Barritt J A, Brenner C A, Willadsen S, Cohen J
Gamete and Embryo Research Laboratory, The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, New Jersey 07052, USA.
Hum Reprod. 2000 Jul;15 Suppl 2:207-17. doi: 10.1093/humrep/15.suppl_2.207.
Our research has focused on promoting the development of compromised embryos by transferring presumably normal ooplasm, including mitochondria, to oocytes during intracytoplasmic insemination. Because of the enigma of mitochondrial heteroplasmy, the mixing of populations of oocyte cytoplasm has provoked considerable debate. We are currently investigating oocyte mitochondrial (mt) DNA mutations and the effects of ooplasmic transplantation on mitochondrial inheritance and mitochondrial functionality. Ageing human oocytes could accumulate mtDNA deletions, which might lead to detrimental development. Elimination of abnormal, rearranged mtDNA, such that the offspring inherit only normal mitochondria, is postulated to occur by a mtDNA 'bottleneck'. Among compromised human oocytes (n = 74) and early embryos (n = 137), investigations have shown the occurrence of deltamtDNA4977, the so-called common deletion, to be 33% among oocytes and 8% among embryos. Using a nested polymerase chain reaction (PCR) strategy of long followed by short PCR, another 23 novel mtDNA rearrangements were found: various rearrangements were present in 51% of the oocytes (n = 295) and 32% of early embryos (n = 197). The difference in the percentage of mtDNA rearrangements between oocytes and embryos was significant (P < 0.0001) and implies that there could be a process of selection as fertilized oocytes become embryos. There was no significant relationship between the percentage of human oocytes or embryos that contained mtDNA rearrangements and age. The first series of ooplasmic transfers have been performed in women with repeated implantation failure associated with slow and morphologically abnormal development of their embryos. In a total of 23 attempts in 21 women, eight healthy babies have been born and other pregnancies are ongoing. By examining the donor and recipient blood samples it is possible to distinguish differences in their mtDNA fingerprint. A small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (six out of 13), amniocytes (one out of four), placenta (two out of four), and fetal cord blood (two out of four). Ooplasmic transfer can thus result in sustained mtDNA heteroplasmy representing both the donor and recipient.
我们的研究重点是，在胞浆内受精过程中，通过将包括线粒体在内的推测正常的卵质转移到卵母细胞中，来促进受损胚胎的发育。由于线粒体异质性的谜团，卵母细胞细胞质群体的混合引发了相当多的争论。我们目前正在研究卵母细胞线粒体（mt）DNA突变以及卵质移植对线粒体遗传和线粒体功能的影响。衰老的人类卵母细胞可能会积累mtDNA缺失，这可能导致有害的发育。据推测，通过mtDNA“瓶颈”可消除异常、重排的mtDNA，从而使后代仅继承正常的线粒体。在受损的人类卵母细胞（n = 74）和早期胚胎（n = 137）中进行的研究表明，所谓的常见缺失deltamtDNA4977在卵母细胞中的发生率为33%，在胚胎中的发生率为8%。采用先长后短的巢式聚合酶链反应（PCR）策略，又发现了23种新的mtDNA重排：51%的卵母细胞（n = 295）和32%的早期胚胎（n = 197）中存在各种重排。卵母细胞和胚胎之间mtDNA重排百分比的差异具有显著性（P < 0.0001），这意味着随着受精卵发育成胚胎，可能存在一个选择过程。含有mtDNA重排的人类卵母细胞或胚胎的百分比与年龄之间没有显著关系。已对胚胎发育缓慢且形态异常、反复植入失败的女性进行了首批卵质转移。在21名女性的总共23次尝试中，已出生8名健康婴儿，其他妊娠仍在进行中。通过检测供体和受体的血样，可以区分它们mtDNA指纹的差异。在以下样本中检测到了一小部分供体线粒体DNA，频率如下：胚胎（13个中有6个）、羊膜细胞（4个中有1个）、胎盘（4个中有2个）和胎儿脐带血（4个中有2个）。因此，卵质转移可导致代表供体和受体的持续mtDNA异质性。
