormalities in 1,421 IVM babies born from 1,187 pregnancies

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  Development of IVM Treatment Top

As mentioned above, immature oocyte retrieval followed by IVM was initially shown to be a successful treatment for infertile women with unovulatory PCOS, because there are numerous antral follicles within the ovaries in this group of patients, which makes them prone to develop ovarian OHSS.[66] In women, although only a single follicle usually grows to the preovulatory stage and releases its oocytes for potential fertilization, several small follicles also develop during the same follicular phase of the menstrual cycle. It seems that approximately 20 antral follicles are selected and carried through to the preovulatory stages of development during each menstrual cycle.[115] Two or three waves of ovarian follicular development in women during menstrual cycle have been reported based on daily transvaginal ultrasonography, challenging the traditional theory of a single cohort of antral follicles that grows only during the follicular phase of the menstrual cycle.[116],[117] The quality and early embryonic developmental competence of immature oocyte following IVM are not detrimentally impacted by the presence of the dominant follicle in the ovaries.[118],[119] Based on those findings, the clinical application of IVM technology has continued to evolve.

Natural cycle in vitro fertilization combined with IVM

It has been demonstrated in women that atresia does not occur in the nondominant follicles even after the dominant follicle is selected in the ovary during follicular phase, because immature oocytes retrieved from nondominant follicles have been successfully matured in vitro, fertilized, and have resulted in several pregnancies and healthy live births.[34] Therefore, one very attractive possibility for increase of success rate of natural cycle of IVF treatment is the combination of immature oocyte retrieval and IVM. If the mature oocyte from the dominant follicle and the immature oocytes from the small follicles were all collected, the chances of a pregnancy may be greatly increased when we manage to mature these immature oocytes and produce several viable embryos.

It seems very important to prevent ovulation from the dominant follicle due to a natural LH surge that occurs when the patients are treated with natural cycle IVF combined with IVM. Our experiences indicate that 10,000 IU hCG can be administered 36 h before oocyte retrieval when the size of the dominant follicle reached 10–14 mm in diameter. Most oocytes collected from the dominant follicles are at the mature M-II stage. A pilot study showed that the clinical pregnancy rate may reach approximately 45%–50% per embryo transfer following natural cycle IVM combined with IVM in a selected group of patients.[35] It has been shown that more than a half of all infertile women who seek out IVF treatment can be treated with natural cycle IVF/M or IVM alone when these treatments have been chosen initially and that natural cycle IVF/M is an efficient treatment, especially for women under the age of 35 years.[36]

The strategy that has been successfully explored using natural IVF is the one combined with IVM for women without PCOS, indicating that a selected group of ovulatory women may benefit from natural cycle IVF/M with acceptable pregnancy rates.[35] More recently, we reported that natural cycle IVF/M treatment might be offered to more than 50% of the total infertile women who were seeking infertility treatment and with more than 40% pregnancy rates.[36],[37] Interestingly, it has been reported that although the clinical pregnancy rates are not different in terms of mature oocytes being retrieved or the time of egg retrieval with natural cycle IVF combined with IVM treatment, the live birth rate is higher when the transferred embryos were produced from the IVM oocytes.[38] Therefore, natural cycle IVF combined with IVM may be the most suitable treatment for younger women with regular menstrual cycles.

Although treatment with natural cycle IVF combined with IVM together with IVM-alone treatments can achieve acceptable pregnancy and implantation rates in more than 50% of infertile women, immature oocyte retrieval from the small follicles encounters technical difficulty most of the time after the retrieval of mature oocytes from the dominant and leading follicles. Therefore, we have proposed the modified treatment based on natural cycle IVF combined with IVM, namely mild stimulation IVF combined with IVM treatment.

Mild stimulation in vitro fertilization combined with IVM

Today, given the efficiency of IVF and improvements in the culture system, natural cycle IVF or mild stimulation may be more suitable for women undergoing IVF treatment. In contrast to conventional IVF treatment, the aim of mild stimulation is to develop safer and more patient-friendly protocols where the risks of the treatment are minimized. Mild stimulation is defined as administration of low-dose exogenous gonadotropins, a shorter duration in gonadotropin-releasing hormone antagonist cotreated cycles, or when oral compounds, such as CC are used for ovarian stimulation, with the aim of retrieving fewer than 8 oocytes.[120],[121] Mild stimulation using CC in combination with low doses of gonadotropins can also be considered a realistic option for patients undergoing IVF with a good prognosis.[122]

A protocol for the mild stimulation combined with IVM treatment has been developed [Figure 2]. If the patient has more than several antral follicles seen on day-2 baseline ultrasound scan, the patient will be given 50–100 mg CC daily for 5 days. The second ultrasound will be performed on day 8 to measure the size of follicular growth. At this stage, based on the follicular size, hMG will be administered at 70–150 IU daily for 3 days. Once a leading follicle reaches 18 mm and another reaches 16 mm in diameter, 5,000 IU of hCG will be given. At 36 h after hCG injection, oocyte retrieval will be performed using a 19 G single aspiration needle. The leading follicles will be aspirated first, after which the subleading follicles will be aspirated to determine whether they are mature oocytes under a dissecting microscope. The mature oocytes can be inseminated with IVF, if the parameters of sperm quality are normal; otherwise, ICSI can be applied. To identify the immature oocytes, both the size of follicles derived from and the morphology of expansion of cumulus–oocyte complexes (COCs) must be analyzed. COCs with few inner layers of compacting cumulus cells with a good pattern of outer layer cumulus expansion appeared as they can be denuded 4–6 h after culture in fertilization medium or IVM medium to determine oocyte maturity. When the oocytes have matured, they can be inseminated by IVF or ICSI, and the remaining immature oocytes can be cultured overnight in IVM medium. Those immature oocytes will be checked again 24 h after IVM for possible insemination. If they become mature, they can be inseminated by ICSI using the sperm sample prepared on the previous day, which needs to be maintained at room temperature with tightly capped tube. Our preliminary data revealed the effectiveness of this mild stimulation IVF combined with IVM treatment approach.
Figure 2: Protocol for mild stimulation cycle IVF combined with IVM treatment. IVF: In vitro fertilization.

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  Conclusion Top

The source of immature oocytes is an important feature for the subsequent embryonic development and pregnancy, but the developmental competence of oocytes derived from the small antral follicles seems not to be adversely affected by the presence of a dominant follicle. Priming with FSH or hCG before immature oocyte retrieval improves oocyte maturation and pregnancy rates. The application of IVM technology can thereby be broadened to treat women suffering from all causes of infertility with acceptable pregnancy and live birth rates. Although IVM has led to the birth of several thousands of healthy babies, IVM technology is still considered experimental by society. It may be time to reconsider the effectiveness and practicality of IVM.

As treatment with IVM continues to evolve, the combination of natural cycle IVF with immature oocyte retrieval followed by IVM of those immature oocytes is now a promising strategy for increasing the rate of successful outcomes. If the treatment processes can be simplified, especially for immature oocyte retrieval, a greater number of infertile women may be able to take advantage of these treatments. Mild stimulation IVF combined with IVM treatment represents a viable alternative to standard treatment, and as we accumulate more experience and outcome data over time, it may prove to be an optimal, first-line treatment choice.

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Conflicts of interest

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  [Figure 1], [Figure 2]

  [Table 1]

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