State of the art in in-vitro oocyte maturation

PURPOSE OF REVIEW: The recovery of immature oocytes followed by in-vitro maturation (IVM) and in-vitro fertilization is an attractive alternative to conventional in-vitro fertilization treatment in which controlled ovarian stimulation with gonadotropins is used to increase the number of available oocytes and embryos. Significant progress has been made to improve pregnancy and implantation rates from in-vitro matured oocytes. This review summarizes current knowledge and achievements in human oocyte in-vitro maturation for clinical application, and will highlight recent advances reported in in-vitro maturation treatment. RECENT FINDINGS: It has been demonstrated that priming of ovarian immature oocytes with follicle-stimulating hormone or human chorionic gonadotropin prior to immature oocyte retrieval improves oocyte maturation rates and embryo quality as well as pregnancy rates in infertile women with polycystic ovaries or polycystic ovary syndrome. The size of follicles may be important for the subsequent embryonic development, but the developmental competence of oocytes derived from the small antral follicles is not adversely affected by the presence of a dominant follicle. However oocyte maturation in vitro is profoundly affected by culture conditions. Currently more than 300 healthy infants have been born following immature oocyte retrieval and in-vitro maturation. In general, the clinical pregnancy and implantation rates have reached 30-35% and 10-15% respectively in infertile women with polycystic ovaries or polycystic ovary syndrome. SUMMARY: In-vitro maturation treatment can now be offered as a successful option to infertile women with polycystic ovaries or polycystic ovary syndrome. It is possible to combine natural cycle in-vitro fertilization with immature oocyte retrieval followed by in-vitro maturation, and thus offer women with various causes of infertility reasonable pregnancy and implantation rates without recourse to ovarian stimulation. Further research remains to be done to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of oocytes matured in vitro.     

Endocrinology of in vitro maturation of oocytes

In-vitro maturation (IVM) of oocytes is an alternative to conventional in-vitro fertilization (IVF) treatment. Significant progress has been made to improve pregnancy and implantation rates from in-vitro matured oocytes. However oocyte maturation in vitro is profoundly affected by culture conditions. Most IVM protocols supplement gonadotropins--folicle stimulating hormone (FSH) and luteinizing hormone (LH) into a culture medium for oocyte maturation. The addition of these hormones, as well as some growth factors is based on their physiological role in oocyte maturation in vivo. However, it is possible, they not to play the same role in promoting oocyte maturation in vitro. The aim of this survey is to examine the hormones and growth factors, which participate in oocyte maturation in vivo and to summarize briefly the available information about the effect they have on the in vitro maturation. The researches on effects of these endocrine factors on oocyte maturation and subsequent fertilization, as well as on early embryonal development proceed.     

超排卵周期未成熟卵体外培养的研究

目的:研究来源于超排卵周期中的未成熟卵在拆除卵丘细胞后进行体外成熟培养(IVM)的成熟、受精及胚胎发育能力,探讨IVM技术的临床应用。方法:选取46名体外受精/卵胞浆内单精子显微注射-胚胎移植(IVF/ICSI-ET)患者为研究对象,比较MI和GV期不成熟卵的体外成熟情况,并比较体内成熟卵和体外成熟卵进行ICSI后的正常受精、异常受精、卵裂和优质胚胎形成情况。结果:体外培养中69.8%的MI期卵和77.2%的GV期卵均在24小时内达到成熟,其24小时和48小时的成熟率、总成熟率均无明显差异(P>0.05)。体外成熟卵与体内成熟卵相比较,正常受精率、异常受精率和卵裂率均无明显差异(P>0.05),优质胚胎形成率较低,差异有显著性(P<0.05)。结论:常规超排卵周期中的未成熟卵在拆除卵丘细胞后能够继续体外发育成熟,具有与体内成熟卵相似的ICSI受精、卵裂能力。虽然优质胚胎的形成率低于体内成熟卵,但增加了可移植胚胎和冷冻胚胎数量,提高了助孕成功率。     

Pre-implantation developmental potential from in vivo and in vitro matured mouse oocytes: a cytoskeletal perspective on oocyte quality

PurposeIn the present study, fertilization and developmental potential of mouse oocytes matured in different conditions were tested. The efficiency of in vitro fertilization (IVF), pre-implantation development and some important aspects of cytokinesis during early cleavages are discussed.MethodsIn vivo matured (IVO), in vitro matured (IVM) and roscovitine-treated (IVM-Rosco) mouse oocytes were subjected to IVF under identical conditions. Three replicates per group were analyzed. Fertilization was identified by the presence of two pronuclei at 6–8 h post-fertilization. Evaluation of pre-implantation embryonic development was done daily from day 2 to day 5 and embryos were processed for analyses of chromatin, nuclear lamina, microtubules and centrosomal proteins by conventional and confocal fluorescence microscopy.ResultsBoth IVM groups displayed lower fertilization rates when compared to in vivo controls. While IVO-derived embryos exhibit efficient and synchronous progression to the blastocyst stage, both IVM-derived embryos exhibit a delay in embryonic progression, and a lower blastocyst rate. Interestingly, IVM-Rosco M-II oocytes exhibited more blastomere symmetries and higher number of cells at the blastocyst stage than the IVM group with the most notable influence being on the centrosome-microtubule complex of blastomeres.ConclusionOur study strongly indicates that when compared to spontaneously in vitro matured oocytes, treatment with roscovitine may partially enhance developmental competence by maintaining coordination between nuclear and cytoplasmic events. Further evidence is given of cytoskeletal biomarkers that can be identified during in vitro oocyte maturation conditions.     

人类未成熟卵母细胞体外培养成熟的研究进展

本文综述了人类未成熟卵母细胞体外培养成熟的现状和未来的发展方向。未成熟卵母细胞体外培养成熟包括二种方法 :卵泡体外培养 ( IVC)和卵母细胞体外成熟 ( IVM)。从原始卵泡发育到成熟卵母细胞 ,受精形成胚胎 ,胚胎移植后妊娠 ,仅在小鼠成功 ,有关人类此方面的研究正在进行中。人类卵母细胞体外成熟已成功应用于治疗不孕症 ,但成功率低。卵母细胞体外成熟受自身大小以及所处月经周期的影响。培养系统添加卵泡液、表皮生长因子、激活素 /抑制素、以及共培养系统有助于卵母细胞体外成熟。根据卵母细胞所处发育阶段选择不同的培养系统 ,探索卵母细胞的生长和成熟是未来的重要研究方向     

骨髓间充质干细胞条件培养液促进小鼠卵母细胞体外成熟的研究

目的:研究骨髓间充质干细胞(mesenchymal stem cell,MSC)条件培养液对小鼠未成熟卵母细胞体外成熟、受精及胚胎发育的影响。方法:分离、培养小鼠MSC,获取MSC条件培养液(conditioned medium of MSC,CM)。收集小鼠生殖泡(germinal vesicle,GV)期卵母细胞,根据有无完整颗粒细胞层包裹颗粒细胞-卵母细胞复合物(cumulus-oocyte complex,COC)分为2类,分别在4种培养基(CM、DMEM、α-MEM、HTF)中培养不同时间,观察卵母细胞核成熟情况,荧光标记后激光共聚焦检测皮质颗粒(cortical granules,CG)分布和含量变化;体外受精后观察受精率和囊胚形成率。结果:2类COC卵母细胞的成熟率CM组明显高于DMEM组、α-MEM组、HTF组(P<0.01);体外受精率和囊胚形成率CM组明显高于DMEM组、HTF组(P<0.01),与α-MEM组和体内成熟组间差异无统计学意义(P>0.05);CM组卵母细胞内CG分布和含量变化与核成熟同步。结论:CM能使小鼠卵母细胞核、质同步成熟,提高了成熟率和卵母细胞质量。     

Follicular factors regulating oocyte maturation and quality

Maturation of the oocyte can be divided into two different aspects: nuclear maturation and cytoplasmic maturation. The spontaneous nature of nuclear maturation in oocytes removed from the follicle and cultured in vitro was observed in mammals as early as 1935. However, oocytes cultured in basic conditions are deficient in some cytoplasmic factors and are, therefore, developmentally incompetent. Data from large domestic species indicate that although oocytes matured in vitro in supplemented media can develop after fertilization, they require the presence of follicular factors during culture to ensure their developmental competence. The importance of follicular maturation on the capacity of oocytes to achieve fertilization and early embryonic development can be studied by reproducing some important events in vitro. Follicular supplementation may be in the form of follicular fluid, granulosa cells or follicle-conditioned media, and there is evidence that the maturational status of the follicle used for co-culture influences subsequent male pronuclear formation. Studies in this laboratory on the prolific Chinese Meishan pig, which has significantly higher early embryonic survival than conventional European breeds, indicate crucial differences in the pattern of follicle development and, therefore, the intrafollicular environment in which the oocytes are nurtured. It is suggested that this produces oocytes of improved 'quality' and this hypothesis is supported by experiments both in vivo and in vitro. Ultimately, it is hoped that these studies on large domestic animals will lead to identification of the follicular factors that influence oocyte quality.     

人未成熟卵母细胞培养体系研究进展

未成熟卵母细胞体外培养成熟(in vitro maturation,IVM)是指将GV期或MI期的卵母细胞在体外培养发育到第二次减数分裂中期(MII期),能够正常发育、受精和着床。人未成熟卵母细胞体外培养成熟(in vitro maturation,IVM)技术已有几十年历史,作为治疗不孕症的新手段,为赠卵以及女性生育力保存提供了新途径。近年随着细胞分子生物科学的迅猛发展,IVM取得了很大进展,向IVM培养系统添加促性腺激素、甾体激素、抗氧化剂、减数分裂抑制剂、生长因子、抑制素/激活素等有助于卵母细胞体外成熟。但仍面临卵母细胞成熟率不高、成熟后体外受精率低、妊娠率低的问题,完善IVM培养系统及相关辅助治疗是IVM的关键,如何获得稳定、有较高成功率的培养体系是亟需解决的问题。现对近几年一些较新的研究发现进行总结,主要从基础培养液、添加成分、培养条件的选择等方面予以综述。     

Direct effect of gonadotropin-releasing hormone agonists on the rabbit ovarian follicle.

OBJECTIVE: To determine if gonadotropin-releasing hormone agonist (GnRH-a)-induced oocyte maturation and degeneration can be attributed to the direct actions on the follicle. DESIGN: Mature rabbit follicle culture. INTERVENTIONS: The mature follicles were cultured with human chorionic gonadotropin (hCG) (100 ng/mL), buserelin acetate (10(-9) to 10(-6) M), leuprolide acetate (10(-9) to 10(-6) M), or buserelin acetate (10(-7) M) with a GnRH antagonist (10(-8) to 10(-6) M) for 14 hours. MAIN OUTCOME MEASURES: The percentage of oocytes achieving germinal vesicle breakdown, the oocyte degeneration rate, prostaglandins (PG) production by mature follicles, and the frequency of fertilization and embryonic development. RESULTS: Gonadotropin-releasing hormone agonist induced the meiotic maturation of follicle-enclosed oocytes in a dose-dependent manner while concomitantly increasing oocyte degeneration. The simultaneous addition of GnRH antagonist inhibited significantly GnRH-a-induced oocyte maturation and PG production by the mature follicles. Furthermore, a GnRH antagonist reversed the oocyte degeneration rate that had been increased by GnRH-a. The rates of normal fertilization and early embryonic development were significantly reduced in the oocytes matured by GnRH-a as compared with those matured by hCG. CONCLUSIONS: Gonadotropin-releasing hormone agonist acts directly on mature rabbit follicles to trigger the oocytes to undergo meiotic maturation, but oocytes matured in vitro by GnRH-a are not necessarily cytoplasmically mature.     

多囊卵巢综合征患者中未成熟卵母细胞体外培养成熟后体外受精的初步研究

目的:探讨未成熟卵母细胞体外培养成熟后体外受精、胚胎培养技术在多囊卵巢综合征患者中的初步应用及其影响因素。方法:用小剂量促性腺激素促使卵泡生长后,根据优势卵泡直径分为2组,直径6～8mm者为组1,10—12mm者为组2。采集未成熟卵母细胞,经体外培养成熟后,再进行体外授精和胚胎培养。结果:组1的GV期卵的成熟率和受精率低于组2者,但两组的MI期卵的成熟率、受精率没有明显差别。两组卵裂率没有明显差别,但形成胚胎的质量组2优于组1。总计成熟率69.3％,成熟卵中正常受精率61.5％。结论:可以用小荆量Gn促使卵泡生长后,采集未成熟卵,用体外成熟-体外授精(IVM/IVF)的方法使多囊卵巢患者在避免OHSS的情况下获得质量良好的胚胎。     

Timed IVM followed by ICSI in a patient with immature ovarian oocytes

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF-intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)-ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.     

Effect of single-oocyte culture system on in vitro maturation and developmental competence in mice

Purpose

To investigate whether single-culture systems influence the quality of in vitro-matured oocytes, we examined the maturation and developmental competence of oocytes obtained by grouped in vitro maturation (IVM) or single IVM.

Methods

In vitro-matured oocytes were obtained using the culture drop (CD) method for the grouped IVM experiments, and the CD and hanging drop (HD) method for the single IVM experiments. To evaluate oocyte developmental competence, we performed in vitro fertilization and culture, and counted the number of blastocysts. To evaluate the oocyte cytoplasmic maturation, we measured the maturation promoting factor (MPF) expression levels.

Results

Oocytes cultured singly had lower maturity and developmental competence than the grouped IVM oocytes. However, enhanced oocyte fertility and blastocyst quality was achieved by the HD single IVM method. Additionally, the MPF activity level increased in all culture methods, compared to the control; however, it lagged behind nuclear maturation.

Conclusions

These results suggest that the HD method is efficient for single IVM.     

Oocyte maturation and ovum quality in pigs

Oocyte quality in pigs is defined as the potential of that oocyte to develop into a viable offspring. There is increasing evidence that the programming of the oocyte must be completed before leaving the ovarian follicle, including both nuclear and cytoplasmic maturation. Pig oocytes matured in vitro under basic conditions are deficient in some, as yet unidentified, cytoplasmic factors and thus developmentally incompetent. This developmental incompetence can be overcome to some extent by follicular supplementation (with follicular fluid or granulosa cells) of the culture medium, emphasizing the importance of somatic signals during oocyte maturation. Furthermore, evidence is accumulating that the status of the follicle has a critical impact on the competence of the oocyte in vitro and in vivo and this has been demonstrated by co-culture with follicles at different maturational stages or from breeds with enhanced embryo survival. It now also appears that manipulation of maternal nutrition before mating or oocyte collection can enhance embryo survival in vivo and oocyte developmental competence in vitro, presumably by altering follicular secretions and hence the environment in which the oocyte is nurtured. Identification of both the key follicular factors influencing pig oocyte quality and reliable markers of oocyte quality will undoubtedly yield major improvements in embryo survival in vivo and embryo production in vitro.     

The rate of in vitro maturation of primary follicles from adult mice and the quality of oocytes is improved in the absence of anti-mullerian hormone

Anti-Müllerian hormone (AMH) inhibits the recruitment of primordial follicles into the growing pool, but its role in primary and secondary follicles is not clear. We isolated primary follicles from the ovaries of 9- to 10-week old mice and examined whether AMH affected follicular development. Follicles were matured in media that was prepared using unsexed fetal bovine serum (FBS) or female FBS (FFBS) with or without added AMH for approximately 2 weeks and maturation rates to secondary follicles and metaphase II (MII) oocytes were measured by standard morphological criteria. Rates of parthenogenetic activation and in vitro fertilization (IVF) were assessed by cleavage and blastocyst development, respectively. Whereas addition of AMH blocked primary to secondary follicle transition, the primary to secondary and secondary to MII follicle maturation rates was significantly improved with FFBS. Folliculogenesis resumed once AMH was removed from the media of the arrested primary follicles. The rates of IVF and parthenogenesis of oocytes after in vitro maturation (IVM) without AMH were also improved compared to controls. The results indicate that removal of AMH from culture conditions during IVM from primary follicular stages should be considered to improve outcome.     

In-vitro-Maturation

The ability to rescue oocytes and mature them in vitro would provide invaluable information about folliculogenesis and oocyte maturation and could provide oocytes for infertile women. In vitro growth (IVG) of follicles and in vitro maturation (IVM) of oocytes are challenging especially in the human because folliculogenesis is a lengthy process with many complex cellular changes in the oocyte and its surrounding follicle cells. Reports have been published on live births in mice after maturation and fertilization. This technique is still in its infancy especially for use in humans. A few live births have resulted from IVM of immature human oocytes aspirated from small antral follicles. Furthermore, it is possible to grow primordial follicles to preantral stages in slices of ovarian tissue and support antrum formation in isolated preantral follicles. Today we are still a long way from growing and maturing preantral follicles to preovulatory stages in vitro, but these techniques may revolutionize assisted reproduction in the future.     

溶血磷脂酸在卵母细胞体外成熟过程中作用的研究进展

卵母细胞体外成熟(IVM)已成为一项热门的辅助生殖技术(ART),有广泛的应用和发展前景。但其体外成熟率和受精后的发育能力还有待提高,培养条件需要进一步优化。溶血磷脂酸(LPA)是磷脂激素家族的重要成员,有生长因子和激素样活性,可产生广泛的生物学效应。研究表明LPA存在于卵泡液中,对卵泡发育、卵母细胞IVM、受精、胚胎发育、种植等产生重要作用。本文就LPA特性及其在卵母细胞体外成熟过程中的作用做一简要的阐述。     

Oocyte maturation

The oocyte is dependent on granulosa cells to provide nutrients and regulatory signals. Granulosa cells must be at the appropriate stage of differentiation to initiate these signals and transmit them to the oocyte. Studies have shown that in vitro-matured oocytes from follicles in early stages of atresia are more competent to support embryonic development than those from actively growing follicles. The acquisition of developmental competence appears to occur prior to in vitro maturation and can be induced by gonadotropin-free coasting in vivo or postmortem ovary incubation in vitro. The acquisition of developmental competence is probably a common signaling or differentiation pathway that occurs in the oocyte and/or associated granulosa regardless of whether the oocyte is destined to ovulate or degenerate. Early follicle atresia is the visually discernible characteristic in vitro that is associated with increased developmental potential.     

In-vitro maturation of eggs: is it really useful?

In in-vitro maturation (IVM), immature oocytes are collected from small antral follicles and allowed to mature in the laboratory before routine in-vitro fertilization or micro-injection. The authors' experience in IVM is based on the treatment of two main groups of patients: women with polycystic ovaries and women with normal ovaries. Patients with polycystic ovarian syndrome have irregular, mostly anovulatory cycles and are at increased risk for ovarian hyperstimulation syndrome because of their higher sensitivity to gonadotropins. Women with normal ovarian function may wish to avoid the side-effects of hormone injections, and therefore IVM has also been offered to couples with tubal, male factor and unexplained infertility. In all these groups of patients, immature oocytes have successfully been matured, fertilized and embryos transferred. Pregnancy rates have been reported to be between 4% and 54%. More than 300 children have been born and follow-up studies have reported no major concerns about the pregnancies, deliveries or health of the babies. There are still many questions concerning IVM. As the factors regulating follicle selection are poorly understood, no specific markers for the optimal time of immature oocyte collection have been defined. Furthermore, basic knowledge on the complex intracellular processes involved in the cytoplasmic maturation of human oocyte is lacking, making the design of optimal culture conditions for maturation difficult. The possible long-term effects of IVM on the health and development of children needs future study.     

Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles

The present experiments compared the maturational and developmental competence of immature oocytes derived from stimulated cycles, following culture in a newly designed in-vitro maturation medium (IVM-medium) or in standard tissue culture medium (TCM-199; control). The results indicated that maturation and fertilization rates were comparable when the cumulus-free M-I stage oocytes were matured in the IVM-medium (78.6%) or the control medium (70.8%). However, there was a significant difference in blastocyst development (P < 0.05) when M-I oocytes were matured in these two media (19.6 versus 7.7%). Both maturation and early embryonic development rates of GV-stage oocytes were significantly higher (P < 0.01) in the IVM-medium (maturation: 75.7%; blastocyst: 12.9%) compared with control (maturation 55.7%; blastocyst: 0.0%). Moreover, embryos developed to the blastocyst stage at a higher rate in both media if GV-stage oocytes had matured within 24 h compared with 48 h of culture. These results demonstrate that immature human oocytes derived from stimulated ovaries can achieve maturation and early embryonic development in vitro, especially in the new IVM-medium, which may allow additional embryos to be produced for clinical use at embryo transfer.     

Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development

The exact role of polyvinylpyrrolidone (PVP) in culture medium for oocyte maturation is still largely unknown. Bovine cumulus-oocyte complexes (COC) were cultured in in-vitro maturation (IVM) medium supplemented with 10% fetal bovine serum (FBS), 0.3% PVP (K-90) or 10% serum substitute supplement (SSS) respectively. The rates of oocyte maturation, fertilization and early embryonic development were evaluated. In addition, the status of DNA fragmentation in the oocytes was determined by comet assay, and the ratio of trophectoderm (TE) cells and inner cell mass (ICM) in blastocysts was determined by differential staining. Furthermore, the percentage of apoptotic cells in the blastocysts was examined by TUNEL assay. The results indicated that the effect of PVP in IVM medium was similar to FBS in terms of oocyte maturation and subsequent embryonic development. However, the addition of SSS in IVM medium retarded further embryonic development and resulted in more oocyte DNA fragmentation and a higher ratio of TE cells and ICM in the blastocysts. However, the number of apoptotic cells in blastocysts was similar among the three groups. These results suggest for the first time that the addition of PVP in oocyte maturation medium is not only a suitable substitute for serum but is also beneficial to in-vitro oocyte maturation.