Significant decrease of maternal mitochondria carryover using optimized spindle-chromosomal complex transfer
Xiaoyu LiaoWenzhi LiKaibo LinWeilin JinShaozhen ZhangYao WangMeng MaYating XieWeina YuZhiguang YanHongyuan GaoLeiwen ZhaoJiqiang SiYun WangJiaying LinChen ChenLi ChenYanping KuangQifeng Lyu
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Abstract:
Mutations in mitochondrial DNA (mtDNA) contribute to a variety of serious multi-organ human diseases, which are strictly inherited from the maternal germline. However, there is currently no curative treatment. Attention has been focused on preventing the transmission of mitochondrial diseases through mitochondrial replacement (MR) therapy, but levels of mutant mtDNA can often unexpectedly undergo significant changes known as mitochondrial genetic drift. Here, we proposed a novel strategy to perform spindle-chromosomal complex transfer (SCCT) with maximal residue removal (MRR) in metaphase II (MII) oocytes, thus hopefully eliminated the transmission of mtDNA diseases. With the MRR procedure, we initially investigated the proportions of mtDNA copy numbers in isolated karyoplasts to those of individual oocytes. Spindle-chromosomal morphology and copy number variation (CNV) analysis also confirmed the safety of this method. Then, we reconstructed oocytes by MRR-SCCT, which well developed to blastocysts with minimal mtDNA residue and normal chromosomal copy numbers. Meanwhile, we optimized the manipulation order between intracytoplasmic sperm injection (ICSI) and SCC transfer and concluded that ICSI-then-transfer was conducive to avoid premature activation of reconstructed oocytes in favor of normal fertilization. Offspring of mice generated by embryos transplantation in vivo and embryonic stem cells derivation further presented evidences for competitive development competence and stable mtDNA carryover without genetic drift. Importantly, we also successfully accomplished SCCT in human MII oocytes resulting in tiny mtDNA residue and excellent embryo development through MRR manipulation. Taken together, our preclinical mouse and human models of the MRR-SCCT strategy not only demonstrated efficient residue removal but also high compatibility with normal embryo development, thus could potentially be served as a feasible clinical treatment to prevent the transmission of inherited mtDNA diseases.Keywords:
Oocyte activation
Oocyte activation
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Oocyte activation
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Oocyte activation is a series of events triggered by the fertilizing spermatozoon and necessary for the beginning of the embryonic development. Calcium plays a pivotal role in this process. Here we used confocal laser scanning microscopy to examine the changes in the concentration of intra-cellular free calcium ([Ca2+]) in human oocytes after intracytoplasmic sperm injection (ICSI). The first considerable but short (<2 min) increase in [Ca2+]1 was detected immediately after the penetration of the micro-injection needle into the ooplasm. This rise by itself did not provoke oocyte activation and was also obtained after the injection of medium without spermatozoa. After a lag period of 4–12 h, oocytes that were subsequently activated initiated a second period of [Ca2+]1 changes. These changes were sperm-dependent and followed one of two alternative patterns, a non-oscillatory one and an oscillatory one. The non-oscillatory pattern resembled the changes described previously during parthenogenetic activation of mammalian oocytes. The oscillatory pattern was similar to the changes accompanying normal fertilization in different mammalian species. It is concluded that the initial [Ca2+]1 rise provoked by the ICSI procedure is not responsible for oocyte activation, and that a release of a sperm factor(s) is required to initiate this process.
Oocyte activation
Spermatozoon
Pronucleus
Penetration (warfare)
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Oocyte activation
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The intracytoplasmic sperm injection (ICSI) has significantly improved male factor infertility treatment; however, complete fertilization failure still occurs in 1-5% of ICSI treatment cycles mainly due to oocyte activation failure. It is estimated that around 40-70% of oocyte activation failure is associated with sperm factors after ICSI. Assisted oocyte activation (AOA) as an effective approach to avoid total fertilization failure (TFF) has been proposed following ICSI. In the literature, several procedures have been described to overcome failed oocyte activation. These include mechanical, electrical, or chemical stimuli initiating artificial Ca2+ rises in the cytoplasm of oocytes. AOA in couples with previous failed fertilization and those with globozoospermia has resulted in varying degrees of success. The aim of this review is to examine the available literature on AOA in teratozoospermic men undergoing ICSI-AOA and determine whether the ICSI-AOA should be considered as an adjunct fertility procedure for these patients.
Oocyte activation
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Oocyte activation
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Total or near-total fertilization failure after intracytoplasmic sperm injection (ICSI) is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 (A23187) activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that "rescue oocyte activation" on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.
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Oocyte activation
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Fertilization failure is a serious challenge of assisted reproduction treatment especially for intracytoplasmic sperm injection (ICSI) cycles, in recent years, artificial oocyte activation (AOA) technology had shined a light on this problem. But the mechanism of oocyte activation failure is still a lack of clear research, as AOA cannot recover all types of fertilization failure, the clinical indications and safety of AOA is still the focus of attention. Although the current study did not show the AOA technology could significantly increase their offspring birth defects, this technology should be cautiously used for selected cases with appropriate indications.This article summarized recent progress of mechanisms and types of ICSI fertilization failure, also the application of AOA and its safety, hoping to give a general view of AOA technique for the ART clinicians and embryologists.
Key words:
Intracytoplasmic sperm injection (ICSI); Fertilization failure; Artificial oocyte activation (AOA)
Oocyte activation
Assisted Reproductive Technology
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Intracytoplasmic sperm injection (ICSI) has increased the potential of the assisted reproductive technologies to propagate mammalian species and has provided an opportunity for research into cell cycle control and the mechanisms involved in sperm-induced oocyte activation. We have investigated the efficacy of ICSI in the rhesus monkey, the mechanism of fertilization following sperm injection and the cytoskeletal rearrangement that occurs upon oocyte activation. These studies were conducted on mature, and to a lesser extent, immature oocytes. Ejaculated spermatozoa, washed, capacitated and activated before immobilization, were injected into oocytes using conventional ICSI methodology. Sperm injection into mature oocytes induced oocyte activation (19/22; 86%) and pronuclear formation. In contrast, sham-injected oocytes did not activate readily (2/16; 13%). To localize oocyte activation factor(s), spermatozoa were separated mechanically into heads and tails which were then injected individually into mature oocytes. Activation occurred in 87% (20/23) of oocytes receiving heads. After tail injection, a single microtubule aster was nucleated and one pronucleus (PN) was seen in four of 21 oocytes. Intracytoplasmic injection of sperm extract (SE) resulted in oocyte activation at a significantly higher rate than occurred following sham injection (76 versus 13%). Sperm-induced oocyte activation was also evaluated in immature metaphase (MI) oocytes; activation occurred in 46% (12/26) of cases; however, only 8% of the activated oocytes exhibited 2 PN. Finally, beta-tubulin localization in untreated and taxol-treated oocytes was established as a marker for cytoplasmic changes associated with oocyte activation. These results are consistent with the hypothesis that spermatozoa contain an oocyte activating factor(s) which is primarily localized in the sperm head. Moreover, an activation response is limited to mature oocytes and is accompanied by cytoskeletal changes analogous to those seen following conventional fertilization.
Oocyte activation
Pronucleus
Male pronucleus
Maturation promoting factor
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