The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine ...The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 mg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blas-tocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, re-spectively). The development rates to the blastocyst stage of NT embryos were significantly dif-ferent among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal ovi-duct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo develop-mental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.展开更多
Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for so...Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had notshown difference significantly (P】0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment展开更多
Background： Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization （IVF） therapy, it is not yet clear which blastocyst morphological paralneters best...Background： Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization （IVF） therapy, it is not yet clear which blastocyst morphological paralneters best predict the outcomes of single blastocyst transfer. In addition. most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozcn euploid blastocysts. Methods： The study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking U laivcrsity Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm （TE）. and inner cell mass （ICM） quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between ＂excellent＂, ＂good＂, ＂average＂, and ＂poor＂-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests. Results： For embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%. 50.3%, 50.3% and 33.3%. respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy ratc （x2= 21.28. P = 0.001） and live-birth rate （x2 = 13.50, P 〈 0.001） increased with the overall blastocyst grade. Both rates were significanlly higher after the transfer era blastocyst that exhibited either an A-grade or B-grade TE, and similarly, an A-grade ICM. than after the transfer el a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocysl expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length （years） of infertility history, and infertility type. Conclusions： A higher overall euploid blas展开更多
文摘The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear trans-fer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 mg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blas-tocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, re-spectively). The development rates to the blastocyst stage of NT embryos were significantly dif-ferent among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal ovi-duct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo develop-mental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.
基金This work was supported by the National "863" Project (Grant No. Z21-01- 01).
文摘Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had notshown difference significantly (P】0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment
文摘目的比较研究冻融囊胚移植(frozen-thawed blastocyst transfer,FBT)周期移植第5日(D5)与第6日(D6)高质量囊胚的临床结局差异。方法回顾性分析2013年1月—2017年8月期间在本中心接受FBT的365例共387个周期不孕症患者的临床结局,根据形成扩张囊胚的天数及移植胚胎的个数分4组,分别为第5日双囊胚移植组(double D5 blastocysts transfer group,DET5组)88个周期、第5日单囊胚移植组(single D5 blastocyst transfer group,SET5组)69个周期、第6日双囊胚移植组(double D6 blastocysts transfer group,DET6组)129个周期及第6日单囊胚移植组(single D6 blastocyst transfer group,SET6组)101个周期。结果四组患者的平均年龄、不孕年限、内膜厚度、基础卵泡刺激素(FSH)及冻融胚胎的复苏率比较,组间差异无统计学意义(P>0.05);SET5组的临床妊娠率(62.3%)和种植率(62.3%)显著高于SET6组(46.5%,46.5%)(P均为0.04),与DET5组和DET6组比较差异无统计学意义(P>0.05);DET5组及DET6组的多胎率均为42.6%,分别与SET5组及SET6组(均为0.0%)比较,差异有统计学意义(P均为0.00)。结论 FBT周期选择高质量的D5囊胚进行单胚胎移植,可以确保临床妊娠率和种植率,并显著降低多胎率。
文摘Background： Despite recent advances that have improved the pregnancy success rates that can be achieved via in vitro fertilization （IVF） therapy, it is not yet clear which blastocyst morphological paralneters best predict the outcomes of single blastocyst transfer. In addition. most of the previous studies did not exclude the effect of embryo aneuploidy on blastocysts transfer. Thus, the present study investigated the predictive value of various parameters on the pregnancy outcomes achieved via the transfer of frozcn euploid blastocysts. Methods： The study retrospectively analyzed 914 single euploid blastocyst transfer cycles that were performed at the Peking U laivcrsity Third Hospital Reproductive Medical Center between June 2011 and May 2016. The expansion, trophectoderm （TE）. and inner cell mass （ICM） quality of the blastocysts were assessed based on blastocyst parameters, and used to differentiate between ＂excellent＂, ＂good＂, ＂average＂, and ＂poor＂-quality embryos. The relationship between these embryo grades and the achieved pregnancy outcomes was then analyzed via the Chi-square and logistic regression tests. Results： For embryo grades of excellent, good, average and poor, the clinical pregnancy rates were 65.0%. 50.3%, 50.3% and 33.3%. respectively; and the live-birth rates were 50.0%, 49.7%, 42.3% and 25.0%, respectively. Both the clinical pregnancy ratc （x2= 21.28. P = 0.001） and live-birth rate （x2 = 13.50, P 〈 0.001） increased with the overall blastocyst grade. Both rates were significanlly higher after the transfer era blastocyst that exhibited either an A-grade or B-grade TE, and similarly, an A-grade ICM. than after the transfer el a blastocyst that exhibited a C-grade TE and/or ICM. The degree of blastocysl expansion had no apparent effect on the clinical pregnancy or live-birth rate. All odds ratio were adjusted for patient age, body mass index, length （years） of infertility history, and infertility type. Conclusions： A higher overall euploid blas
