TY - JOUR
AU - Heindryckx,, B
AB - Abstract STUDY QUESTION Can pronuclear transfer (PNT) or maternal spindle transfer (ST) be applied to overcome poor embryo development associated with advanced maternal age or early embryo arrest in a mouse model? SUMMARY ANSWER Both PNT and ST may have the potential to restore embryonic developmental potential in a mouse model of reproductive ageing and embryonic developmental arrest. WHAT IS KNOWN ALREADY Germline nuclear transfer (NT) techniques, such as PNT and ST, are currently being applied in humans to prevent the transmission of mitochondrial diseases. Yet, there is also growing interest in the translational use of NT for treating infertility and improving IVF outcomes. Nevertheless, direct scientific evidence to support such applications is currently lacking. Moreover, it remains unclear which infertility indications may benefit from these novel assisted reproductive technologies. STUDY DESIGN, SIZE, DURATION We applied two mouse models to investigate the potential of germline NT for overcoming infertility. Firstly, we used a model of female reproductive ageing (B6D2F1 mice, n = 155), with ages ranging from 6 to 8 weeks (young), 56 (aged) to 70 weeks (very-aged), corresponding to a maternal age of <30, ∼36 and ∼45 years in humans, respectively. Secondly, we used NZB/OlaHsd female mice (7–14 weeks, n = 107), as a model of early embryo arrest. This mouse strain exhibits a high degree of two-cell block. Metaphase II (MII) oocytes and zygotes were retrieved following superovulation. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian reserve was assessed by histological analysis in the reproductive-aged mice. Mitochondrial membrane potential (△Ψm) was measured by JC-1 staining in MII oocytes, while spindle-chromosomal morphology was examined by confocal microscopy. Reciprocal ST and PNT were performed by transferring the meiotic spindle or pronuclei (PN) from unfertilised or fertilised oocytes (after ICSI) to enucleated oocytes or zygotes between aged or very-aged and young mice. Similarly, NT was also conducted between NZB/OlaHsd (embryo arrest) and B6D2F1 (non-arrest control) mice. Finally, the effect of cytoplasmic transfer (CT) was examined by injecting a small volume (∼5%) of cytoplasm from the oocytes/zygotes of young (B6D2F1) mice to the oocytes/zygotes of aged or very-aged mice or embryo-arrest mice. Overall, embryonic developmental rates of the reconstituted PNT (n = 572), ST (n = 633) and CT (n = 336) embryos were assessed to evaluate the efficiency of these techniques. Finally, chromosomal profiles of individual NT-generated blastocysts were evaluated using next generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Compared to young mice, the ovarian reserve in aged and very-aged mice was severely diminished, reflected by a lower number of ovarian follicles and a reduced number of ovulated oocytes (P < 0.001). Furthermore, we reveal that the average △Ψm in both aged and very-aged mouse oocytes was significantly reduced compared to young mouse oocytes (P < 0.001). In contrast, the average △Ψm in ST-reconstructed oocytes (very-aged spindle and young cytoplast) was improved in comparison to very-aged mouse oocytes (P < 0.001). In addition, MII oocytes from aged and very-aged mice exhibited a higher rate of abnormalities in spindle assembly (P < 0.05), and significantly lower fertilisation (60.7% and 45.3%) and blastocyst formation rates (51.4% and 38.5%) following ICSI compared to young mouse oocytes (89.7% and 87.3%) (P < 0.001). Remarkably, PNT from zygotes obtained from aged or very-aged mice to young counterparts significantly improved blastocyst formation rates (74.6% and 69.2%, respectively) (P < 0.05). Similarly, both fertilisation and blastocyst rates were significantly increased after ST between aged and young mice followed by ICSI (P < 0.05). However, we observed no improvement in embryo development rates when performing ST from very-aged to young mouse oocytes following ICSI (P > 0.05). In the second series of experiments, we primarily confirmed that the majority (61.8%) of in vivo zygotes obtained from NZB/OlaHsd mice displayed two-cell block during in vitro culture, coinciding with a significantly reduced blastocyst formation rate compared to the B6D2F1 mice (13.5% vs. 90.7%; P < 0.001). Notably, following the transfer of PN from the embryo-arrest (NZB/OlaHsd) zygotes to enucleated non-arrest (B6D2F1) counterparts, most reconstructed zygotes developed beyond the two-cell stage, leading to a significantly increased blastocyst formation rate (89.7%) (P < 0.001). Similar findings were obtained after implementing ST between NZB/OlaHsd and B6D2F1 mice, followed by ICSI. Conversely, the use of CT did not improve embryo development in reproductive-age mice nor in the embryo-arrest mouse model (P > 0.05). Surprisingly, chromosomal analysis revealed that euploidy rates in PNT and ST blastocysts generated following the transfer of very-aged PN to young cytoplasts and very-aged spindles to young cytoplasts were comparable to ICSI controls (with young mouse oocytes). A high euploidy rate was also observed in the blastocysts obtained from either PNT or ST between young mice. Conversely, the transfer of young PN and young spindles into very-aged cytoplasts led to a higher rate of chromosomal abnormalities in both PNT and ST blastocysts. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The limited number of blastocysts analysed warrants careful interpretation. Furthermore, our observations should be cautiously extrapolated to humans given the inherent differences between mice and women in regards to various biological processes, including centrosome inheritance. The findings suggest that ST or PNT procedures may be able to avoid aneuploidies generated during embryo development, but they are not likely to correct aneuploidies already present in some aged MII oocytes. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study to evaluate the potential of PNT and ST in the context of advanced maternal age and embryonic developmental arrest in a mouse model. Our data suggest that PNT, and to a lesser extent ST, may represent a novel reproductive strategy to restore embryo development for these indications. STUDY FUNDING/COMPETING INTEREST(S) M.T. is supported by grants from the China Scholarship Council (CSC) (Grant no. 201506160059) and the Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF) (Grant no. 01SC2916 and no. 01SC9518). This research is also supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051017N, G051516N and G1507816N). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A pronuclear transfer, spindle transfer, infertility, reproductive age, embryo arrest, cytoplasmic transfer, embryo development, mouse model Introduction Due to the well-established decline in female fecundity with advancing age, reproductively aged women represent a challenging group of infertility patients. This is largely attributed to ovarian ageing, leading to a decrease in both the quality and quantity of oocytes, particularly in women over the age of 35 years (Nelson et al., 2013; May-Panloup et al., 2016). While current IVF and ICSI procedures together with other assisted reproductive techniques, such as cryopreservation, time-lapse imaging, preimplantation genetic testing for aneuploidies and assisted oocyte activation have been widely used to treat infertility, their success remains limited in women of advanced maternal age. This is primarily due to the reduced number of embryos available for transfer and the increased rate of embryo aneuploidy (Franasiak et al., 2014; Ubaldi et al., 2017). Moreover, a proportion of patients are also faced with complete embryo arrest, inherently leading to poor IVF outcomes (Benkhalifa et al., 2003; Zhang et al., 2016). As such, the development of novel technologies aimed at improving embryo development, particularly in cases of advanced maternal age, remains an alluring prospect. In addition to nuclear factors, cytoplasmic factors also play an essential role in oocyte competence (Zhang and Liu, 2015; Tanaka and Watanabe, 2019). Mitochondria are important organelles within the cytoplasm responsible for generating ATP and have their own genome (mtDNA). Mitochondrial-derived ATP supports microtubule organisation, meiotic spindle assembly and chromosome alignment during oocyte maturation (Schatten et al., 2014). Subsequent fertilisation and preimplantation development largely rely on the energy provided solely by the mitochondrial pool present in mature oocytes (St John et al., 2010; Schatten et al., 2014). Consequently, mitochondrial defects may hamper these crucial processes, leading to reduced oocyte quality and ultimately poor developmental outcomes (Zhang et al., 2006). Increasing evidence suggests that maternal ageing may be associated with mitochondrial defects in oocytes and embryos (Tilly and Sinclair, 2013; Wilding, 2015). Mitochondrial dysfunction resulting from advanced maternal age may contribute to oocyte or embryo aneuploidies, partly owing to an insufficient ATP supply, required for correct meiotic divisions (Eichenlaub-Ritter et al., 2004; Pan et al., 2008). Furthermore, maternal ageing is reportedly associated with disturbances in the levels of mtDNA copy number and the incidences of mtDNA deletions or mutations in oocytes and embryos (Pallotti et al., 1996; Barritt et al., 2000; Chan et al., 2005; Fragouli et al., 2015). Nevertheless, these findings remain inconsistent, and a causal link between mitochondrial factors and maternal age-related infertility remains controversial. Successful mammalian preimplantation development also heavily relies on the orchestrated regulation of maternal-effect genes and the embryonic genome (Li et al., 2010). Following fertilisation, early embryos are transcriptionally quiescent, and their development is mainly regulated by maternal RNAs and proteins stored in the cytoplasm, which are encoded by maternal-effect genes during oogenesis (Gao et al., 2017). At the cleavage-stage (e.g. two-cell stage in mouse), a major wave of embryonic genome activation occurs, which coincides with a gradual degradation of maternally derived transcripts and proteins (Li et al., 2010; Xue et al., 2013). Any perturbations in these processes may ultimately lead to embryo arrest. Cytoplasmic transfer (CT) has been previously attempted for oocyte rejuvenation, with an aim to improve IVF outcomes. This procedure involves the introduction of a small portion of cytoplasm from a competent oocyte into an incompetent one (Cohen et al., 1997; Van Blerkom et al., 1998). Nevertheless, the benefit and safety of this method remains obscure, primarily due to some abnormalities observed in the resulting offspring and the lack of further validation studies (Chen et al., 2016; Labarta et al., 2019a). Alternatively, mitochondrial supplementation has been reported to improve oocyte competence, offered as ‘Autologous Germline Mitochondrial Energy Transfer’ (AUGMENT). Unlike CT, this approach requires isolation of autologous mitochondria, followed by injection into the patient’s oocytes during ICSI (Labarta et al., 2019a). These mitochondria can be harvested from oocyte precursor cells or oogonial stem cells present in the patient’s ovary (White et al., 2012). However, it remains difficult to validate the efficiency of this methodology owing to the small number of patients treated and the ongoing controversy regarding the existence of oogonial stem cells (Oktay et al., 2015; Labarta et al., 2019a). Recently, a randomised experimental study revealed that AUGMENT treatment did not improve embryo quality in infertile women with multiple IVF failures (Labarta et al., 2019b). Germline nuclear transfer (NT) has been proposed to prevent the transmission of mtDNA diseases. This technique involves the transfer of the nuclear genome from the patient’s oocytes or zygotes into enucleated donor oocytes or zygotes with healthy mtDNA (Craven et al., 2017). Until now, maternal spindle transfer (ST) and pronuclear transfer (PNT) are the most well-studied, and have been demonstrated to be effective in animal and human models (Tachibana et al., 2009; Neupane et al., 2014; Hyslop et al., 2016; Zhang et al., 2017). Besides its use for mtDNA diseases, NT has also been proposed as a novel reproductive technology to overcome certain infertility disorders (Zhang et al., 2016). The rationale is that replacement of a low-quality cytoplasm with a competent one by means of NT may enhance oocyte capacity, in turn improving embryo development (Craven et al., 2017; Labarta et al., 2019a). Nevertheless, this experimental treatment has led to substantial controversy, primarily due to the lack of validation studies. Ultimately, the benefit and safety of this technology remains unknown, while scientific evidence regarding the infertility indications for which this technology may be beneficial is currently lacking. Here, we aimed to determine whether PNT and ST could be applied to improve fertilisation rates and embryo developmental potential using a reproductive-age and an embryo-arrest mouse model. Additionally, we examined CT as a technique for oocyte rejuvenation. Finally, we evaluate the efficiency of these three methodologies to restore oocyte and/or embryo competence. Materials and methods Animal models and ethical approval B6D2F1 hybrid female mice (Charles River Laboratories, Brussels, Belgium) of advancing age were used as a reproductive-age model. Based on maternal age, ranging from 6–8 weeks to 56 weeks and 70 weeks, these mice were subdivided into young controls, aged and very-aged groups, respectively. Moreover, NZB/OlaHsd inbred female mice (Harlan laboratories), 7–14 weeks-old, were used as a model of early embryo arrest, with most embryos displaying two-cell block during in vitro development. All animal experiments were approved by the Animal Ethics Committee of the Ghent University Hospital (ECD No. 17/67), Ghent, Belgium. Oocyte and zygote recovery B6D2F1 and NZB/OlaHsd females were stimulated with pregnant mare’s serum gonadotrophin (7.5 IU), followed by human chorionic gonadotropin (hCG, 7.5 IU) 46–48 h later. Metaphase II (MII) oocytes were collected from females 12–14 h post-hCG injection. Zygotes were collected from females mated with B6D2F1 or NZB/OlaHsd males 19–21 h post-hCG injection. The collection procedures were performed in HEPES-buffered potassium simplex optimised medium (KSOM-HEPES), containing 4 mg/ml bovine serum albumin (Calbiochem, Belgium). Cumulus cells surrounding the oocytes or zygotes were removed by a brief exposure to 200 IU/ml hyaluronidase in KSOM-HEPES. All oocytes and zygotes were cultured in KSOM medium under mineral oil, at 37°C in 6% CO2 and 5% O2 (hereafter termed standard culture conditions). Mouse ICSI Frozen sperm samples, from B6D2F1 and NZB/OlaHsd males, were warmed at room temperature for 15 min and selected using a swim-up protocol. The sperm were firstly immobilised with decapitation, and a single sperm head was injected into the ooplasm using piezo-driven ICSI, as described previously (Vanden Meerschaut et al., 2013). Fertilisation outcomes were examined 5–8 h after ICSI, based on the presence of 2PN. Pronuclear transfer PNT was performed using an inverted microscope (OLYMPUS, IX71) equipped with micromanipulators and a laser objective (ZILOS-tk, HAMILTON THORNE) (Fig. 1A; Supplementary Fig. S2A). The zygotes were exposed to KSOM-HEPES containing cytochalasin D (2 μg/ml) and nocodazole (1 μg/ml) for 15 min at 37°C, before micromanipulation and during PNT procedures. A small ablation was made in the zona pellucida using the laser for insertion of the biopsy pipette. The 2PN were aspirated from a nuclear donor zygote into a biopsy pipette with an inner diameter of 15 μm and moved to a droplet containing haemagglutinating virus of Japan envelope (HVJ-E) (GenomONE-CFEX, Cosmo Bio Co). Following a brief exposure, the karyoplast (2PN) along with a small volume of HVJ-E were gently released into the perivitelline space (PV) of an enucleated recipient zygote (Fig. 1A;Supplementary Fig. S2A). Karyoplast-cytoplast fusion was achieved 30–60 min after PNT. The PNT-reconstructed embryos were washed in KSOM-HEPES and subsequently transferred to KSOM medium for culture in standard conditions. Figure 1. Open in new tabDownload slide Representative images showing the procedures of PNT, ST and CT in mice. See Supplementary Fig. S2. For the PNT procedure, MII oocytes were obtained from aged/very-aged (nuclear donor) and young (cytoplast recipient) mice. Following fertilisation via ICSI, two pronuclei (PN) from (I) donor zygote were transferred into (II) enucleated recipient zygote. Notably, for PNT in the embryo-arrest mouse model, we used in vivo zygotes from the NZB/OlaHsd females mated with males. For the ST procedure, the (V, VI, VII) spindle was visualised using a polarised microscopy. The spindle-chromosome complex from MII oocytes obtained from aged/very-aged mice (V) was transferred into enucleated MII oocytes from young mice (VI). For the CT procedure, a small volume of cytoplasm, which occupied ∼5% of the total oocyte volume (IX), was extracted from young mouse oocytes and then injected into aged/very-aged recipient counterparts along with a single sperm head (X). Finally, all (III, VII, XI) reconstructed embryos were cultured in standard culture conditions. Blastocysts formed after 96 h in PNT (IV), ST (VIII) and CT (XII). Figure 1. Open in new tabDownload slide Representative images showing the procedures of PNT, ST and CT in mice. See Supplementary Fig. S2. For the PNT procedure, MII oocytes were obtained from aged/very-aged (nuclear donor) and young (cytoplast recipient) mice. Following fertilisation via ICSI, two pronuclei (PN) from (I) donor zygote were transferred into (II) enucleated recipient zygote. Notably, for PNT in the embryo-arrest mouse model, we used in vivo zygotes from the NZB/OlaHsd females mated with males. For the ST procedure, the (V, VI, VII) spindle was visualised using a polarised microscopy. The spindle-chromosome complex from MII oocytes obtained from aged/very-aged mice (V) was transferred into enucleated MII oocytes from young mice (VI). For the CT procedure, a small volume of cytoplasm, which occupied ∼5% of the total oocyte volume (IX), was extracted from young mouse oocytes and then injected into aged/very-aged recipient counterparts along with a single sperm head (X). Finally, all (III, VII, XI) reconstructed embryos were cultured in standard culture conditions. Blastocysts formed after 96 h in PNT (IV), ST (VIII) and CT (XII). Spindle transfer Before ST, the MII oocytes were incubated in droplets of KSOM-HEPES containing cytochalasin D (2 μg/ml) at 37°C for 15 min in a glass-bottom dish. The dish was then placed on the warmer stage of an inverted microscope (OLYMPUS, IX71) fitted with an Oosight™ Imaging System (for spindle visualisation), micromanipulators and a laser objective (ZILOS-tk, HAMILTON THORNE). A small opening was made near the spindle with the laser. The biopsy pipette was inserted through the opening and the karyoplast (spindle) was aspirated into the pipette. Subsequently, the karyoplast was briefly exposed to the HVJ-E and gently expelled into the PV of an enucleated recipient oocyte (Fig. 1B;Supplementary Fig. S2B). Karyoplast-cytoplast fusion usually occurred 30–60 min after ST. Finally, the ST-reconstituted oocytes were rinsed with KSOM-HEPES and transferred to KSOM medium. Further culture was performed in standard conditions. Cytoplasmic transfer During the ICSI procedure, a small portion of cytoplasm, which occupied ∼5% of the total oocyte or zygote volume, was extracted from the donor oocytes or zygotes and injected into recipient counterparts along with a single sperm head (Fig. 1C;Supplementary Fig. S2C). Embryo culture and parthenogenetic activation Non-manipulated control and reconstructed embryos following PNT, ST and CT were cultured in KSOM medium (Day 1–3), followed by Cook Blastocyst medium (Cook; Sydney IVF Blastocyst medium) (Day 3–5) in standard culture conditions. Intact MII oocytes were parthenogenetically activated in Ca2+-free KSOM medium containing 10 mM SrCl2 and 2 μg/ml cytochalasin D for 4 h, serving as controls for culture conditions. Blastocyst grading Mouse blastocysts were graded based on their degrees of expansion and hatching, as grade I (cavitating), grade II (expanded), grade III (hatching) and grade IV (completely hatched) (Supplementary Fig. S3) (Cheng et al., 2004). Assessment of ovarian reserve Murine ovaries were fixed in 4% paraformaldehyde (24 h), processed and embedded in paraffin. Serial sections (5 μm) through the whole ovary were prepared and mounted on coverslips for staining with haematoxylin and eosin (HE). The number of follicles at various stages of development was determined by systematic counts under a light microscope (OLYMPUS, CX31) by investigators who were blinded to the origin of the sections. Measurement of inner mitochondrial membrane potentials by JC-1 staining The mitochondrial membrane potential is closely related to the capability of ATP synthesis. JC-1 (Invitrogen) is a dual-emission, potential-sensitive dye that accumulates preferentially in mitochondria within the oocyte cytoplasm. When the mitochondrial membrane potential (△Ψm) is <100 mV, JC-1 remains a monomer and emits green fluorescence (excitation/emission: 514/529 nm) (low polarised mitochondria), while at △Ψm > 140 mV, it forms J-aggregates and emits red fluorescence (excitation/emission: 585/590 nm) (high polarised mitochondria). For JC-1 staining, MII oocytes were cultured in KSOM medium containing JC-1 (15 μg/ml) at 37°C for 10 min. To include a pharmacological control, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, Belgium) was used to inhibit the oxidative phosphorylation activity of mitochondria in oocytes. Initially, a standard concentration was optimised by examining oocyte survival rates after treating the oocytes with different concentrations of CCCP (0, 1, 10, 100 μM). Oocytes in this control group were incubated with a standard concentration of CCCP for 20 min and then stained with JC-1. Following washes, the MII oocytes were examined by fluorescence microscopy (EVOS FL Color Imaging Systems). The captured images were processed using Image J software to measure the fluorescence intensity (Schindelin et al., 2012). Finally, the △Ψm level was assessed by calculating the ratios of red to green fluorescence intensity. Immunocytochemistry Oocytes were fixed with microtubule-stabilising buffer (0.1 M PIPES, 5 Mm MgCl2, 2.5 mM EGTA, 0.01% aprotinin, 1 mM dithiothreitol, 50% deuterium oxide, 1 pM taxol, 0.1% Triton X-100 and 3% formalin) for 30 min at 37°C. For fluorescence staining, oocytes were incubated overnight at 4°C with primary antibodies anti-α and β-tubulin (1:200). After washing three times, samples were treated with secondary antibodies Alexa Fluor 488 goat anti-mouse IgG for 1 h at 37°C, followed by extended washing. Chromosomes were stained with 20 μg/ml Hoechst-33258 for 30 min. Finally, samples were mounted in Mowiol containing 0.01% phenylenediamine and examined using a laser scanning confocal microscope (Leica, SP8). Next generation sequencing Whole genome amplification (WGA) of embryo samples was performed using the SurePlex DNA Amplification System as per manufacturer’s instructions. Samples were only being included following successful amplification, assessed by fragment analysis. Next generation sequencing (NGS) was performed as described previously (Popovic et al., 2018, 2019). Briefly, library preparation was performed using the NEXTflex™ Rapid DNA-Seq Library Prep Kit for Illumina Sequencing, using Agencourt AMPure XP beads for purification. Libraries were quantified using the NEBNext® Library Quant Kit for Illumina®. Subsequent data analysis was performed using the QDNAseq algorithm (Scheinin et al., 2014). Reads were aligned to the Genome Reference Consortium Mouse Build 38 (GRCm38, Mus musculus, mm10). Statistical analysis All data are shown as mean ± SD. All statistical analyses were performed using the Prism 6.0 statistical analysis programme (GraphPad). Differences between means were assessed by Student’s t-test. For the embryo development rates, chi-square test was performed. Results were considered statistically significant if P < 0.05. Results Effects of maternal ageing on ovarian reserve and the number of ovulated oocytes in mouse Initially, we examined the general condition of young, aged and very-aged mice (Supplementary Fig. S1A–C). We found that the body weights of aged and very-aged mice were significantly higher than those of young mice (35.60 ± 6.35 vs. 39.93 ± 4.82 vs. 22.68 ± 2.60; P < 0.001; Supplementary Fig. S1A). However, the ovarian weights of very-aged mice were markedly lower than those of aged and young mice (P < 0.05; Supplementary Fig. S1B). We further adopted the ovarian follicle pool, a source of developing follicles, as a parameter to evaluate ovarian reserve (Fig. 2B–D). Histological analysis of ovaries revealed that the number of primordial, primary and secondary follicles dramatically decreased with age (Fig. 2A). Moreover, following superovulation, oocyte yield reduced significantly with age, from 35.7 ± 9.2 per mouse in young mice to 11.6 ± 5.2 per mouse in aged mice and 4.3 ± 3.8 in very-aged mice (Fig. 3A). Moreover, we observed that the morphology of MII oocytes was comparable between young and aged mice, while MII oocytes obtained from very-aged mice often presented with a granular cytoplasm (Fig. 3B–E). These results indicate that advanced maternal age negatively affects both ovarian reserve and the number of ovulated oocytes in mice. Figure 2. Open in new tabDownload slide Effects of maternal ageing on the ovarian reserve in mice. (A) Numbers of primordial, primary and secondary follicles in aged, very-aged and young mice. Values represent average number of follicles per ovary ± SD; n shows the total number of ovaries from each group. Differences between means were determined by the Mann–Whitney U test. P < 0.05 was considered significant. Representative haematoxylin and eosin (HE) staining images of primordial (B), primary (C), and secondary (D) follicles, denoted by black arrow. Scale bar, 20 μm. Figure 2. Open in new tabDownload slide Effects of maternal ageing on the ovarian reserve in mice. (A) Numbers of primordial, primary and secondary follicles in aged, very-aged and young mice. Values represent average number of follicles per ovary ± SD; n shows the total number of ovaries from each group. Differences between means were determined by the Mann–Whitney U test. P < 0.05 was considered significant. Representative haematoxylin and eosin (HE) staining images of primordial (B), primary (C), and secondary (D) follicles, denoted by black arrow. Scale bar, 20 μm. Figure 3. Open in new tabDownload slide Effects of maternal ageing on the number of ovulated oocytes in mice. (A) Numbers of ovulated oocytes collected after hormonal stimulation of young, aged and very-aged mice. Values represent average number of follicles per female ± SD; n shows the total number of females from each group. Differences between means were determined by the Mann–Whitney U test. P < 0.05 was considered significant. Morphological evaluation of ovulated oocytes from young (B), aged (C) and very-aged (D) mice. (E) Representative images with high-magnification showed granular cytoplasm in very-aged mouse oocytes compared to young mouse oocytes. Scale bar, 50 μm. Figure 3. Open in new tabDownload slide Effects of maternal ageing on the number of ovulated oocytes in mice. (A) Numbers of ovulated oocytes collected after hormonal stimulation of young, aged and very-aged mice. Values represent average number of follicles per female ± SD; n shows the total number of females from each group. Differences between means were determined by the Mann–Whitney U test. P < 0.05 was considered significant. Morphological evaluation of ovulated oocytes from young (B), aged (C) and very-aged (D) mice. (E) Representative images with high-magnification showed granular cytoplasm in very-aged mouse oocytes compared to young mouse oocytes. Scale bar, 50 μm. Effects of maternal ageing on the mitochondrial membrane potential and meiotic spindle assembly in MII oocytes Using JC-1 staining, we further examined the effects of maternal age on the inner mitochondrial membrane potential (△Ψm) of MII oocytes. The staining results indicated that the △Ψm level was significantly decreased in aged and very-aged mouse MII oocytes, compared to those obtained from young mice (1.23 vs. 1.04 vs. 1.92, P < 0.01; Fig. 4A and B). In addition, we tested the specificity of the JC-1 dye signal using an inhibitor of mitochondrial activity (CCCP). Using young mouse oocytes, we observed that 10 and 100 μM of CCCP impaired oocyte survival rates (14.8% and 11.1%, respectively), while 1 μM of CCCP did not compromise oocytes survival rates, comparable to the 0 μM of CCCP treatment (98.3% and 100%, respectively). Accordingly, 1 μM of CCCP was used as a standard concentration to treat young mouse oocytes followed by JC-1 staining. As expected, the △Ψm level was markedly reduced in young mouse oocytes following CCCP treatment (Supplementary Fig. S4A and B). Furthermore, while a majority of MII oocytes obtained from young mice contained barrel-shaped spindles and normally aligned chromosomes (96.8%), higher rates of abnormal spindles were observed in aged and very-aged mice compared to young controls (18.8% vs. 23.9% vs. 3.2%; P < 0.05; Fig. 5A and B). The abnormal spindle-chromosome structures included disintegrated spindle poles, as well as irregularly scattered chromosomes (Fig. 5B;Supplementary Fig. S5). These findings suggest an age-associated alteration in both △Ψm and meiotic spindle assembly in MII oocytes. Figure 4. Open in new tabDownload slide Effects of maternal ageing on mitochondrial membrane potential of mouse MII oocytes. (A) Representative images displaying young (upper panel), aged (middle panel) and very-aged (lower panel) mouse oocytes stained with JC-1 dye. Green and red fluorescence suggest JC-1 monomer and JC-1 aggregate fluorescence, respectively. Scale bar, 50 μm. (B) Ratios of red to green JC-1 fluorescence in the MII oocytes of young, aged and very-aged mice. Values represent mean ± SD of at least three independent experiments; n shows the total number of oocytes tested from each group. Differences between means were determined by the Mann–Whitney U test. P < 0.05 was considered significant. Figure 4. Open in new tabDownload slide Effects of maternal ageing on mitochondrial membrane potential of mouse MII oocytes. (A) Representative images displaying young (upper panel), aged (middle panel) and very-aged (lower panel) mouse oocytes stained with JC-1 dye. Green and red fluorescence suggest JC-1 monomer and JC-1 aggregate fluorescence, respectively. Scale bar, 50 μm. (B) Ratios of red to green JC-1 fluorescence in the MII oocytes of young, aged and very-aged mice. Values represent mean ± SD of at least three independent experiments; n shows the total number of oocytes tested from each group. Differences between means were determined by the Mann–Whitney U test. P < 0.05 was considered significant. Figure 5. Open in new tabDownload slide Effects of maternal ageing on the meiotic spindle-chromosome assembly in mouse MII oocytes. (A) Percentage of abnormal spindle and misaligned chromosomes in MII oocytes of young, aged and very-aged mice. Data represent mean ± SD of at least three independent experiments; n shows the number of oocytes. Differences between means were determined by the chi-square test. P < 0.05 was considered significant. (B) Representative images of spindle-chromosomal structures in young, aged and very-aged groups. The timing of oocyte fixation was 15 h post-hCG injection. Oocytes were stained with Hoechst 33258 (blue) for DNA, and α-tubulins and β-tubulins (green) for microtubules. Normal spindle (barrel shaped) and aligned chromosomes (toothbrush appearance) are shown in the young group. Misshaped spindle and misaligned chromosomes were detected in aged and very-aged groups. Scale bars, 10 μm. Figure 5. Open in new tabDownload slide Effects of maternal ageing on the meiotic spindle-chromosome assembly in mouse MII oocytes. (A) Percentage of abnormal spindle and misaligned chromosomes in MII oocytes of young, aged and very-aged mice. Data represent mean ± SD of at least three independent experiments; n shows the number of oocytes. Differences between means were determined by the chi-square test. P < 0.05 was considered significant. (B) Representative images of spindle-chromosomal structures in young, aged and very-aged groups. The timing of oocyte fixation was 15 h post-hCG injection. Oocytes were stained with Hoechst 33258 (blue) for DNA, and α-tubulins and β-tubulins (green) for microtubules. Normal spindle (barrel shaped) and aligned chromosomes (toothbrush appearance) are shown in the young group. Misshaped spindle and misaligned chromosomes were detected in aged and very-aged groups. Scale bars, 10 μm. Assessment of mitochondrial membrane potential in reconstructed oocytes following ST between very-aged and young mice We further tested whether ST could improve the levels of mitochondrial membrane potential (△Ψm) in reconstituted embryos. Using JC-1 staining, the △Ψm was examined in ST-oocytes obtained following transfer of very-aged spindles to young cytoplasts. The intact MII oocytes obtained from young mice and very-aged mice served as controls. The results showed that the △Ψm level was significantly increased in ST-reconstructed oocytes, compared to those obtained from very-aged mice (P < 0.001; Supplementary Fig. S4A and B). Moreover, no significant difference was found in the △Ψm level between ST (very-aged spindle and young cytoplast) and intact control (with young oocytes) groups (Supplementary Fig. S4A and B). Together, these findings suggest that ST may have a positive impact on the △Ψm of the reconstructed embryos. The use of PNT, ST and CT to improve embryonic developmental potential in a reproductive-age mouse model Prior to NT, we collected MII oocytes from young, aged and very-aged mice for ICSI. Our findings reveal that both fertilisation and blastocyst formation rates were significantly lower in the aged and very-aged groups compared to young mice (P < 0.001; Table I). Table I Comparisons of embryonic development after PNT, ST and CT between aged or very-aged and young mice, and ICSI controls. Groups . No. of nuclear donor and recipient cytoplast . Reconstructed embryos . Fertilisation (%) . Two-cell (%) . Blastocysts (%) . ICSI control (aged oocytes) 79 37/61 (60.7%)** 37/61 (60.7%)** 19/37 (51.4%)** ICSI control (very-aged oocytes) 67 24/53 (45.3%)** 26/53 (49.1%)** 10/26 (38.5%)** ICSI control (young oocytes) 87 70/78 (89.7%)C c 71/78 (91.0%)C c 62/71 (87.3%)C c PNT (nuclear from aged to young cytoplast) 144 70 59/70 (84.3%)C b 44/59 (74.6%)B a PNT (nuclear from very-aged to young cytoplast) 138 67 52/67 (77.6%)* B 36/52 (69.2%)* A PNT (nuclear from young to young cytoplast) 96 44 40/44 (90.9%)C c 35/40 (87.5%)C b ST (nuclear from aged to young cytoplast) 203 98 78/98 (79.6%)Ca 80/98 (81.6%)C b 61/80 (76.3%)C a ST (nuclear from very-aged to young cytoplast) 185 81 38/81 (46.9%)** 37/81 (45.7%)** 13/37 (35.1%)** ST (nuclear from young to young cytoplast) 192 91 76/91 (83.5%) C b 76/91 (83.5%)C b 62/76 (81.6%)C b CT (young cytoplasm into aged oocytes) 130 55 32/55 (58.2%)** 31/55 (56.4%)** 16/31 (51.6%)** CT (young cytoplasm into very-aged oocytes) 108 45 20/45 (44.4%)** 22/45 (48.9%)** 7/22 (31.8%)** CT (young cytoplasm into young oocytes) 144 62 54/62 (87.1%) C b 55/62 (88.7%)C c 47/55 (85.5%)C Groups . No. of nuclear donor and recipient cytoplast . Reconstructed embryos . Fertilisation (%) . Two-cell (%) . Blastocysts (%) . ICSI control (aged oocytes) 79 37/61 (60.7%)** 37/61 (60.7%)** 19/37 (51.4%)** ICSI control (very-aged oocytes) 67 24/53 (45.3%)** 26/53 (49.1%)** 10/26 (38.5%)** ICSI control (young oocytes) 87 70/78 (89.7%)C c 71/78 (91.0%)C c 62/71 (87.3%)C c PNT (nuclear from aged to young cytoplast) 144 70 59/70 (84.3%)C b 44/59 (74.6%)B a PNT (nuclear from very-aged to young cytoplast) 138 67 52/67 (77.6%)* B 36/52 (69.2%)* A PNT (nuclear from young to young cytoplast) 96 44 40/44 (90.9%)C c 35/40 (87.5%)C b ST (nuclear from aged to young cytoplast) 203 98 78/98 (79.6%)Ca 80/98 (81.6%)C b 61/80 (76.3%)C a ST (nuclear from very-aged to young cytoplast) 185 81 38/81 (46.9%)** 37/81 (45.7%)** 13/37 (35.1%)** ST (nuclear from young to young cytoplast) 192 91 76/91 (83.5%) C b 76/91 (83.5%)C b 62/76 (81.6%)C b CT (young cytoplasm into aged oocytes) 130 55 32/55 (58.2%)** 31/55 (56.4%)** 16/31 (51.6%)** CT (young cytoplasm into very-aged oocytes) 108 45 20/45 (44.4%)** 22/45 (48.9%)** 7/22 (31.8%)** CT (young cytoplasm into young oocytes) 144 62 54/62 (87.1%) C b 55/62 (88.7%)C c 47/55 (85.5%)C Fertilisation was performed using ICSI with B6D2F1 sperm. Blastocyst rates were calculated based on the cleaved embryos (two-cell). The percentages were compared using the chi-square test and P < 0.05 was considered significant. Within the same column, the percentages with asterisk marks were statistically different when compared to the ICSI control (young oocytes); * P < 0.05, ** P < 0.001. The percentages with superscript capital letters were statistically different when compared to the ICSI control (very-aged oocytes); A P < 0.05, B P < 0.01, C P < 0.001. The percentages with superscript lowercase letters were statistically different when compared to the ICSI control (aged oocytes); a P < 0.05, b P < 0.01, c P < 0.001. PNT, pronuclear transfer; ST, maternal spindle transfer; CT, cytoplasmic transfer. Open in new tab Table I Comparisons of embryonic development after PNT, ST and CT between aged or very-aged and young mice, and ICSI controls. Groups . No. of nuclear donor and recipient cytoplast . Reconstructed embryos . Fertilisation (%) . Two-cell (%) . Blastocysts (%) . ICSI control (aged oocytes) 79 37/61 (60.7%)** 37/61 (60.7%)** 19/37 (51.4%)** ICSI control (very-aged oocytes) 67 24/53 (45.3%)** 26/53 (49.1%)** 10/26 (38.5%)** ICSI control (young oocytes) 87 70/78 (89.7%)C c 71/78 (91.0%)C c 62/71 (87.3%)C c PNT (nuclear from aged to young cytoplast) 144 70 59/70 (84.3%)C b 44/59 (74.6%)B a PNT (nuclear from very-aged to young cytoplast) 138 67 52/67 (77.6%)* B 36/52 (69.2%)* A PNT (nuclear from young to young cytoplast) 96 44 40/44 (90.9%)C c 35/40 (87.5%)C b ST (nuclear from aged to young cytoplast) 203 98 78/98 (79.6%)Ca 80/98 (81.6%)C b 61/80 (76.3%)C a ST (nuclear from very-aged to young cytoplast) 185 81 38/81 (46.9%)** 37/81 (45.7%)** 13/37 (35.1%)** ST (nuclear from young to young cytoplast) 192 91 76/91 (83.5%) C b 76/91 (83.5%)C b 62/76 (81.6%)C b CT (young cytoplasm into aged oocytes) 130 55 32/55 (58.2%)** 31/55 (56.4%)** 16/31 (51.6%)** CT (young cytoplasm into very-aged oocytes) 108 45 20/45 (44.4%)** 22/45 (48.9%)** 7/22 (31.8%)** CT (young cytoplasm into young oocytes) 144 62 54/62 (87.1%) C b 55/62 (88.7%)C c 47/55 (85.5%)C Groups . No. of nuclear donor and recipient cytoplast . Reconstructed embryos . Fertilisation (%) . Two-cell (%) . Blastocysts (%) . ICSI control (aged oocytes) 79 37/61 (60.7%)** 37/61 (60.7%)** 19/37 (51.4%)** ICSI control (very-aged oocytes) 67 24/53 (45.3%)** 26/53 (49.1%)** 10/26 (38.5%)** ICSI control (young oocytes) 87 70/78 (89.7%)C c 71/78 (91.0%)C c 62/71 (87.3%)C c PNT (nuclear from aged to young cytoplast) 144 70 59/70 (84.3%)C b 44/59 (74.6%)B a PNT (nuclear from very-aged to young cytoplast) 138 67 52/67 (77.6%)* B 36/52 (69.2%)* A PNT (nuclear from young to young cytoplast) 96 44 40/44 (90.9%)C c 35/40 (87.5%)C b ST (nuclear from aged to young cytoplast) 203 98 78/98 (79.6%)Ca 80/98 (81.6%)C b 61/80 (76.3%)C a ST (nuclear from very-aged to young cytoplast) 185 81 38/81 (46.9%)** 37/81 (45.7%)** 13/37 (35.1%)** ST (nuclear from young to young cytoplast) 192 91 76/91 (83.5%) C b 76/91 (83.5%)C b 62/76 (81.6%)C b CT (young cytoplasm into aged oocytes) 130 55 32/55 (58.2%)** 31/55 (56.4%)** 16/31 (51.6%)** CT (young cytoplasm into very-aged oocytes) 108 45 20/45 (44.4%)** 22/45 (48.9%)** 7/22 (31.8%)** CT (young cytoplasm into young oocytes) 144 62 54/62 (87.1%) C b 55/62 (88.7%)C c 47/55 (85.5%)C Fertilisation was performed using ICSI with B6D2F1 sperm. Blastocyst rates were calculated based on the cleaved embryos (two-cell). The percentages were compared using the chi-square test and P < 0.05 was considered significant. Within the same column, the percentages with asterisk marks were statistically different when compared to the ICSI control (young oocytes); * P < 0.05, ** P < 0.001. The percentages with superscript capital letters were statistically different when compared to the ICSI control (very-aged oocytes); A P < 0.05, B P < 0.01, C P < 0.001. The percentages with superscript lowercase letters were statistically different when compared to the ICSI control (aged oocytes); a P < 0.05, b P < 0.01, c P < 0.001. PNT, pronuclear transfer; ST, maternal spindle transfer; CT, cytoplasmic transfer. Open in new tab To mimic the clinical context in humans more accurately, we selected fertilised oocytes post-ICSI for PNT, rather than from harvested zygotes. Two pronuclei (2PN) from the zygotes obtained from aged or very-aged mice were transferred into the enucleated zygotes from young mice (Fig. 1; Supplementary Fig. S2A). Notably, these reconstructed PNT zygotes yielded a marked improvement in blastocyst formation rates compared to ICSI controls (aged or very-aged mouse oocytes) (74.6% vs. 69.2% vs. 51.4% vs. 38.5%; P < 0.05; Table I;Supplementary Fig. S6E and F). Remarkably, the PNT zygotes (aged PN and young cytoplast) showed comparable blastocyst rates to those of other PNT zygotes obtained following transfer of young PN into a young cytoplast, as well as ICSI controls (young oocytes) (74.6% vs. 87.5% vs. 87.3%; Table I). Alternatively, we transferred 2PN from young zygotes into enucleated aged or very-aged mouse zygotes (young PN and aged/very-aged cytoplast). Remarkably, this led to a sharp decline in blastocyst rates compared to the PNT zygotes obtained following the transfer of young PN into a young cytoplast (12.5% vs. 9.1% vs. 87.5%; P < 0.001; Table I and Supplementary Table SI). Moreover, most of the generated blastocysts from PNT (PN from aged or very-aged zygotes to young cytoplasts) (72.7% and 66.7%, respectively) were classified as grade III. These percentages were comparable to the ICSI controls (young oocytes) (71.0%; Supplementary Table SIII). Our data suggest that PNT is highly efficient in rescuing poor embryo development, which showed to be mainly driven by the cytoplasm. We then performed ST by transferring the spindle-chromosome complex from MII oocytes obtained from aged or very-aged mice to the enucleated oocytes of young mice (Fig. 1; Supplementary Fig. S2B). Following ICSI, the reconstituted ST oocytes (aged spindle and young cytoplast) displayed a significant increase in both fertilisation (79.6%) and blastocyst formation rates (76.3%) in comparison to matched ICSI controls (aged or very-aged oocytes) (P < 0.05; Table I;Supplementary Fig. S6A and E). These rates were similar to those of other ST oocytes obtained following the transfer of a young spindle into a young cytoplast (83.5% and 81.6%, respectively; Table I). However, the reconstituted oocytes, obtained following ST from very-aged mouse oocytes to the young mouse cytoplast did not show an improvement in fertilisation (46.9%) and blastocyst (35.1%) rates after ICSI (Table I;Supplementary Fig. S6B and F). Notably, the ST group (aged spindle and young cytoplast) showed a high rate of hatching (grade III) blastocysts, which was similar to the ICSI controls (young oocytes) (68.9% vs. 71.0%; Supplementary Table SIII). However, the other ST group (very-aged spindle and young cytoplast) revealed a significantly lower rate of hatching (grade III) blastocysts in comparison to the ICSI controls (young oocytes) (15.4% vs. 71.0%; Supplementary Table SIII). Reciprocally, we also transferred the spindle-chromosome complex from young mouse oocytes into the aged or very-aged mouse cytoplast. Both the fertilisation and blastocyst formation rates in these reconstituted oocytes were found to be significantly reduced compared to other ST oocytes obtained following the transfer of a young spindle to a young cytoplast (P < 0.001; Table I;Supplementary Table SI). In parallel, CT was carried out by injecting a small volume (∼5%) of cytoplasm from young mouse oocytes into aged or very-aged mouse oocytes along with a single B6D2F1 sperm (Fig. 1; Supplementary Fig. S2C). We found that neither fertilisation nor blastocyst formation rates were improved in the CT-generated oocytes compared to matched ICSI controls (aged or very-aged oocytes) (P > 0.05; Table I;Supplementary Fig. S6C, D, G and H). Furthermore, these rates were significantly lower compared to those following CT between young mouse oocytes (P < 0.01; Table I). Together, our results suggest that PNT and ST represent feasible approaches for improving oocyte competence, but ST has limited potential for rejuvenating very-old mouse oocytes. Moreover, CT alone was not sufficient to overcome age-related deficiencies in mouse oocytes. The use of PNT, ST and CT to restore embryo developmental competence in an embryo-arrest mouse model We primarily evaluated preimplantation development in an embryo-arrest model (NZB/OlaHsd female mice). Compared to B6D2F1 mice, NZB/OlaHsd zygotes displayed significantly higher rates of two-cell block following culture. This was the case for both in vivo produced zygotes (mated with B6D2F1 males) and in vitro zygotes obtained following ICSI with B6D2F1 sperm (4.1% and 5.8% vs. 61.8% and 57.8%; P < 0.001). Accordingly, blastocyst rates were also significantly lower in these groups compared to controls (90.7% and 80.8% vs. 13.5% and 9.4%; P < 0.001; Table II). In addition, we used NZB/OlaHsd males or sperm for mating or ICSI, however, observed no significant difference in embryo development compared to zygotes generated using B6D2F1 sperm (Table II). We then conducted reciprocal PNT on in vivo produced zygotes (mated with B6D2F1 males) from NZB/OlaHsd and B6D2F1 females (Fig. 1; Supplementary Fig. S2A). Most PNT-reconstructed zygotes (NZB/OlaHsd PN and B6D2F1 cytoplast) developed beyond the two-cell stage, resulting in significantly higher blastocyst rates compared to other PNT zygotes generated following the transfer of NZB/OlaHsd PN into NZB/OlaHsd cytoplasts, as well as NZB/OlaHsd in vivo produced zygotes (89.7% vs. 7.8% vs. 13.5%; P < 0.001; Tables II and III; Supplementary Fig. S6I). Moreover, similar blastocyst rates were observed when comparing zygotes following the transfer of NZB/OlaHsd PN into B6D2F1 cytoplasts and PNT zygotes constructed from B6D2F1 PN and B6D2F1 cytoplasts (89.7% and 91.7%, respectively; Table III and Supplementary Table SII). Notably, transfer of 2PN from B6D2F1 zygotes into NZB/OlaHsd cytoplasts resulted in a significant decrease in blastocyst formation rates compared to PNT within B6D2F1 zygotes (50.8% vs. 91.7%; P < 0.001; Supplementary Table SII). Furthermore, 63.9% of the generated blastocysts from PNT (NZB/OlaHsd PN to B6D2F1 cytoplasts) were classified as grade III, which was comparable to the ICSI control (B6D2F1 oocytes) (76.2%) and in vivo B6D2F1 zygote control (65.9%) (Supplementary Table SIV). These observations indicate that PNT was efficient in overcoming embryo arrest in NZB/OlaHsd mice. Table II Comparisons of in vitro embryonic development of NZB/OlaHsd and B6D2F1 mice. Mouse strain . Groups . No. of oocytes or zygotes . Two-cell (%) . Arrest at two-cell stage (%) . Blastocyst (%) . NZB/OlaHsd (embryo arrest) In vivo zygotes (mated with B6D2F1 males) 111 89/111 (80.2%) 55/89 (61.8%) 12/89 (13.5%) In vivo zygotes (mated with NZB/OlaHsd males) 79 59/79 (74.7%) 35/59 (59.3%) 7/59 (11.9%) ICSI of oocytes (with B6D2F1 sperm) 88 64/88 (72.7%) 37/64 (57.8%) 6/64 (9.4%) ICSI of oocytes (with NZB/OlaHsd sperm) 74 52/74 (70.3%) 30/52 (57.7%) 4/52 (7.7%) Parthenogenetic activation of oocytes 90 76/90 (84.4%) 49/76 (64.5%) 8/76 (10.5%) B6D2F1 (non-arrest) In vivo zygotes (mated with B6D2F1 males) 108 97/108 (89.8%) 4/97 (4.1%) 88/97 (90.7%) In vivo zygotes (mated with NZB/OlaHsd males) 78 63/78 (80.8%) 6/63 (9.5%) 52/63 (82.5%) ICSI of oocytes (with B6D2F1 sperm) 64 52/64 (81.3%) 3/52 (5.8%) 42/52 (80.8%) ICSI of oocytes (with NZB/OlaHsd sperm) 56 44/56 (78.6%) 3/44 (6.8%) 34/44 (77.3%) Parthenogenetic activation of oocytes 71 58/71 (81.7%) 1/58 (1.7%) 54/58 (93.1%) Mouse strain . Groups . No. of oocytes or zygotes . Two-cell (%) . Arrest at two-cell stage (%) . Blastocyst (%) . NZB/OlaHsd (embryo arrest) In vivo zygotes (mated with B6D2F1 males) 111 89/111 (80.2%) 55/89 (61.8%) 12/89 (13.5%) In vivo zygotes (mated with NZB/OlaHsd males) 79 59/79 (74.7%) 35/59 (59.3%) 7/59 (11.9%) ICSI of oocytes (with B6D2F1 sperm) 88 64/88 (72.7%) 37/64 (57.8%) 6/64 (9.4%) ICSI of oocytes (with NZB/OlaHsd sperm) 74 52/74 (70.3%) 30/52 (57.7%) 4/52 (7.7%) Parthenogenetic activation of oocytes 90 76/90 (84.4%) 49/76 (64.5%) 8/76 (10.5%) B6D2F1 (non-arrest) In vivo zygotes (mated with B6D2F1 males) 108 97/108 (89.8%) 4/97 (4.1%) 88/97 (90.7%) In vivo zygotes (mated with NZB/OlaHsd males) 78 63/78 (80.8%) 6/63 (9.5%) 52/63 (82.5%) ICSI of oocytes (with B6D2F1 sperm) 64 52/64 (81.3%) 3/52 (5.8%) 42/52 (80.8%) ICSI of oocytes (with NZB/OlaHsd sperm) 56 44/56 (78.6%) 3/44 (6.8%) 34/44 (77.3%) Parthenogenetic activation of oocytes 71 58/71 (81.7%) 1/58 (1.7%) 54/58 (93.1%) Blastocyst rates were calculated based on the cleaved embryos (two-cell). Open in new tab Table II Comparisons of in vitro embryonic development of NZB/OlaHsd and B6D2F1 mice. Mouse strain . Groups . No. of oocytes or zygotes . Two-cell (%) . Arrest at two-cell stage (%) . Blastocyst (%) . NZB/OlaHsd (embryo arrest) In vivo zygotes (mated with B6D2F1 males) 111 89/111 (80.2%) 55/89 (61.8%) 12/89 (13.5%) In vivo zygotes (mated with NZB/OlaHsd males) 79 59/79 (74.7%) 35/59 (59.3%) 7/59 (11.9%) ICSI of oocytes (with B6D2F1 sperm) 88 64/88 (72.7%) 37/64 (57.8%) 6/64 (9.4%) ICSI of oocytes (with NZB/OlaHsd sperm) 74 52/74 (70.3%) 30/52 (57.7%) 4/52 (7.7%) Parthenogenetic activation of oocytes 90 76/90 (84.4%) 49/76 (64.5%) 8/76 (10.5%) B6D2F1 (non-arrest) In vivo zygotes (mated with B6D2F1 males) 108 97/108 (89.8%) 4/97 (4.1%) 88/97 (90.7%) In vivo zygotes (mated with NZB/OlaHsd males) 78 63/78 (80.8%) 6/63 (9.5%) 52/63 (82.5%) ICSI of oocytes (with B6D2F1 sperm) 64 52/64 (81.3%) 3/52 (5.8%) 42/52 (80.8%) ICSI of oocytes (with NZB/OlaHsd sperm) 56 44/56 (78.6%) 3/44 (6.8%) 34/44 (77.3%) Parthenogenetic activation of oocytes 71 58/71 (81.7%) 1/58 (1.7%) 54/58 (93.1%) Mouse strain . Groups . No. of oocytes or zygotes . Two-cell (%) . Arrest at two-cell stage (%) . Blastocyst (%) . NZB/OlaHsd (embryo arrest) In vivo zygotes (mated with B6D2F1 males) 111 89/111 (80.2%) 55/89 (61.8%) 12/89 (13.5%) In vivo zygotes (mated with NZB/OlaHsd males) 79 59/79 (74.7%) 35/59 (59.3%) 7/59 (11.9%) ICSI of oocytes (with B6D2F1 sperm) 88 64/88 (72.7%) 37/64 (57.8%) 6/64 (9.4%) ICSI of oocytes (with NZB/OlaHsd sperm) 74 52/74 (70.3%) 30/52 (57.7%) 4/52 (7.7%) Parthenogenetic activation of oocytes 90 76/90 (84.4%) 49/76 (64.5%) 8/76 (10.5%) B6D2F1 (non-arrest) In vivo zygotes (mated with B6D2F1 males) 108 97/108 (89.8%) 4/97 (4.1%) 88/97 (90.7%) In vivo zygotes (mated with NZB/OlaHsd males) 78 63/78 (80.8%) 6/63 (9.5%) 52/63 (82.5%) ICSI of oocytes (with B6D2F1 sperm) 64 52/64 (81.3%) 3/52 (5.8%) 42/52 (80.8%) ICSI of oocytes (with NZB/OlaHsd sperm) 56 44/56 (78.6%) 3/44 (6.8%) 34/44 (77.3%) Parthenogenetic activation of oocytes 71 58/71 (81.7%) 1/58 (1.7%) 54/58 (93.1%) Blastocyst rates were calculated based on the cleaved embryos (two-cell). Open in new tab Table III Comparisons of embryonic development after reciprocal PNT, ST and CT between NZB/OlaHsd and B6D2F1 mice. Groups . No. of nuclear donor and recipient cytoplast . No. of reconstructed embryos . Two-cell (%) . Blastocyst (%) . PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 189 92 51/92 (55.4%) 4/51 (7.8%) PNT (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 167 81 68/81 (84.0%)** 61/68 (89.7%)A ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 154 75 45/75 (60.0%) 6/45 (13.3%) ST (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 166 77 60/77 (77.9%)* 41/60 (68.3%)B CT (B6D2F1 cytoplasm into B6D2F1 oocytes) 123 56 50/56 (89.3%) 46/50 (92.0%) CT (B6D2F1 cytoplasm into NZB/OlaHsd oocytes) 137 64 46/64 (71.9%)a 4/46 (8.7%)C CT (NZB/OlaHsd cytoplasm into NZB/OlaHsd oocytes) 119 54 37/54 (68.5%)b 4/37 (10.8%)D Groups . No. of nuclear donor and recipient cytoplast . No. of reconstructed embryos . Two-cell (%) . Blastocyst (%) . PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 189 92 51/92 (55.4%) 4/51 (7.8%) PNT (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 167 81 68/81 (84.0%)** 61/68 (89.7%)A ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 154 75 45/75 (60.0%) 6/45 (13.3%) ST (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 166 77 60/77 (77.9%)* 41/60 (68.3%)B CT (B6D2F1 cytoplasm into B6D2F1 oocytes) 123 56 50/56 (89.3%) 46/50 (92.0%) CT (B6D2F1 cytoplasm into NZB/OlaHsd oocytes) 137 64 46/64 (71.9%)a 4/46 (8.7%)C CT (NZB/OlaHsd cytoplasm into NZB/OlaHsd oocytes) 119 54 37/54 (68.5%)b 4/37 (10.8%)D Both PNT and CT were performed using in vivo zygotes (mated with B6D2F1 males). ST was performed using MII oocytes followed by ICSI (B6D2F1 sperm). Blastocyst rates were calculated based on the cleaved embryos (two-cell). The percentages were compared using the chi-square test. Within the column of two-cell (%). * P < 0.05 versus ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast). ** P < 0.001 versus PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast); a, b P < 0.05 versus CT (B6D2F1 cytoplasm into B6D2F1 oocytes). Within the column of blastocyst (%), A P < 0.001 versus PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast); B P < 0.001 versus ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast); C, D P < 0.001 versus CT (B6D2F1 cytoplasm into B6D2F1 oocytes). Open in new tab Table III Comparisons of embryonic development after reciprocal PNT, ST and CT between NZB/OlaHsd and B6D2F1 mice. Groups . No. of nuclear donor and recipient cytoplast . No. of reconstructed embryos . Two-cell (%) . Blastocyst (%) . PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 189 92 51/92 (55.4%) 4/51 (7.8%) PNT (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 167 81 68/81 (84.0%)** 61/68 (89.7%)A ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 154 75 45/75 (60.0%) 6/45 (13.3%) ST (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 166 77 60/77 (77.9%)* 41/60 (68.3%)B CT (B6D2F1 cytoplasm into B6D2F1 oocytes) 123 56 50/56 (89.3%) 46/50 (92.0%) CT (B6D2F1 cytoplasm into NZB/OlaHsd oocytes) 137 64 46/64 (71.9%)a 4/46 (8.7%)C CT (NZB/OlaHsd cytoplasm into NZB/OlaHsd oocytes) 119 54 37/54 (68.5%)b 4/37 (10.8%)D Groups . No. of nuclear donor and recipient cytoplast . No. of reconstructed embryos . Two-cell (%) . Blastocyst (%) . PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 189 92 51/92 (55.4%) 4/51 (7.8%) PNT (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 167 81 68/81 (84.0%)** 61/68 (89.7%)A ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast) 154 75 45/75 (60.0%) 6/45 (13.3%) ST (NZB/OlaHsd nuclear to B6D2F1 cytoplast) 166 77 60/77 (77.9%)* 41/60 (68.3%)B CT (B6D2F1 cytoplasm into B6D2F1 oocytes) 123 56 50/56 (89.3%) 46/50 (92.0%) CT (B6D2F1 cytoplasm into NZB/OlaHsd oocytes) 137 64 46/64 (71.9%)a 4/46 (8.7%)C CT (NZB/OlaHsd cytoplasm into NZB/OlaHsd oocytes) 119 54 37/54 (68.5%)b 4/37 (10.8%)D Both PNT and CT were performed using in vivo zygotes (mated with B6D2F1 males). ST was performed using MII oocytes followed by ICSI (B6D2F1 sperm). Blastocyst rates were calculated based on the cleaved embryos (two-cell). The percentages were compared using the chi-square test. Within the column of two-cell (%). * P < 0.05 versus ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast). ** P < 0.001 versus PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast); a, b P < 0.05 versus CT (B6D2F1 cytoplasm into B6D2F1 oocytes). Within the column of blastocyst (%), A P < 0.001 versus PNT (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast); B P < 0.001 versus ST (NZB/OlaHsd nuclear to NZB/OlaHsd cytoplast); C, D P < 0.001 versus CT (B6D2F1 cytoplasm into B6D2F1 oocytes). Open in new tab Likewise, we carried out reciprocal ST using MII oocytes from NZB/OlaHsd or B6D2F1 mice (Fig. 1; Supplementary Fig. S2B). Following ICSI (with B6D2F1 sperm), the ST-reconstituted oocytes, with NZB/OlaHsd spindle and B6D2F1 cytoplast, yielded a higher blastocyst rate compared to other ST oocytes obtained following the transfer of NZB/OlaHsd spindle into a NZB/OlaHsd cytoplast and ICSI (NZB/OlaHsd) oocytes (68.3% vs. 13.3% vs. 9.4%; P < 0.001; Supplementary Fig. S6I). Moreover, we observed a comparable rate of hatching (grade III) blastocysts amongst the ST (NZB/OlaHsd spindles to B6D2F1 cytoplasts) and both the ICSI (B6D2F1 oocytes) and in vivo B6D2F1 zygote control groups (70.7% vs. 76.2% vs. 65.9%; Supplementary Table SIV). Nevertheless, blastocyst rates were lower compared to ST amongst B6D2F1 oocytes (B6D2F1 spindles and B6D2F1 cytoplasts) (68.3% vs. 88.6%; P < 0.05; Tables II and III; Supplementary Table SII). Additionally, compared to ST amongst B6D2F1 oocytes, blastocyst rates were dramatically reduced when transferring the spindle-chromosome complex from B6D2F1 oocytes to enucleated NZB/OlaHsd oocytes (88.6% vs. 14.3%; P < 0.001; Supplementary Table SII). Furthermore, we compared the overall efficiency of ST and PNT based on blastocyst rates of reconstituted embryos (NZB/OlaHsd PN/spindle and B6D2F1 cytoplast). ST showed a significantly lower blastocyst rate compared to PNT (P < 0.01; Table III). Finally, using CT, we supplemented NZB/OlaHsd in vivo produced zygotes (mated with B6D2F1 males) with a small portion of cytoplasm from the B6D2F1 zygotes. Blastocyst formation rates were not enhanced after CT compared with NZB/OlaHsd in vivo produced zygotes (8.7% vs. 13.5%; P > 0.05; Tables II and III; Supplementary Fig. S6J). Overall, our data demonstrate that ST may rescue embryo arrest, but with a lower efficiency compared to PNT. In contrast, zygotic CT was not effective for improving embryo development in NZB/OlaHsd mice. Chromosomal analysis of the reconstructed embryos following PNT and ST To evaluate the effects of PNT and ST procedures on embryo aneuploidy, we assessed the chromosomal status of individual blastocysts generated following either PNT or ST using NGS. We evaluated the blastocysts obtained following PNT or ST from (i) young mouse zygotes/oocytes to young mouse cytoplasts, (ii) young mouse zygotes/oocytes to very-aged mouse cytoplasts and (iii) very-aged mouse zygotes/oocytes to young mouse cytoplasts. Blastocysts generated after piezo-driven ICSI using either young mouse oocytes or very-aged mouse oocytes served as controls. The sequencing data revealed that all blastocysts (n = 10) generated from ICSI controls (using young mouse oocytes) were euploid (Fig. 6; Supplementary Fig. S7A). Conversely, three (out of ten) blastocysts generated from very-aged mouse oocytes after ICSI were abnormal, presenting with monosomies and deletions (Fig. 6; Supplementary Fig. S7B). All blastocysts generated following PNT (n = 10) and ST (n = 10) between young mice were euploid, comparable to the young ICSI controls (Fig. 6; Supplementary Fig. S7C and F). Interestingly, all blastocysts (n = 10) obtained following PNT from very-aged mouse zygotes to young mouse cytoplasts were euploid, while the transfer of young PN into very-aged mouse cytoplasts resulted in a higher rate of chromosomal abnormalities (four abnormal blastocysts out of nine), comparable to the very-aged ICSI controls (Fig. 6; Supplementary Fig. S7B, D and E). In the ST group, 9 (out of 10) blastocysts generated following the transfer of very-aged spindles to young cytoplasts were normal, while one blastocyst presented with a 15q deletion (Fig. 6; Supplementary Fig. S7H). Conversely, ST from young mouse oocytes into very-aged mouse cytoplasts resulted in a higher rate of chromosomal aberrations (six abnormal blastocysts out of nine). Interestingly, a high proportion of the abnormal blastocysts presented with mosaicism or distinct structural aberrations (Fig. 6; Supplementary Fig. S7G). These findings suggest that maternal age-related aneuploidy in mice may also be attributed to cytoplasmic defects, leading to an increase in mitotic errors during preimplantation development. Nevertheless, given the limited number of blastocysts examined, our observations require careful interpretation. Figure 6. Open in new tabDownload slide Bar graph showing euploidy rates in the blastocysts obtained from different PNT, ST and control groups. See Supplementary Fig. S7. Figure 6. Open in new tabDownload slide Bar graph showing euploidy rates in the blastocysts obtained from different PNT, ST and control groups. See Supplementary Fig. S7. Discussion To our knowledge, this is the first preclinical study to comparatively evaluate the effectiveness of PNT, ST and CT for improving oocyte quality and embryo development in mammals. Notably, experimental evidence regarding the effectiveness and safety of these approaches, particularly for improving IVF outcomes remains remarkably limited (Craven et al., 2017). To assess the benefit of these techniques, we used two distinct mouse models: a model of reproductive ageing and a model of early embryo arrest. Age-related infertility and poor embryo quality are regularly encountered in a clinical IVF setting. Nevertheless, these indications remain challenging to treat and oocyte donation often represents the only possible therapeutic approach. Our results suggest that both PNT and ST may be effective for improving oocyte quality and competence in such cases. These novel reproductive technologies may thus provide new potential avenues for genetic parenthood for patients facing age-related infertility and early embryo developmental arrest. In contrast, we demonstrate that CT was not efficient in restoring the developmental potential of mouse embryos in this context. However, until more is known about the efficiency and safety of PNT and ST, these techniques remain experimental. Mouse models have been extensively used to study ageing in vivo, being applied to investigate age-related alterations in several cell types, including oocytes (Pan et al., 2008; Haverfield et al., 2016). In this study, we primarily evaluated reproductive ageing in female mice, with ages ranging from 56 to 70 weeks, corresponding to ∼36 and ∼45 years in humans, respectively (Dutta and Sengupta, 2016). In mammals, advancing maternal age leads to a progressive decline in ovarian follicular reserve, mainly attributed to ovarian ageing (Coxworth and Hawkes, 2010; Tilly and Sinclair, 2013). Accordingly, we observed an increased loss of the ovarian follicular pool, including primordial, primary and secondary follicles, in aged mice. Similarly, the number of ovulated oocytes also decreased significantly with age, supporting the correlation between maternal ageing and a reduction in the ovarian reserve. Several studies have reported an age-related decline in mitochondrial membrane potential in both murine and human oocytes, leading to impaired embryo development (Wilding et al., 2001; Thouas et al., 2004; Igarashi et al., 2016). Moreover, errors in maternal meiosis characteristic of age-related infertility have also been attributed to mitochondrial dysfunction in aged oocytes (Hamatani et al., 2004; Pan et al., 2008; Ben-Meir et al., 2015). In line with these observations, our data reveal that the mitochondrial membrane potential in MII oocytes of aged and very-aged mice was significantly reduced compared to that of young mouse oocytes, coinciding with a higher rate of spindle defects and chromosomal misalignment. In contrast, the membrane potential was improved in ST-reconstituted oocytes obtained following the transfer of very-aged spindles to young cytoplasts, compared to very-aged mouse oocytes. Nevertheless, future work is required to determine the causal role of mitochondrial membrane potential on oocyte competence. Mitochondrial DNA content has been suggested as a potential viability marker for oocytes and embryos (Duran et al., 2011; Fragouli et al., 2015). Future work involving mtDNA quantification may therefore provide valuable evidence for the role of mitochondria in aged oocytes. Additionally, following ICSI and in vitro culture, the oocytes from aged and very-aged mice showed a significant decrease in both fertilisation and blastocyst formation rates compared to young controls. As such, our results confirm previous reports, suggesting that advanced maternal age may be, at least in part, associated with mitochondrial dysfunctions within the oocytes, affecting their ability to develop further. As cytoplasmic factors, including the mitochondria and maternal RNAs and proteins, are crucial for supporting the zygote and embryo development prior to embryonic genome activation (Li et al., 2010; Zhang and Liu, 2015). PNT or ST may be applied with the aim of replacing a patient’s cytoplasm with a donated healthy one. This may ultimately improve preimplantation development, serving as a potential treatment strategy for overcoming poor embryo development (Spikings et al., 2006). Our study, using different mouse models, suggests that both PNT and ST may be effective in rescuing poor embryo development. We primarily reveal that embryo developmental potential was improved following the transfer of 2PN or the meiotic spindle from zygotes or oocytes obtained from aged or very-aged mice into the counterpart cytoplast of young mice. Interestingly, in the very-aged mice, PNT appeared to be more efficient compared to ST. As the spindle-chromosome complex is not surrounded by a nuclear membrane, chromosomes may be more readily lost or damaged during ST (Tachibana et al., 2013). This issue is likely exacerbated in aged and very-aged mouse oocytes, due to the higher rate of spindle abnormalities. It is well established that approximately 50% of human embryos arrest during the first week of in vitro culture (Hardy et al., 2001). However, a proportion of patients recurrently experience higher rates of embryo arrest, and in some instances all embryos across multiple IVF cycles may be affected (Zhang et al., 2016). Animal studies have also revealed that certain inbred mouse strains and F1 hybrids exhibit two-cell block during in vitro development (Shire and Whitten, 1980; Muggleton-Harris et al., 1982). We further performed both PNT and ST to investigate the value of these approaches for embryo arrest. To this end, 2PN or meiotic spindles from zygotes or oocytes obtained from embryo-arrest mice were transferred to non-arrest counterparts. Overall, we observed a significant improvement in embryo development following both PNT and ST, with PNT again proving more efficient. However, to date, experimental evidence from animals and humans for the application of NT treatment for overcoming embryo arrest is still lacking (Craven et al., 2017). Only in one case report, a 30-year-old patient, who experienced two failed IVF cycles due to embryo arrest at the two-cell stage, was reported to obtain a pregnancy after PNT treatment, but without a successful live birth (Zhang et al., 2016). Several cytoplasmic factors, including mitochondria, cytoplasmic organelles, metabolites, maternal RNAs and proteins, are involved in oocyte competence (Reader et al., 2017; Tanaka and Watanabe, 2019). For instance, earlier mouse studies have reported an impaired intracellular calcium homeostasis (regulated by the endoplasmic reticulum) in in vitro aged oocytes (Igarashi et al., 1997; Takahashi et al., 2009). Consequently, as a means of ‘cytoplasmic rescue’, the PNT and ST techniques may have the potential to rejuvenate dysfunctional oocytes resulting from cytoplasmic deficiencies. Nevertheless, as the molecular mechanism of these effects remains unclear, the use of PNT or ST for treating infertility should be approached with caution. Notably, maternal obesity may be a confounding factor in the interpretation of our data, as the body weights of aged and very-aged mice were significantly higher than those of young mice. This is possible because mitochondrial dysfunction and reduced oocyte developmental potential have both been associated with maternal obesity (Igosheva et al., 2010; Wu et al., 2015). In the future, it would be useful to curtail age-related weight gain by providing an enriched cage environment. The embryo aneuploidy rate is known to increase with maternal age, primarily due to maternal meiosis I or II errors arising during oogenesis (Hassold and Hunt, 2001; Nagaoka et al., 2012; Franasiak et al., 2014). In addition, mitotic errors during preimplantation development may lead to the mosaicism within the embryo (Mantikou et al., 2012; McCoy et al., 2015). It has been reported that in older women, the incidence of aneuploidy may exceed 80% (Nagaoka et al., 2012; Franasiak et al., 2014). In contrast, in aged mice, the levels of aneuploidy are generally not as high as those in humans, but vary widely depending on the mouse strain (Camlin et al., 2017). Previous mouse studies assessing oocyte aneuploidy at the MII stage have revealed lower levels of oocyte aneuploidy in C57Bl6/J (17–19 months, 9%) and B6D2F1 mice (16–18 months, 25%), and higher oocyte aneuploidy rates in MF1 (15–17 months, 33%) and CD1 mice (19–25 months, 43%) (Pan et al., 2008; Sebestova et al., 2012; Shomper et al., 2014; Yun et al., 2014). In our study, we used B6D2F1 mice as a model of female reproductive ageing. NGS was applied to assess the prevalence of aneuploidy in individual blastocysts following PNT and ST. Our analysis revealed that the blastocysts obtained following standard ICSI using very-aged mouse oocytes showed an increased rate of aneuploidy, comparable to previous observations regarding oocyte aneuploidy in the B6D2F1 mouse strain (Pan et al., 2008). Surprisingly, however, we observed a reduced rate of aneuploidy in PNT and ST blastocysts generated following the transfer of very-aged PN or spindles into young mouse cytoplasts. These were comparable to ICSI controls (with young mouse oocytes) and those following PNT and ST from young mouse zygotes or oocytes to young mouse cytoplasts. Strikingly, blastocysts generated following PNT and ST from young mouse zygotes or oocytes to very-aged mouse cytoplasts, both showed a high rate of chromosomal aberrations, including whole chromosomal abnormalities, segmental defects and mosaicism. These data support that the oocyte cytoplasm of old mice may contribute to chromosomal aberrations, at least in our mouse model. It is known that abnormalities in both the nucleus and the cytoplasm may compromise oocyte competence (Liu and Keefe, 2004; Conti and Franciosi, 2018). However, PNT and ST only offer to replace a potentially deficient cytoplasm with a more competent one (Wolf et al., 2015). As it is implausible that PNT or ST can correct an abnormal PN or spindle after transferring it into a young mouse cytoplast, our findings imply that maternal age-related blastocyst aneuploidy may arise primarily from mitotic errors following fertilisation. Mitotic errors have been recognised as a feature of preimplantation development, leading to chromosomal mosaicism and structural aberrations (Delhanty et al., 1993, 1997). While this hypothesis is somewhat contradictory to the spindle defects shown in Fig. 5, it may be assumed that a high proportion of uniformly abnormal oocytes following ICSI do not reach the blastocyst stage and were thus not available for analysis. This is in line with the reduced blastocyst rate observed for our very-aged ICSI controls (Table I). Conversely, segregation errors leading to mosaicism and structural defects are less detrimental to preimplantation development in mice (Lightfoot et al., 2006; Mashiko et al., 2020). As such, the abnormalities observed in blastocysts derived from very-aged cytoplasts may be related to a cytoplasmic defect rather than a nuclear one. Previous research suggests that the quality of the cytoplasm, which includes the transcriptome is compromised in aged mouse oocytes. Oocyte obtained from old mice showed perturbations in the expression of genes involved in mitotic cell cycle regulation, as well as spindle assembly checkpoint regulation (Pan et al., 2008). Dysregulation of these processes coupled with the age-related decline in cytoplasmic quality may result in a disruption of homeostasis, leading to chromosomal instability during preimplantation development. A previous study in humans employing ST at the MII stage from in vitro matured (IVM) oocytes into in vivo matured MII oocytes suggests that this technique may have the potential to alleviate ooplasmic deficiency (Tanaka et al., 2009). This work revealed improved fertilisation, cleavage and blastocyst formation rates following ICSI in ST-reconstituted oocytes compared to controls (IVM oocytes). Furthermore, the analysis of two blastomeres of five ST-generated embryos at the eight-cell stage revealed chromosomally normal karyotypes. Nevertheless, the control IVM oocytes showed a low aneuploidy rate of 4.5%, while only a small proportion of the reconstructed embryos were cytogenetically analysed (Tanaka et al., 2009). These results and our findings are in line with those of Mitsui et al. (2009), who reported significantly improved blastocyst rates and development to term following ST from aged mice into young cytoplasts, compared with oocytes reconstructed with young mouse spindles and aged mouse cytoplasts. As no abnormalities were reported in the resultant pups, the authors suggest that nuclear competence (the integrity of chromosome alignment) was normal in the MII oocytes from aged females and that the cytoplasm itself led to the low development rate of aged mouse oocytes (Mitsui et al., 2009). An alternative hypothesis may be that the young cytoplasm can potentially repair spindle defects, given that the ploidy of the abnormal spindles is not affected. The young cytoplasm may replenish key cell cycle checkpoint factors, allowing for potential errors to be more readily identified and corrected, ultimately leading to normal division (Potapova and Gorbsky, 2017). Nevertheless, there is currently no compelling evidence that supports PNT nor ST for correcting aneuploidy associated with maternal ageing or embryo arrest (Tanaka and Watanabe, 2019). Accordingly, a study in mouse indicated that germinal vesicle transfer from aged mouse oocytes to young mouse cytoplasts could not rescue age-related chromosome misalignment in the reconstituted MII oocytes (Cui et al., 2005). Owing to the scarcity of research involving NT techniques and the limited numbers of samples analysed in our study, further work will be necessary to establish the true benefits of PNT and ST for improving cytoplasm quality and reducing chromosomal instability, particularly in humans. Unlike meiotic errors, age-related effects do not appear to influence the occurrence of mitotic errors in humans (Munne and Wells, 2017). Embryos obtained from aged B6D2F1 female mice may be more prone to chromosomal segregation defects during in vitro preimplantation development, acting as the underlying cause of blastocyst aneuploidy in this mouse model. In humans, recent studies have indicated that both PNT and ST procedures using in vivo matured oocytes did not lead to an increased rate of aneuploidy in the generated blastocysts (Hyslop et al., 2016; Kang et al., 2016). Another recent study in humans by our group has also revealed comparable euploidy rates in the generated blastocysts following second polar body transfer (using human IVM oocytes) and ICSI controls (with human IVM oocytes) (Tang et al., 2019). Our results are in line with these findings, indicating that NT techniques themselves may not promote chromosomal instability during preimplantation development. Nevertheless, our conclusion is limited due to a small number of individual blastocysts analysed. Early animal studies have reported the use of CT for oocyte rejuvenation by increasing the intracellular ATP concentrations or preventing apoptosis in mouse oocytes (Van Blerkom et al., 1998; Perez et al., 2000). In cows, oocytes artificially depleted of mitochondrial DNA could be rescued by CT and these gave rise to normal offspring (Chiaratti et al., 2011). However, in our study using CT, the addition of ∼5% cytoplasm from young mice oocytes into the oocytes of aged and very-aged mice did not enhance fertilisation or embryo developmental outcomes. Likewise, supplementation of the embryo-arrest mouse zygotes with a limited cytoplasm from non-arrest mouse zygotes did not improve embryo development. Our data thus suggest that CT is not effective for improving oocyte quality. Further preclinical studies may shed light on the benefit of this methodology in humans. Due to the lack of published studies on the use of these technologies for treating infertility in both animals and humans (Labarta et al., 2019a), it is rather difficult to compare our results with those of others. Additionally, considering that the pattern of centrosome inheritance is different between mouse (maternal inheritance) and humans (paternal inheritance) (Schatten et al., 1991; Schatten and Sun, 2009), our observations should be cautiously extrapolated to humans. Overall, our data attest to the therapeutic potential of PNT and ST for improving oocyte competence and overcoming embryo developmental arrest. However, future research including embryo transfer following PNT and ST treatments will be crucial to ascertain the effects of these techniques on pregnancy outcomes and live birth rates. Further studies will be critical to definitively confirm the efficiency and safety of these methodologies prior to clinical application. Acknowledgements We thank Leen Pieters for her assistance in the HE staining. We thank Dr Elise Vantroys for her kind support by providing us with JC-1 dye. We thank Bieke Bekaert for her assistance in mouse stimulation. Authors’ roles M.T. and B.H. designed and performed the experiments, collected and analysed data and wrote the manuscript. M.P., A.B., B.M. and M.V. performed the data acquisition and analysis. P.S. conducted experiments. P.C., P.D., D.D., D.S. and R.V. conceived, designed and supervised the experiments. All authors contributed to the interpretation of the results and revised the manuscript. Funding This study was supported by grants from the China Scholarship Council (CSC) awarded to M.T. (Grant no. 201506160059), the Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF) awarded to M.T. (Grant no. 01SC2916 and no. 01SC9518), and FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051017N). P.D.S. is holder of a fundamental clinical research mandate by FWO-Vlaanderen. Grants from the Flemish Foundation of Scientific Research (FWO, Grant no. G051516N and G1507816N) were awarded to B.H. Conflict of interest All authors declare no conflict of interest. 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TI - Germline nuclear transfer in mice may rescue poor embryo development associated with advanced maternal age and early embryo arrest
JF - Human Reproduction
DO - 10.1093/humrep/deaa112
DA - 2020-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/germline-nuclear-transfer-in-mice-may-rescue-poor-embryo-development-Qr5rg9Mzxn
SP - 1562
EP - 1577
VL - 35
IS - 7
DP - DeepDyve
ER -