目的:原核转移(PNT)是否能够恢复体外生长(IVG)小鼠卵母细胞的细胞质次优引起的胚胎发育停滞?
结论:PNT到体内成熟的细胞质显着改善了IVG小鼠卵母细胞的胚胎发育,导致生活,肥沃的后代。
背景:体外卵泡培养已被认为是癌症患者的生育力保存选择。描述人类卵泡培养的研究仍然很少,由于组织的可用性低。小鼠模型已广泛用于研究和优化卵泡培养。虽然取得了重要成就,包括在老鼠身上生产健康的后代,与体内生长的卵母细胞相比,IVG卵母细胞的质量较差,可能是因为细胞质功能不全。
■该研究于2020年9月至2022年2月进行。总的来说,使用120只15日龄B6D2小鼠进行二次卵泡培养并评估IVG卵母细胞的质量。从85只8至12周龄的B6D2小鼠中获得体内生长的对照卵母细胞,卵巢刺激后。为了收集精子,使用了四名10至14周龄的B6D2男性。对于胚胎移植,14名8至12周龄的CD1女性用作代孕母亲,10名10-24周龄的CD1输精管切除男性用于产生假孕女性。最后,为了交配,使用四只8-10周龄的B6D2雌性小鼠和两只10周龄的B6D2雄性小鼠来确认核转移(NT)来源的幼崽的生育力。
方法:从卵巢中分离15天大的B6D2小鼠的次级卵泡,并培养9天,在给予成熟刺激之前。成熟16-18小时后,收集卵母细胞并评估成熟率,卵母细胞直径,激活率,纺锤形态,钙释放能力,和线粒体膜电位.对于每一个实验,使用体内生长的卵母细胞作为对照进行比较。当在IVG卵母细胞中证实细胞质不成熟和胚胎发育不良时,进行PNT。为此,IVG卵母细胞的原核,在孤雌生殖激活和试管婴儿后产生,被转移到受精的细胞质中,体内生长的卵母细胞。对产生的胚胎进行遗传分析和胚胎移植以确认该技术的安全性。
结果:卵泡培养9天后,收集703个卵母细胞,其中76%显示成熟到中期II期。IVG卵母细胞直径明显较低,在对照组中测量为67.4μm与73.1μm(P<0.001)。纺锤形态在IVG和对照卵母细胞之间没有显着差异,但IVG组钙释放能力受损。在IVG卵母细胞中观察到1.62个任意单位的平均钙释放,显著低于对照卵母细胞的5.74(P<0.001)。最后,与对照组相比,IVG的线粒体膜电位较差,达到0.95对2.27的平均值(P<0.001)。在用氯化锶(SrCl2)进行孤雌生殖激活后,评估了IVG卵母细胞的发育潜力。只有59.4%的IVG卵母细胞分裂成两个细胞,36.3%的卵母细胞达到胚泡期,显著低于对照卵母细胞的89.5%和88.2%,(P<0.001和0.001)。PNT和纺锤体转移(ST)在孤雌生殖激活卵母细胞的试验实验中进行了探索,作为克服胚胎发育不良的手段。确认NT的附加值后,我们继续通过PNT产生双亲胚胎。为此,IVG和对照卵母细胞首先接受IVF。只有15.5%的IVG卵母细胞正常受精,对照组为45.5%(P<0.001),导致IVG组囊胚形成失败(0对86.2%,P<0.001)。当IVG受精卵的原核转移到对照受精卵的细胞质中时,囊胚率恢复到86.9%,与控件类似的级别。PNT胚胎的遗传分析显示染色体正常,率达到80%。最后,活着的一代,胚胎移植给代孕母亲后,PNT的可育后代是可能的。
方法:不适用。
结论:来自PNT的分析胚胎的遗传特征来源于那些太小而无法得出具体结论的群体,而ST,这将是首选的NT方法,由于技术限制,不能用于生成双亲胚胎。即使有希望,PNT的使用应该被认为是实验性的。此外,结果是在小鼠模型中获得的,因此,需要在人IVG卵母细胞中对该技术进行验证,以评估该技术的临床相关性.IVG卵母细胞的遗传图谱,这将是染色体异常的最终特征,由于单细胞可靠分析的局限性,未进行分析。
结论:PNT具有克服IVG小鼠卵母细胞胞质质量差的能力。考虑到人IVG卵母细胞的成熟效率低和长期体外培养后潜在的有害影响,NT可用于挽救胚胎发育,并可能导致高质量胚胎移植的可用性增加。
背景:A.C.是FWO(FondsvoorWetenschappelijkOnderzoek)赠款(1S80220N和1S80222N)的持有者。B.H.和A.V.S.已获得特别的BOF(BijzonderOnderzoeksfonds),GOA(Geconcerteerdeonderzoeksacties)2018000504(GOA030-18BOF)资助。B.H.一直在从FerringPharmaceuticals(Aalst,比利时)。作者声明他们没有利益冲突。
OBJECTIVE: Is pronuclear transfer (PNT) capable of restoring embryo developmental arrest caused by cytoplasmic inferiority of in vitro-grown (IVG) mouse oocytes?
CONCLUSIONS: PNT to in vivo matured cytoplasm significantly improved embryo development of IVG mouse oocytes, leading to living, fertile offspring.
BACKGROUND: In vitro follicle culture has been considered as a fertility preservation option for cancer patients. Studies describing the culture of human follicles remain scarce, owing to low availability of tissue. Mouse models have extensively been used to study and optimize follicle culture. Although important achievements have been accomplished, including the production of healthy offspring in mice, IVG oocytes are of inferior quality when compared to in vivo-grown oocytes, likely because of cytoplasmic incompetence.
UNASSIGNED: The study was carried out from September 2020 to February 2022. In total, 120 15-day-old B6D2 mice were used to perform secondary follicle culture and assess the quality of IVG oocytes. In vivo-grown control oocytes were obtained from 85 8- to 12-week-old B6D2 mice, following ovarian stimulation. For sperm collection, four B6D2 males between 10 and 14 weeks old were used. For embryo transfer, 14 8- to 12-week-old CD1 females served as surrogate mothers and 10 CD1 vasectomized males 10-24 weeks old were used to generate pseudo-pregnant females. Finally, for mating, four B6D2 female mice aged 8-10 weeks and two B6D2 male mice aged 10 weeks old were used to confirm the fertility of nuclear transfer (NT)-derived pups.
METHODS: Secondary follicles from 15-day-old B6D2 mice were isolated from the ovaries and cultured for 9 days, before a maturation stimulus was given. Following 16-18 h of maturation, oocytes were collected and evaluated on maturation rate, oocyte diameter, activation rate, spindle morphology, calcium-releasing ability, and mitochondrial membrane potential. For every experiment, in vivo-grown oocytes were used as a control for comparison. When cytoplasmic immaturity and poor embryo development were confirmed in IVG oocytes, PNT was performed. For this, the pronuclei from IVG oocytes, created following parthenogenetic activation and IVF, were transferred to the cytoplasm of fertilized, in vivo-grown oocytes. Genetic analysis and embryo transfer of the generated embryos were implemented to confirm the safety of the technique.
RESULTS: Following 9 days of follicle culture, 703 oocytes were collected, of which 76% showed maturation to the metaphase II stage. Oocyte diameters were significantly lower in IVG oocytes, measuring 67.4 μm versus 73.1 μm in controls (P < 0.001). Spindle morphology did not differ significantly between IVG and control oocytes, but calcium-releasing ability was compromised in the IVG group. An average calcium release of 1.62 arbitrary units was observed in IVG oocytes, significantly lower than 5.74 in control oocytes (P < 0.001). Finally, mitochondrial membrane potential was inferior in IVG compared to the control group, reaching an average value of 0.95 versus 2.27 (P < 0.001). Developmental potential of IVG oocytes was assessed following parthenogenetic activation with strontium chloride (SrCl2). Only 59.4% of IVG oocytes cleaved to two cells and 36.3% reached the blastocyst stage, significantly lower than 89.5% and 88.2% in control oocytes, respectively (P < 0.001 and 0.001). Both PNT and spindle transfer (ST) were explored in pilot experiments with parthenogenetically activated oocytes, as a means to overcome poor embryo development. After the added value of NT was confirmed, we continued with the generation of biparental embryos by PNT. For this purpose, IVG and control oocytes first underwent IVF. Only 15.5% of IVG oocytes were normally fertilized, in contrast to 45.5% in controls (P < 0.001), with resulting failure of blastocyst formation in the IVG group (0 versus 86.2%, P < 0.001). When the pronuclei of IVG zygotes were transferred to the cytoplasm of control zygotes, the blastocyst rate was restored to 86.9%, a similar level as the control. Genetic analysis of PNT embryos revealed a normal chromosomal profile, to a rate of 80%. Finally, the generation of living, fertile offspring from PNT was possible following embryo transfer to surrogate mothers.
METHODS: N/A.
CONCLUSIONS: Genetic profiles of analysed embryos from PNT originate from groups that are too small to draw concrete conclusions, whilst ST, which would be the preferred NT approach, could not be used for the generation of biparental embryos owing to technical limitations. Even though promising, the use of PNT should be considered as experimental. Furthermore, results were acquired in a mouse model, so validation of the technique in human IVG oocytes needs to be performed to evaluate the clinical relevance of the technology. The genetic profiles from IVG oocytes, which would be the ultimate characterization for chromosomal abnormalities, were not analysed owing to limitations in the reliable analysis of single cells.
CONCLUSIONS: PNT has the ability to overcome the poor cytoplasmic quality of IVG mouse oocytes. Considering the low maturation efficiency of human IVG oocytes and potential detrimental effects following long-term in vitro culture, NT could be applied to rescue embryo development and could lead to an increased availability of good quality embryos for transfer.
BACKGROUND: A.C. is a holder of FWO (Fonds voor Wetenschappelijk Onderzoek) grants (1S80220N and 1S80222N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) 2018000504 (GOA030-18 BOF) funding. B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.