胎胚移植在育种工作中的作用

胎胚移植在育种工作中的作用现代农业生产具有多元化的特点。除了大型多种经营和专门化的农场外，又创建了农场联合体，小型的合作社和私人农场。畜牧业形成市场经济，确定不同经济效益的家畜必须合乎需求变化的规律。必须快速培育出高产的家畜品种、品系、品族，以符合市...

原因不明不孕患者3周期宫腔内人工授精失败后行体外受精胚-胎移植受精方式的选择

目的探讨原因不明不孕患者3次宫腔内人工授精(IUI)失败后行体外受精胚-胎移植(IVFET)的最佳受精。方法选取2009年5月—2011年12月于唐山市妇幼保健院生殖遗传科因不明原因不孕行IVF-ET的患者160例,其中行单精子注射(ICSI)的90例作为研究(ICSI)组,同期行常规IVF的70例作为对照组,比较二组患者的一般情况及最终结局。结果 ICSI组的受精率、优质胚胎率、妊娠率高于常规IVF组,差异有统计学意义(P<0.05)。结论对于原因不明的不孕患者3周期IUI失败后行IVF选择ICSI治疗的结局较为理想。

手术取卵日偶发ED男性不同取精方式对助孕结局的影响

目的:探讨在体外受精-胚胎移植(IVF-ET)手术取卵(OPU)当天因男方偶发阴茎勃起功能障碍(ED)造成手淫取精困难的最佳处理方式。方法:回顾性分析2011年1月至2014年5月进入IVF周期偶发ED的男性患者93例,分为新鲜精液组45例(A组),提前冻精子组30例(B组),卵子冷冻组18例(C组),其中新鲜精液组又分口服药物(西地那非)组18例(A1组)和未服用药物组27例(A2组),比较各组受精率、优质胚胎率、妊娠率。结果:A组、B组、C组相比年龄、不孕年限、平均获卵数、成熟卵率、卵裂率均无统计学差异(P>0.05)。正常受精率(80.78%vs 80.43%vs 84.77%)、优质胚胎率(53.27%vs 52.97%vs 47.69%)、临床妊娠率(60.00%vs 56.77%vs 44.44%)也无统计学差异(P>0.05),C组3PN率(0.63%)明显低于A组(9.61%)和B组(4.34%)(P<0.05);新鲜精液组中A1组与A2组年龄、不孕年限、平均获卵数、成熟卵率、受精率、卵裂率、优质胚胎率、妊娠率均无统计学差异(P>0.05)。结论:IVF-ET中取卵当天偶发ED男性口服西地拉非有助于手淫取精,不影响助孕结局;对预知取精可能失败的患者可采用提前冷冻精子,取精失败者可采用冷冻卵子,但尽可能使用新鲜精液,避免睾丸附睾穿刺性损伤。

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Diminution of toxic copper accumulation in toxic milk mice modeling Wilson disease by embryonic hepatocyte intrasplenic transplantation

AIM: To observe the therapeutic effect of intrasplenic transplantation with embryonic hepatocytes on amelioration of hereditary copper accumulation in toxic milk (TX) mouse modeling Wilson disease. METHODS: Donor hepatocytes were harvested from 14-d fetal liver of a pregnant homogeneous DL mouse. These cells were successively cultured, labeled with fluorescein dye Hoechst 33342 for 24 h, and sequentially infused into the spleen parenchyma of the recipient TX mice. No host immunosuppression measures were taken. Two and four weeks after transplantation, the recipients were killed for routine histologic investigation and immunohistochemistry study up to 4 wk after transplantation. The serum copper and ceruloplasmin concentrations of the recipient mice were determined by graphite furnace atomic absorption spectroscopy.RESULTS: In the following 2nd and 4th wk after transplantation, the donor hepatocytes could be visualized in the livers of 47.3% recipients. The serum ceruloplasmin and copper concentrations increased by 1.6-fold after 2 wk and 2.0-fold times after 4 wk respectively, which ultimately rose from about 30% of the normal level to nearly 60%(P＜0.01). The hepatic copper concentration decreased 7.2%, 4 wk after transplantation. Pathologic examination showed that there were many actively proliferative hepatocyte precursor cells with specific embryonic hepatocyte marker AFP migrated into hepatic sinusoidsof the recipients. A large number of cells carrying hepatocytes marker and albumin were observed in the recipient spleen tissues.CONCLUSION: Embryonic hepatocytes are capable of differentiating into mature hepatocytes in vivo. After transplantation, the hereditary abnormalities of copper metabolism in TX mice could be corrected partially by intrasplenic transplantation of homogeneous embryonic hepatocytes.

Research Progress on Technique of Frozen Embryo Transfer in Sheep

The paper introduced the research progress on the technique of frozen embryo transfer in sheep,illustrated selection of donors and receptors,superovulation,synchronization of estrus,embryo cryopreservation and embryo transplantation. Frozen embryo transfer in sheep is another breakthrough in the high-quality sheep raising,and this technique in China is in its infancy recommendation stage,but it will be comprehensively popularized in the future.

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.

Establishment of a transplantation tumor model of human osteosarcoma in chick embryo

Objective: We established a transplantation tumor model of human osteosarcoma in chick embryo, studied its morphological and biological characteristics, and observed its dynamic process of angiogenesis induction so that a simple and practical model can be provided for studying osteosarcoma. Methods: Human osteosarcoma cells at different concentrations were inoculated in chorioallantoic membrane (CAM) of chick embryos at different embryonic ages to observe the factors affecting the survival of the transplanted osteosarcoma in chick embryo, growth characteristics of the transplantation tumor, and the morphological characteristics and biological characteristics of the transplantation tumor. Results: The transplantation tumor model of human osteosarcoma in chick embryo was successfully established. It was found that the transplantation tumor was easy to grow and it showed strong angiogenesis-inducing effects. Under the light microscope, the transplantation tumor showed a similar tissue structure to human osteosarcoma. Conclusion: It is feasible to establish a transplantation tumor model of human osteosarcoma in chick embryo. The model can be easily duplicated with a simple operation, which provides a useful animal model for studying osteosarcoma.

Genetically identified complete hydatidiform mole coexisting with a normal fetus following IVF-ET

A complete hydatidiform mole coexisting with a fetus following in vitro fertilization and embryo transfer (IVF-ET) is a rare event. The diagnosis is often not easy because of the morphological similarity to a partial mole, but important to the treatment. We present a recent case in which STR polymorphism analysis clearly revealed a different genetic origin for the fetal and molar parts. STR polymorphisms on 15 variable number tandem repeat loci and a gender-determination locus, which were detected by polymerase chain reaction, indicating that the cord/placenta and molar tissue were parental and androgenous, respectively. During follow-up, the patient developed persistent gestational trophoblastic tumor (GTT) which was successfully treated with chemotherapy. In this case, STR polymorphism analysis exactly diagnosed a twin pregnancy consisting of a complete hydatidiform mole and a fetus.

Factors influencing the outcome of embryo freezing and thawing program

Objective: To investigate the factors that might influence the success of an embryo freezing and thawing program. Method: The relationship between the pregnancy rate in 73 cycles of embryo freezing and thawing program and the following factors was analyzed: maternal age, E 2 level at the time of HCG trigger, embryo storage time, number of thawed embryos transferred, presence of sponsoring embryos and intact embryos. And the survival rate of thawed embryos with different morphology, cell stage and storage time was evaluated. Result: Transfer with three or more than three thawed embryos resulted in pregnancy rates of 38.5% and 35.7%, respectively, compared with 5.3% for transfer of fewer than three embryos. The presence of sponsoring embryos and intact embryos significantly increases pregnancy rate in embryo freezing and thawing program. No other factor examined had any effect on pregnancy outcome. The survival rate of good morphology embryos was higher than poor ones, but was not influenced by cell stage and storage time. Conclusion: Embryo morphology before freezing, number of thawed embryos transferred and the presence of intact embryos are important to the outcome of embryo freezing and thawing program.

High efficiency DNA delivery into swine oocytes and embryos by electronic pulse delivery (EPD)

The production of transgenic swine for xenotransplantation has been proposed as an optimal option to overcome the chronic shortage of human organ donors. Generation of genetically engineered swine has been elusive due to the difficulties in gene transfer. In order to achieve effective gene delivery, a key step for the genetic modification, we applied electronic pulse delivery (EPD) technology to introduce HZKb-DC DNA construct into swine eggs. Using the developed EPD ProtocolsTM, we have achieved good viability of the EPD treated oocytes, satisfactory embryonic development of the EPD treated embryos, and stable DNA transfer into the swine embryos with high efficiency. Thus, application of the EPD technology promises to effectively facilitate the generation of large trangenic mammals.

Future of liver transplantation: Non-human primates for patient-specific organs from induced pluripotent stem cells

Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.

Effect of Gossypol on Bovine Blastocyst Attachment and Trophoblastic outgrowth in Vitro

The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaughtered cows and were matured and fertilized in vitro. Cleaved oocyte were culturedin CRlaa + BOEC and TC-199 + 10% FCS combined in an 1:1 ratio. After 8 days of co-culture,the hatched blastocysts were randomly allotted to different treatment groups. All were cultured ona fetal fibroblast monolaycr (prepared from bovine fetuses) in TC-199 culture medium supplementedwith 10% fetal calf serum (TCFCS). But the groups differed from one another in the dose ofgossypol given: 0.01 μg, 0.1 μg, 1 μg, 10 μs/ml, and no gossypol as control. All cultures wereperformed in 24-well culture plates at 39℃ with 5% CO_2 in air. The results indicate that the ratesof attached and outgrowing blastocysts in the medium containing 1 μg/ml gossypol were significantlylowcr than the control group (p<0.01) and outgrowth were inhibited by gossypol in a dose-dependent manner.

Double Pulmonary Embolism and Left Common Iliac Vein Thrombosis after in vitro fertilization and embryo transfer： a Case Report

This article discusses the mechanism, diagnosis, and treatment of a case of double pulmonary embolism and left common iliac vein thrombosis following in vitro fertilization （IVF） and embryo transfer （ET） to arouse vigilance for unexpected thrombosis after ovarian hyperstimulation and to serve as a clinical reference of this condition. This paper reports the case of a woman with double pulmonary embolism and left common iliac vein thrombosis after IVF-ET and the successful management of this condition. The woman had primary infertility, and underwent IVF-ET with 14 ooeytes recovered and two embryos transferred. The patient suffered breathing difficulty 17 days after the ET, and was hospitalized 6 days later as her condition had exacerbated. Computed tomographic （CT） angiography of pulmonary arteries showed double pulmonary embolism, left common iliae vein thrombosis, abnormal density shadow in uterine appendages, and ascites. A healthy baby boy and girl were safely delivered by Caesarean section after successful treatment. Thrombosis after IVF-ET is an uncommon but life-threatening complication and concurrent pulmonary embolism is even rarer. It is suggested that careful thrombosis risk assessment be taken before therapy and particular attention be paid to patients with special body constitutions to develop thrombosis. Whenever patients experience breathing difficulty after IVF-ET, it is strongly advised to examine the possibility of pulmonary embolism. The main treatments for thrombosis are anticoagulant therapy by low-molecular-weight heparin and thrombolysis by urokinase.

未成熟卵母细胞体外培养治疗多囊卵巢综合征性不孕症的临床研究

目的探讨在自然周期获取未成熟卵母细胞体外成熟技术(IVM)治疗多囊卵巢综合征(PCOS)性不孕症的临床疗效。方法 41例行体外受精-胚胎移植助孕的PCOS患者,共43个周期。获未成熟卵母细胞482枚,进行体外培养成熟后行单精子卵胞浆内注射,受精后取优质胚胎移植宫腔内。统计其获卵率、受精率、卵裂率和妊娠率。结果 43个周期体外培养成熟卵子320枚、成熟率为66.39%;用于受精363枚、受精329枚、受精率为90.63%卵裂279枚、卵裂率为84.80%,其中40个周期进行了胚胎移植,周期取消率为6.97%,19例患者妊娠,移植周期妊娠率44.18%。结论 IVM技术是治疗多囊卵巢综合征性不孕症的有效方法。

Effect of electroacupuncture on oocyte quality and pregnancy of patients with PCOS undergoing in vitro fertilization and embryo transfer

Objective To observe the impacts of electroacupuncture （EA） on oocyte quality and pregnant outcome for the patients with polycystic ovary syndrome （PCOS） undergoing in vitro fertilization and embryo transfer （IVF-ET） and explore its potential mechanism. Methods Sixty-six patients with PCOS and undergoing IVF-ET were divided into two groups randomly, including an observation group （34 cases） and a control group （32 cases）. Ethinylestradiol and cyproterone acetate tablets and gonadotropin-releasing hormone agonist were administered for long-program superovulation in either group. In the observation group, the intervention of EA was applied to Guānyuán （关元 CV 4）, Zhōngjí （中极 CV 3）, Sānyīnjiāo （三阴交 SP 6）, Zǐgōng （子宫 EX-CA 1） and Tàixī （太溪 KI 3） additionally for 30 min, once daily, 1 menstrual cycle before controlled ovarian hyperstimulation （COH） and during COH. The pregnant outcome, evaluation of kidney deficiency syndrome, blood hormone level on the day of human chorionic gonadotropin injection and the concentrations of stem cell factor （SCF） in the serum and follicular fluid on the day of oocyte collection were compared between the two groups. Results The score of kidney deficiency symptoms was reduced remarkably after treatment in either group and the improvement in the observation group was superior to that in the control group （P0.01）. The fertilization rate [（76.25？±？20.33）% vs （66.34？±？15.44）%], cleavage rate [（98.66？±？3.70）% vs （94.47？±？9.45）%] and the rate of high-quality embryos [（60.20？±？22.20）% vs （50.55？±？16.15）%] in the observation group were all superior to those in the control group separately （all P0.05）. Clinical pregnancy rate （46.67%, 14/30） in the observation group was higher than that （37.93%, 11/29） in the control group, but without statistical difference （P0.05）. SCF concentrations in the serum and follicular fluid on the day of oocyte collection in the observation group were higher obviously than those in the control group （both P0.05）. Conclusion Electroacupuncture plays an active role in the pregnant outcomes of PCOS patients undergoing IVF-ET and it can relieve the symptoms of kidney deficiency in terms of TCM and improve clinical pregnant rate. The mechanism is relevant to the overall adjustment of organic endocrinal system and the local micro-environment of ovary and the improvement of oocyte quality through the up-regulation of SCF concentration.

Effects of electro-acupuncture on embryo implanted potential of patients with infertility of different syndromes

Objective To explore the impacts of electro-acupuncture on embryo implanted potential and its molecular mechanism in patients with infertility of different syndromes. Methods Among infertile patients treated with in vitro fertilization and embryo transplantation （IVF-ET） combined with electro-acupuncture, 82 cases of kidney deficiency syndrome （group A）, 74 cases of liver qi stagnation syndrome （group B） and 54 cases of phlegm dampness syndrome （group C） were selected. Patients in three groups all received a long program of ovarian hyperstimulation. Additionally, electro-acupuncture was applied before controlled ovarian hyperstimulation （COH） and in the process of ovarian hyperstimulation. Contents of human leukocyte antigen G （HLA-G） in the serum were determined in three groups respectively on the 2nd day of menstruation （M2） and the day of human Chorionic Gonadotropin （hCG） injection. Content of HLA-G in the culture solution of three groups was examined separately on the day of embryo transplantation. The fertilization rate, implantation rate and clinical pregnancy rate were observed for patients in three groups. Results Contents of HLA-G in the serum on the day of hCG injection and in the culture solution on the day of embryo transplantation in group A and B were significantly higher than those in group C （all P0.05）. However, there was no significant difference with contents of HLA-G in serum on M2 day among three groups. The high quality embryo rate in either group A （73.6%, 352/478） or group B （70.6%, 379/537） was significantly higher than that in group C （54.2%, 208/384） with significantly statistical difference （all P0.01）. But there were no significant differences in clinical pregnancy rate, fertilization rate and cleavage rate among three groups. Conclusion Electro-acupuncture combined could increase contents of HLA-G in the body and the level of HLA-G secreted from embryos of patients in the process of IVF-ET. Eventually, the pregnancy outcome and the pregnancy rate are improved. It showed better clinical effects of electro-acupuncture for patients with kidney deficiency and liver qi stagnation syndromes than those of phlegm dampness syndome.

Effects of Chinese herbs combined with in vitro fertilization and embryo transplantation on infertility:a clinical randomized controlled trial

OBJECTIVE： To assess the effects of using Chinese herbs in assisted reproductive technology. METHODS： Four hundred and thirty-three subjects aged less than 42 years with infertility due to Fallo- pian tube or male-related factors who were willing to undertake in vitro fertilization and embryo trans- plantation were randomly allocated to a Chinese herb intervention group （n=216） or a conventional treatment control group （n=217）. All subjects re- ceived one of four routine ultra-ovulation-promot- ing therapies at the Reproductive Center in the Third Hospital Affiliated to Beijing University ac- cording to their physician＇s assessments. The sub- jects in the intervention group received various Chi- nese herbs depending on their conventional treat- ment. Endometrial thickness, number of acquired eggs, and rates of normal fertility, high-quality em- bryos, biochemical and clinical pregnancy of sub- jects were assessed in both groups.RESULTS： The high-quality embryo rate of 51.9%, biochemical pregnancy rate of 51.0%, clinical preg- nancy rate of 44.2% and endometrial thickness of （10.84± 1.75） mm in the intervention group were all significantly higher than those in the control group [48.7%, 38.9%, 34.8%, and （10.52±1.50） mm, respec- tively; P〈O.05]. The normal fertility rate of 58.5% in the Chinese herb group was also significantly supe- rior to the 54.7% achieved in the control group （P〈 0.01）. There were no statistically significant differ- ences （P〉0.05） in the average number of acquired eggs within a single cycle, incidence of excessive stimulation of ovary, rates of embryo transplanta- tion or early abortion and birth of living babies be- tween the two groups. CONCLUSION： Our findings indicate that Chinese herbs increase endometrial thickness, improve the quality of fertility and embryo, and promote embry- onic nidation, thus enhancing the success rate of in vitro fertilization/intracytoplasmic sperm injec- tion-embryo transplantation cycle. Using Chinese herbs improves the outcomes and safety of assist- ed reproductive technologies.

Effect of maternal age on the outcomes of in vitro fertilization and embryo transfer(IVF-ET)

This is a retrospective,observational study to evaluate the effect of maternal age on the outcomes of in vitro fertilization and embryo transfer(IVF-ET).11830 IVF-ET cycles from 10268 women were included.Four groups of different maternal age periods were compared.The groups were 21 30 years old group(4549 cycles),31-35 years old group(4424 cycles),36-40 years old group(2429 cycles),and over 40 years old group(428 cycles).The mean starting dose of Gn and mean total dose of Gn in each cycle were significantly higher(P<0.01),while the mean retrieved oocyte number was significantly lower(P<0.01) in groups of higher maternal age period than those in each of the lower groups.The biochemical pregnancy rate and the clinical pregnancy rate were significantly lower(P<0.01),while the miscarriage rate was significantly higher(P<0.01) in groups of higher maternal age period than those in the lower groups.No difference was found in two-pronuclear zygotes(2PN) rate and good quality embryo rate among different groups.Birth defect rate was also comparable in the born babies in different groups.In the group with patients' age over 40 years old,the pregnancy rate was 26.87%,the clinical pregnancy rate was 19.39%,while the miscarriage rate after clinical pregnancy was 36.14%.To draw the conclusion,patients with higher maternal age had worse IVF outcomes.In women of fertile age,patients between 20 and 30 years old have the best IVF outcomes.Patients over 40 years old have poor IVF outcome and high miscarriage rate,which suggested the necessity of preimplantation genetic screening(PGS).

A survey of acupuncture effect on the result of invitro fertilization

Objective This study was made to evaluate the effect of acupuncture on fertilization results in the patients undergoing medical treatment with IVF. Methods This study was conducted on 164 infertile patients who had referred to the infertility clinic of Imam Khomeini Hospital in 2009–2010（82 patients in acupuncture group and 82 in control group）. In the acupuncture group, before embryo transfer on oocyte puncture day, 2 days after puncture and one cycle before embryo transfer the patients were put under electroacupuncture on the Bǎihuì（百会 GV 20）, Yāoyángguān（腰阳关 CV 3）, Tàichōng（太冲 LR 3）, Nèiguān（内关 PC 6）, Sānyīnjiāo（三阴交 SP 6）, Guīlái（归来 ST 29）, Zúsānlǐ（足三里 ST 36）, and Hégǔ（合谷 LI 4） for three times each for 25 minutes. The points on ears include nèi Shēngzhíqì（内生殖器 TF 2）, and Shénmén（神门 TF 4）. The control group received no acupuncture. Positive pregnancy test, clinical pregnancy, ongoing pregnancy were compared between the two groups. Results Pregnancy level in the acupuncture group were 34 pregnancies（41.5%） as compared to 21（25.6%） in the control group（P〈0.05）.Observation level of gestational sacs in the 5th week of pregnancy included 31（37.8%） and 17（20.7%） sacs in the acupuncture and control groups, respectively（P〈0.05）.Level of ongoing pregnancies after 12 weeks included 21（21.6%） and 12（14.6%） pregnancies in the acupuncture and control groups, respectively（P〉0.05）. Conclusion Acupuncture will considerably improve the result of IVF as compared to the control group.