胚胎染色体非整倍体筛查用于平衡易位携带者植入前的遗传学诊断

目的 交信号和1个Y染色体杂交信号者,则诊断为整倍体胚胎;异常杂交信号的胚胎则诊断为非整倍体胚胎.结果 (1)11个平衡易位的PGD周期中,选出杂交信号完整的130个细胞核进行分析,FISH共分析了937个荧光杂交信号,其中整倍体细胞核38个,共有304个杂交信号;其余92个为非整倍体细胞核.(2)在92个非整倍体细胞核中,Ⅰ、Ⅱ及Ⅲ级胚胎的比例分别为20个(22%)、36个(39%)及36个(39%);38个整倍体细胞核中,Ⅰ、Ⅱ及Ⅲ级胚胎的比例分别为13个(34%)、17个(45%)及8个(21%),两者的Ⅰ、Ⅱ及Ⅲ级胚胎数分别比较,差异均无统计学意义(P＞0.05).虽然染色体整倍体率在不同级别胚胎中的分布比较,差异均无统计学意义(P＞0.05),但优质胚胎(Ⅰ级+Ⅱ级)中非整倍体率仍为60%(56/92).(3)平衡胚胎来源的卵裂球细胞核整倍体率(71.4%,30/42)明显高于非平衡胚胎来源的卵裂球细胞核整倍体率(9.1%,8/88),两者比较,差异有统计学意义(P＜0.05).平衡胚胎来源的卵裂球细胞核非整倍体率(包括三体、单体、复杂非整倍体、单倍体、多倍体)明显低于非平衡胚胎来源的卵裂球细胞核非整倍体率(P＜0.05).结论 平衡易位携带者的胚胎中有较高的非整倍体率,因此,胚胎非整倍体筛查在平衡易位携带者的PGD中有重要价值和临床意义.

Abstract：

Objective To determine the importance of aneuploidy screening in preimplantation genetic diagnosis for the couples of chromosome translocation carriers. Methods To perform 11 prenatal genetic disgnosis (PGD) cycles for 7 couples of chromosome translocation carriers from January 2006 to March 2009 in the Reproductive Medical Center, First Affiliated Hospital of Zhengzhou University. To re-analyze the well-fixed, non-multinuclear and non-debris nuclei using the probes of LSI 13, 18, 21,CEPX, CEPY to detect the aneuploidy rate which come from the PGD cycles of the couples of chromosome translocation carriers. The euploid embryo was defined as two fluorescence in situ hybridization (FISH)signals of LSI 13, 18, 21 respectively and two signals of CEPX, or one signal of CEPX and one signal of CEPY. The other abnormal signals were defined as aneuploid embryo. Results (1) A tolal of 130 nuclei from 11 PGD cycles got the integrated re-FISH signals. Nine hundred and thirty-seven FISH signals were analysized, including 304 signals from 38 euploid nuclei and the others from 92 aneuploid nuclei. (2) The number of the aneuploid nuclei from grade Ⅰ , Ⅱ and Ⅲ embryo was 20 (22%), 36(39%), and 36(39%). The number of the euploid nuclei from grade Ⅰ , Ⅱ and Ⅲ embryo was 13(34%), 17(45%),and 8(21%). There was no significant difference of aneupioidy rate between the embryos form different grades (P＞0.05). However, the rate of aneuploid nucleus from good quality embryos (grade Ⅰ + grade Ⅱ) was 60% (59/92). (3) The euploidy rate was 71.4% (30/42) from balanced embryos, while 9.1%(8/88)from unbalanced embryos. There was significant difference between them (x2=53.4, P＜0.05).The rate of aneuploidy from balanced embryos was lower than those from unbalanced embryos (P＜0.05).Conclusions Since higher rate of aneuploidy was detected in embryos of the couples of chromosome translocation carriers. It is advisable to recommend the FISH re-analysis for aneuploidy screening to preimplantation genetic diagnosis for the couples of chromosome translocation carriers.     

染色体易位的植入前遗传学诊断

目的 观察染色体平衡易位和罗伯逊(罗氏)易位基因携带者夫妇进行植入前遗传学诊断(PGD)后的胚胎染色体遗传特征和胚胎着床、妊娠情况,探讨PGD在染色体易位基因携带者夫妇实现正常生育中的意义.方法 用荧光原位杂交(FISH)技术对36对夫妇的胚胎进行PGD,其中14例为染色体平衡易位(平衡易位组),22例为染色体罗氏易位(罗氏易位组),并对诊断结果和胚胎着床、妊娠情况进行分析.结果 36例患者共活检胚胎253个,成功诊断胚胎225个,成功率为88.9%(225/253),获得可供移植的正常或平衡的胚胎共58个.平衡易位组和罗氏易位组PGD后胚胎着床率分别为36%(5/14)和14%(6/44),临床妊娠率分别为4/9和26%(5/19).结论 PGD可有效诊断胚胎染色体平衡易位和罗氏易位,避免反复流产和不必要的非意愿性终止妊娠,并获得理想的胚胎着床率和临床妊娠率.     

减少移植胚胎数量对冻融胚胎移植结局的影响

目的 探讨年龄<35岁妇女,减少冻融胚胎移植数量对移植结局的影响.方法 77例年龄<35岁患者共进行冻融胚胎移植90个周期,采用慢速冷冻快速复苏法对胚胎进行冷冻及解冻,移植胚胎数量分别为2个胚胎(48个周期)和3个胚胎(42个周期),比较其移植结局,包括临床妊娠率、胚胎植入率和多胎妊娠率等.结果 移植2个胚胎患者的胚胎复苏存活率、优质胚胎率、临床妊娠率、胚胎植入率和多胎妊娠率分别为88.9%、89.6%、37.5%、26.0%、38.9%,移植3个胚胎患者分别为88.1%、81.0%、42.9%、18.3%、16.7%;两者间分别比较,差异均无统计学意义(P>0.05).但移植2个胚胎者中无三胎妊娠发生,而移植3个胚胎者中发生2次三胎妊娠.结论 冻融胚胎移植周期中,如果胚胎质量良好,年龄<35岁妇女移植胚胎数量从3个减少至2个,并不会明显降低临床妊娠率,同时可减少三胎妊娠的发生.

Abstract：

Objective To study the impact on pregnant outcome of reducing the number of embryos transferred from three to two in women at age less than 35 who received frozen-thawed embryo transfer (FET). Methods The analysis was performed on 90 FET cycles (77 infertile couples,less than 35 years old) with slow-freezing/rapid-thawing method, including 48 cycles with two embryos transferred and 42 cycles with three embryos transferred. The embryo survival rate, high quality embryo rate, clinical pregnancy rate, implantation rate and multiple pregnancies rate were analyzed. Results No significant differences in embryo survival rate (88.9% versus 88.1%), high quality embryo rate (89.6% versus 81.0%), clinical pregnancy rate (37.5% versus 42.9%), implantation rate (26.0% versus 18.3%) and multiple pregnancy rate (38.9% versus 16.7%) were observed between two and three embryos transferred group (all P > 0.05). However, there were 2 triple pregnancies in three embryos transferred group while none in two embryos transferred group. Conclusion Reducing the number of high quality embryos transferred from three to two in women at age of less than 35 years old who received FET,could decrease the incidence of triple pregnancy and keep the similar clinical pregnancy rate.     

二维电泳分析胎儿酒精综合征斑马鱼模型蛋白表达谱

目的 利用二维电泳分析乙醇诱导斑马鱼胚胎发育畸形的可能分子机制.方法 采用400 mmol/L乙醇在斑马鱼胚胎圆顶期处理3 h,观察乙醇导致的斑马鱼胚胎发育畸形;通过二维凝胶电泳分析受精卵期、圆顶期、胚盾期和5-体节期的胚胎总蛋白,采用差减分析法减少母源性蛋白的干扰,比较5一体节期正常对照组和乙醇处理组斑马鱼胚胎蛋白表达谱的改变;通过原位杂交验证二维电泳的分析结果.结果 400 mmol/L乙醇处理导致62%的胚胎产生体轴发育障碍和60%的胚胎产生独眼畸形.二维电泳分析结果显示,400 mmol/L乙醇处理会导致受精后12 h(12 hours post fertilization,12 hpf)斑马鱼胚胎胶原蛋白2a1和TAR DNA结合蛋白表达分别下调81%和73%.随后原位杂交显示,胶原蛋白2a1在乙醇处理组胚胎体轴的表达明显下调;24 hpf乙醇处理胚胎胶原蛋白2a1在胚胎体轴的表达水平逐渐恢复正常,但神经管结构受到严重影响.结论 乙醇处理后斑马鱼胚胎发育畸形可能是由于乙醇干扰了胚胎发育过程中胶原蛋白2a1和TAR DNA结合蛋白的表达;其中乙醇对胶原蛋白2a1在轴中胚层早期表达的干扰,可能是导致发育后期体轴和神经管畸形的重要原因.

Abstract：

Objective To study the putative mechanisms underlying fetal alcohol syndrome by comparative protein-profile analysis between normal and ethanol-treated zebrafish embryo with twodimensional electrophoresis (2-DE).Methods Zebrafish embryos were exposed in 400 mmol/L ethanol at dome stage for 3 hours,and then ethanol-induced abnormalities were observed.Proteomes of zebrafish embryos at early stages including zygote stage,dome stage,shield stage and 5-somite stage,were separated by 2-DE.The subtraction analysis method was applied to eliminate the interference from maternal derived proteins.The ethanol-treated embryos at 5-somite stage was analyzed by 2-DE,and the protein profile was compared with that generated from control embryos at the same stage.The data obtained from 2-DE analysis were verified by in-situ hybridization.Results 400 mmol/L ethanol treatment caused axial malformation (62%) and cyclopia (60%) in zebrafish embryos.The 2-DE analysis showed that the expression of Collagen2al (Col2a1) and TAR DNA binding protein (TDP) was decreased in 12 hours post fertilization (12 hpf) ethanol-treated embryos by 81% and 73%,respectively.The in-situ hybridization also demonstrated that the expression of Col2al in axial mesoderm was reduced by ethanol treatment at the same stage.But for 24 hpf ethanoltreated embryos,the expression of Col2al in axis recovered to a comparable level to that in control embryos,while the structure of neural tube was disrupted severely by ethanol exposure.Conclusions It is suggested that the expressions of Col2al and TDP were disrupted by ethanol during early stage,which might induce the zebrafish developmental abnormalities.The ethanol interference on early expression of Col2al is supposed to be one of the major reasons leading to later abnormalities of axis and neutral tube.     

荧光原位杂交技术不同方案用于染色体易位携带者胚胎植入前遗传学诊断的效率比较

目的 探讨荧光原位杂交(FISH)技术不同方案用于染色体易位携带者胚胎植入前遗传学诊断(PGD)的效率.方法 根据FISH检测方案的不同进行分组:采用全染色体涂抹探针对染色体易位携带者8个周期的109个卵母细胞第一极体进行诊断(A组),采用联合端粒和着丝粒探针对染色体易位携带者29个周期的357个卵裂球进行诊断(B组),比较两组的活检成功率、固定时细胞丢失率、无核细胞数等.结果 A组的109个卵母细胞中72个受精,受精率为66.1%(72/109),B组的357个卵裂球中304个受精,受精率为85.2%(304/357),A组的受精率显著低于B组,差异有统计学意义(P<0.05).固定时细胞丢失率A组为9.6%(12/104),B组为1.6%(4/252),两组比较,差异也有统计学意义(P<0.05).杂交后核的无信号率A组为11.2%(10/89),B组为3.0%(7/233),两组比较,差异有统计学意义(P<0.05).A组的诊断率为72.5%(79/109),显著低于B组的89.8%(230/256),差异有统计学意义(P<0.05).A组的临床妊娠率和胚胎植入率分别为3/7和22.2%(4/18),均高于B组的30.4%(7/23)和15.7%(8/51),但差异均无统计学意义(P>0.05).结论 FISH两种方案均可有效地进行染色体平衡易位的PGD,卵裂球PGD的诊断效率更高.     

人乳头状瘤病毒与妇科其他常见病原微生物感染关系的调查

Objective To Investigate correlation between screening assay of human papillomavirus (HPV) and microbial pathogens in gynecology. Methods Cervical samples were collected to search for HPV, bacteria and yeast infections in gynecologic outpatients. HPV typing was carried out by PCR and sequencing on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification (SDA) and the other microorganisms were detected by conventional methods. All data were analyzed to investigate the correlation among them. Results In this cross-sectional study, among 857 enrolled outpatients, there were 266 cases with positive HPV DNA, and the rate of infection was 31.0%(266/857). HPV genotype showed that thirty-five different HPV types were identified, of which HPV16 was the most prevalent ( 14.5%, 38/262), followed by HPV58 (9.2%, 24/262), HPV53 (8.0%, 21/262)and HPV42 (6.1%, 16/262); while other genotypes were present in less than 5% of HPV positive women.According to the reclassification, the aggregated percentage (high-risk and probably high-risk) of detected HPV was 58.8% ( 154/262), 27.9% (73/262) for low-risk and 13.4% (35/262) for unknown-risk HPV types. Among HPV positive women, cervical brush specimens results showed that more than 60% cases with normal cytology, 3.8% (10/266) with high-grade squamous intraepithelial lesions (HSIL), 29. 7% (79/266) with low-grade squamous intraepithelial lesions (LSIL) and 3.0% (8/266) with atypical squamous cells of undetermined significance (ASCUS), respectively. Statistical analyses revealed there was a significant association between the infected HPV and Chlamydia trachomatis or Ureaplasma urealyticum ( >10000 CCU/ml; all P <0.01), while no correlation was found between HPV infection and bacterial vaginosis, streptococcus agalactiae, candida, Trichomonas vaginalis or Ureaplasma urealyticum ( ≤ 10000CCU/ml; all P > 0.05 ). Among the cases with bacterial vaginosis, the positive rate of HPV infected was42.6%. Chlamydia trachomatis was one of the high-risk factors for the infection of HPV ( OR = 2.82, 95%CI: 1.74 -4.57). Mycoplasma hominis was isolated only in 2 cases, no patient was infected with Neisseria gonorrhocae. Conclusions Although bacterial vaginosis was not significantly associated with HPV, it was more common among the HPV positive women. There is the significant association between HPV and Chlamydia trachomatis or Ureaplasma urealyticum which may be increase the infection of HPV. These data suggest that it may be important to screen for the simultaneous presence of different microorganisms which may have synergistic pathological effects.     

人乳头状瘤病毒与妇科其他常见病原微生物感染关系的调查

Objective To Investigate correlation between screening assay of human papillomavirus (HPV) and microbial pathogens in gynecology. Methods Cervical samples were collected to search for HPV, bacteria and yeast infections in gynecologic outpatients. HPV typing was carried out by PCR and sequencing on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification (SDA) and the other microorganisms were detected by conventional methods. All data were analyzed to investigate the correlation among them. Results In this cross-sectional study, among 857 enrolled outpatients, there were 266 cases with positive HPV DNA, and the rate of infection was 31.0%(266/857). HPV genotype showed that thirty-five different HPV types were identified, of which HPV16 was the most prevalent ( 14.5%, 38/262), followed by HPV58 (9.2%, 24/262), HPV53 (8.0%, 21/262)and HPV42 (6.1%, 16/262); while other genotypes were present in less than 5% of HPV positive women.According to the reclassification, the aggregated percentage (high-risk and probably high-risk) of detected HPV was 58.8% ( 154/262), 27.9% (73/262) for low-risk and 13.4% (35/262) for unknown-risk HPV types. Among HPV positive women, cervical brush specimens results showed that more than 60% cases with normal cytology, 3.8% (10/266) with high-grade squamous intraepithelial lesions (HSIL), 29. 7% (79/266) with low-grade squamous intraepithelial lesions (LSIL) and 3.0% (8/266) with atypical squamous cells of undetermined significance (ASCUS), respectively. Statistical analyses revealed there was a significant association between the infected HPV and Chlamydia trachomatis or Ureaplasma urealyticum ( >10000 CCU/ml; all P <0.01), while no correlation was found between HPV infection and bacterial vaginosis, streptococcus agalactiae, candida, Trichomonas vaginalis or Ureaplasma urealyticum ( ≤ 10000CCU/ml; all P > 0.05 ). Among the cases with bacterial vaginosis, the positive rate of HPV infected was42.6%. Chlamydia trachomatis was one of the high-risk factors for the infection of HPV ( OR = 2.82, 95%CI: 1.74 -4.57). Mycoplasma hominis was isolated only in 2 cases, no patient was infected with Neisseria gonorrhocae. Conclusions Although bacterial vaginosis was not significantly associated with HPV, it was more common among the HPV positive women. There is the significant association between HPV and Chlamydia trachomatis or Ureaplasma urealyticum which may be increase the infection of HPV. These data suggest that it may be important to screen for the simultaneous presence of different microorganisms which may have synergistic pathological effects.     

子宫内膜异位症患者盆腔粘连与疼痛的相关性

Objective To study the incidence of pelvic adhesions in endometriosis(EM) and the relationship between pelvic adhesions and pain symptoms. Methods The incidence of pelvic adhesions, dysmenorrhea, chronic pelvic pain, dyspareunia, dyschizia in 480 patients with EM were studied retrospectively to evaluate the correlation between pelvic adhesions and the degree of pain symptoms. In accordance with the revised American Fertility Society classification (r-AFS), it was observed that 155 cases were in Stage Ⅰ,33 cases were in stage Ⅱ,108 cases were in stage Ⅲ and 184 were cases in stage Ⅳ. Results (1) Among 480 cases with EM, 72.3% (347/480) of patients have pelvic adhesions simultaneously, of which 25.2% (39/155) patients were in Stage Ⅰ, 78.8% (26/33) patients were in Stage Ⅱ, 90.7% (98/108) patients were in Stage Ⅲ and 100.0% (184/184) patients were in Stage Ⅳ. It was found that positive correlation existed between the degree of pelvic adhesions and clinical staging of EM(rs=0.870,P<0.01).(2) 61.0%(293/480) of patients presented dysmenorrhoea, of which the percentages of mild, moderate and severe dysmenorrhea were 52.2%(153/293), 26.6%(78/293), 21.2% (62/293), respectively. The rate of patients presenting chronic pelvic pain (CPP), dyspareunia and dyschezia were 23.8% (114/480), 15.4% (74/480) and 7.1% (34/480), respectively. (3) Ovarian adhesions was positively correlated with dysmenorrhea and CPP(rs=0.367 and 0.267,P<0.01). Adhesion of the bottom and posterior wall of uterus was positively correlated with dysmenorrhea and CPP (rs=0.336, 0.164,P<0.01); adhesions of fallopian tubes were positively correlated with dysmenorrhea, CPP and dyschezia (rs=0.283, 0.225 and 0.159, P<0.01). Adhesions of rectum was positively correlated with dysmenorrhea (rs=0.101,P<0.05). In addition to dyspareunia, the degree of pelvic adhesions was positively correlated with dysmenorrhea, CPP and dyschezia (rs=0.470, 0.273, 0.132, P<0.01). Conclusion Pelvic adhesions are characteristic lesions of endometriosis, the site and degree pelvic adhesions are closely correlated with pain symptoms.     

子宫内膜异位症患者盆腔粘连与疼痛的相关性

Objective To study the incidence of pelvic adhesions in endometriosis(EM) and the relationship between pelvic adhesions and pain symptoms. Methods The incidence of pelvic adhesions, dysmenorrhea, chronic pelvic pain, dyspareunia, dyschizia in 480 patients with EM were studied retrospectively to evaluate the correlation between pelvic adhesions and the degree of pain symptoms. In accordance with the revised American Fertility Society classification (r-AFS), it was observed that 155 cases were in Stage Ⅰ,33 cases were in stage Ⅱ,108 cases were in stage Ⅲ and 184 were cases in stage Ⅳ. Results (1) Among 480 cases with EM, 72.3% (347/480) of patients have pelvic adhesions simultaneously, of which 25.2% (39/155) patients were in Stage Ⅰ, 78.8% (26/33) patients were in Stage Ⅱ, 90.7% (98/108) patients were in Stage Ⅲ and 100.0% (184/184) patients were in Stage Ⅳ. It was found that positive correlation existed between the degree of pelvic adhesions and clinical staging of EM(rs=0.870,P<0.01).(2) 61.0%(293/480) of patients presented dysmenorrhoea, of which the percentages of mild, moderate and severe dysmenorrhea were 52.2%(153/293), 26.6%(78/293), 21.2% (62/293), respectively. The rate of patients presenting chronic pelvic pain (CPP), dyspareunia and dyschezia were 23.8% (114/480), 15.4% (74/480) and 7.1% (34/480), respectively. (3) Ovarian adhesions was positively correlated with dysmenorrhea and CPP(rs=0.367 and 0.267,P<0.01). Adhesion of the bottom and posterior wall of uterus was positively correlated with dysmenorrhea and CPP (rs=0.336, 0.164,P<0.01); adhesions of fallopian tubes were positively correlated with dysmenorrhea, CPP and dyschezia (rs=0.283, 0.225 and 0.159, P<0.01). Adhesions of rectum was positively correlated with dysmenorrhea (rs=0.101,P<0.05). In addition to dyspareunia, the degree of pelvic adhesions was positively correlated with dysmenorrhea, CPP and dyschezia (rs=0.470, 0.273, 0.132, P<0.01). Conclusion Pelvic adhesions are characteristic lesions of endometriosis, the site and degree pelvic adhesions are closely correlated with pain symptoms.     

子宫内膜异位症患者盆腔粘连与疼痛的相关性

Objective To study the incidence of pelvic adhesions in endometriosis(EM) and the relationship between pelvic adhesions and pain symptoms. Methods The incidence of pelvic adhesions, dysmenorrhea, chronic pelvic pain, dyspareunia, dyschizia in 480 patients with EM were studied retrospectively to evaluate the correlation between pelvic adhesions and the degree of pain symptoms. In accordance with the revised American Fertility Society classification (r-AFS), it was observed that 155 cases were in Stage Ⅰ,33 cases were in stage Ⅱ,108 cases were in stage Ⅲ and 184 were cases in stage Ⅳ. Results (1) Among 480 cases with EM, 72.3% (347/480) of patients have pelvic adhesions simultaneously, of which 25.2% (39/155) patients were in Stage Ⅰ, 78.8% (26/33) patients were in Stage Ⅱ, 90.7% (98/108) patients were in Stage Ⅲ and 100.0% (184/184) patients were in Stage Ⅳ. It was found that positive correlation existed between the degree of pelvic adhesions and clinical staging of EM(rs=0.870,P<0.01).(2) 61.0%(293/480) of patients presented dysmenorrhoea, of which the percentages of mild, moderate and severe dysmenorrhea were 52.2%(153/293), 26.6%(78/293), 21.2% (62/293), respectively. The rate of patients presenting chronic pelvic pain (CPP), dyspareunia and dyschezia were 23.8% (114/480), 15.4% (74/480) and 7.1% (34/480), respectively. (3) Ovarian adhesions was positively correlated with dysmenorrhea and CPP(rs=0.367 and 0.267,P<0.01). Adhesion of the bottom and posterior wall of uterus was positively correlated with dysmenorrhea and CPP (rs=0.336, 0.164,P<0.01); adhesions of fallopian tubes were positively correlated with dysmenorrhea, CPP and dyschezia (rs=0.283, 0.225 and 0.159, P<0.01). Adhesions of rectum was positively correlated with dysmenorrhea (rs=0.101,P<0.05). In addition to dyspareunia, the degree of pelvic adhesions was positively correlated with dysmenorrhea, CPP and dyschezia (rs=0.470, 0.273, 0.132, P<0.01). Conclusion Pelvic adhesions are characteristic lesions of endometriosis, the site and degree pelvic adhesions are closely correlated with pain symptoms.     

Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies

Preimplantation genetic diagnosis (PGD) for translocations has been shown to significantly reduce the risk of recurrent miscarriage, but because the majority of embryos produced are unbalanced, pregnancy rate is relatively low since 20% or more cycles have no normal or balanced embryos to transfer. The purpose of this study was to evaluate whether PGD could improve pregnancy outcome in translocation carriers with a history of two or more consecutive miscarriages and no live births. PGD for translocations was offered to translocation carriers with two or more previous miscarriages (average 3.5) and no live births (0/117 pregnancies) using a combination of distal and proximal probes to the breakpoints. After PGD, only 18.3% of embryos were normal or balanced. Only 5.3% of pregnancies were lost after PGD compared with 100% before PGD (P < 0.001). The cumulative pregnancy rate was 57.6% and the cumulative ongoing pregnancy rate was 54.5% in the short period of time of 1.24 IVF cycles, or 46.3% and 43.9% respectively per cycle. In conclusion, PGD significantly reduced losses and increased the number of viable pregnancies (P < 0.001). IVF plus PGD are a faster method of conceiving a live child than natural conception, at least for translocation carriers with recurrent miscarriages and no previous live births.     

Natural selection between day 3 and day 5/6 PGD embryos in couples with reciprocal or Robertsonian translocations

Purpose

For translocation carriers, preimplantation genetic diagnosis (PGD) provides the opportunity to distinguish between normal/balanced and unbalanced embryos prior to implantation and, as such, increases the likelihood of a successful ongoing pregnancy. The data presented here compares autosomal reciprocal and Robertsonian translocation segregation patterns in day 3 versus day 5/6 IVF-PGD embryos to determine if there is a difference in the chromosome segregation patterns observed at these developmental time points.

Methods

A retrospective analysis on PGD translocation carriers at Monash IVF was performed. Segregation patterns were compared between day 3 and day 5/6 embryos to ascertain whether selection against malsegregants exists.

Results

For reciprocal translocations, 1649 day 3 embryos (139 translocations) from 144 couples and 128 day 5/6 embryos (59 translocations) from 60 couples were analysed. Day 3 segregation analysis showed that 22.3% of embryos were normal/balanced (consistent with 2:2 alternate segregation) and 77.7% were unbalanced (malsegregation). Day 5/6 segregation analysis showed that 53.1% of embryos were normal/balanced and 46.9% were unbalanced. For Robertsonian translocations, 847 day 3 embryos (8 translocations) from 54 couples and 193 day 5/6 embryos (6 translocations) from 31 couples were analysed. Day 3 segregation analysis showed that 38.7% of embryos were normal/balanced (consistent with 2:1 alternate segregation) and 61.3% were unbalanced. Day 5/6 segregation analysis showed that 74.1% of embryos were normal/balanced and 25.9% were unbalanced.

Conclusions

This data demonstrates an increase in the proportion of genetically normal/balanced embryos at day 5/6 of development. This suggests a strong natural selection process between day 3 and day 5/6 in favour of normal/balanced embryos. These findings support performing PGD testing on day 5/6 of embryo development.

     

Efficacy and clinical outcome of preimplantation genetic diagnosis using FISH for couples of reciprocal and Robertsonian translocations: the Korean experience

OBJECTIVE: To evaluate the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridization (FISH) for couples with chromosomal translocations. METHODS: PGD using FISH was performed in 59 cycles of 43 couples with reciprocal translocations, and 11 cycles of 6 couples with Robertsonian translocations. The diagnostic and clinical data were reviewed in a series of 70 treatment cycles of 49 couples from January 2001 to June 2002 at Samsung Cheil Hospital, Korea. RESULTS: A total of 1408 oocytes were retrieved, and 938 (81.7%) out of 1148 matured oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Single blastomere biopsy and FISH analysis were successfully carried out in 99.3% (890/896) and 94.4% (840/890), respectively. Among 193 normal or balanced embryos, 169 embryos were transferred in 64 cycles (91.4% per started cycle). Twenty clinical pregnancies including two ectopic pregnancies and three spontaneous miscarriages (28.6% per started cycle, 31.3% per transfer cycle, 40.8% per couple) were established. Of the three spontaneous miscarriages, one was karyotyped as normal, one had an unbalanced arrangement and one was tetraploid. One case of preterm twin delivery occurred and 16 healthy babies were delivered in 12 single and 2 twin pregnancies. CONCLUSION: The clinical outcome was successful in 28.6% (14/49) of the treated couples with translocations after PGD. The spontaneous abortion rate was significantly reduced from 95.8% (69/72) to 16.7% (3/18) in these couples.     

Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases

The effect of translocations on embryo development was evaluated and results were compared in terms of embryo development with those of embryos obtained from standard intracytoplasmic sperm injection (ICSI) cycles. In 23 translocation carriers with 34 cycles, fertilization, pronuclear morphology scoring (PMS), developmental arrest, cleavage and blastocyst formation were evaluated and compared with embryos obtained from non-translocation cases undergoing ICSI (n = 98 cycles). In 28 cycles, preimplantation genetic diagnosis (PGD) was performed on prezygotes (first and second polar body biopsy for female carriers; n = 3) or on embryos having seven or more blastomeres (blastomere biopsy; n = 25). In six cycles for four couples, probes for translocated chromosomes were not available, so PGD could not be performed. Overall, in translocation cases, a lower fertilization rate, a higher rate of retarded embryo development, and a lower rate of blastocyst formation were observed compared with embryos of non-translocation cases. Fluorescence in-situ hybridization (FISH) analysis showed a 70.9% abnormality rate for reciprocal translocations and 55.0% for Robertsonian translocations respectively. In cases with Robertsonian and reciprocal translocation carriers, the probability of poor embryo development, which may be a result of high segregation abnormalities, may negatively affect the outcome of assisted reproductive techniques. This poor prognosis should also be considered when genetic counselling for translocation is given.     

Clinical outcome of treatment cycles using preimplantation genetic diagnosis for structural chromosomal abnormalities

OBJECTIVES: To explore oocyte recovery, embryo quality, the number of transferable embryos and pregnancy rate after preimplantation genetic diagnosis (PGD) in patients with structural chromosomal aberrations. METHODS: PGD was performed in seven couples with Robertsonian translocations (Rob), eight couples with reciprocal translocations (Rec), two couples with inversions and one couple with a deletion. A total of 43 treatment cycles were carried out. RESULTS: A total of 14.2 oocytes per cycle were retrieved. Fertilisation and cleavage rates were 63% and 58%, respectively. Of the biopsied embryos 20% were transferable. Comparison of the Rob and Rec group revealed no significant differences in number of oocytes, fertilisation or cleavage rates. The number of transferable embryos after biopsy was significantly higher in the Rob group than in the Rec group. When embryo transfer (ET) was performed the pregnancy rate did not differ between the Rob and the Rec groups. Twenty-eight embryo transfers (one or two embryos) were carried out leading to eight clinical pregnancies (29% per ET): two twins, four singletons, one miscarriage and one ectopic pregnancy. All the children are carriers of balanced chromosomal aberrations. CONCLUSION: An acceptable pregnancy rate can be achieved among couples with structural chromosomal abnormalities.     

The application of PGT-A for carriers of balanced structural chromosomal rearrangements

Abstract

The aim of this study was to analyze differences in chromosomal aberrations and euploidy in embryos of each translocation type and gender of carrier in the case series of 10 couples with balanced translocations who underwent IVF with embryos trophectoderm (TE) biopsy and PGT-A to detect chromosomal aberrations. This is a Case Series (Retrospective study). In each case, controlled ovarian hyperstimulation, oocyte insemination with intracytoplasmic sperm injection (ICSI) and cultivation gave multiple blastocysts, that underwent trophectoderm (TE) biopsy with PGT-A analysis using aCGH and NGS. Number of total unbalanced translocations compared to the number of sporadic aneuploid embryos was 39.6% to 39.6% (50% to 50% of all 37 aneuploid embryos). The highest euploidy rate was in male carrier group – 26.7% and the lowest in the Robertsonian translocation carrier group – 18.2%. Sporadic aneuploidy – 68.2% was highest in Robertsonian translocation carrier group and lowest in female group – 11.1%. Chromosomal aberrations related to translocation were highest in female carrier group – 77.8% and lowest in Robertsonian translocation carrier group – 13.6%. Our study showed that expectancy of total embryo aneuploidy rates will be higher in carriers, than in people with normal karyotype. The prevalence of chromosomal aberrations related to translocation was 4.5 times higher in Reciprocal carrier group than in Robertsonian translocation carrier group. Among maternal and paternal carrier groups, the embryos from female carriers had the lowest euploidy rate, unbalanced translocation rate 4.7 times higher than in the male carrier group and higher total aneuploidy rates.     

Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers

We have developed preimplantation genetic diagnosis (PGD) for carriers of chromosomal abnormalities using fluorescent in situ hybridisation (FISH). Here we present the detailed analysis of 64 biopsied, normally developing embryos obtained from four Robertsonian and three reciprocal translocation carriers in 11 treatment cycles of which four resulted in normal pregnancies (three simplex, one duplex). In order to investigate the degree of mosaicism and segregation mode in the embryos, the primary analysis of the biopsied cells was extended with the analysis of all cells from the non-transferred embryos. The analysis also included a second hybridisation with two additional probes, not involved in the translocation (chromosomes 1 and 9), in order to investigate the overall degree of mosaicism. Seventeen out of 64 analysed embryos were balanced for the chromosomes involved in the translocation and 14 of these were transferred. Forty-seven out of 64 embryos (73%) were mosaic regarding the chromosomes involved in the translocation and alternate segregation mode was the most common mode of segregation. Moreover, we have found a higher degree of mosaicism for the chromosomes involved in translocations as compared to control chromosomes. This difference was more pronounced for the embryos from reciprocal translocation carriers. The results, mechanisms, significance and implications of our findings are discussed.     

Preimplantation genetic diagnosis by fluorescence in situ hybridization of reciprocal and Robertsonian translocations

ObjectiveThe presence of reciprocal and Robertsonian chromosomal rearrangement is often related to recurrent miscarriage. Using preimplantation genetic diagnosis, the abortion rate can be decreased. Cases treated at our center were reviewed.Materials and methodsA retrospective analysis for either Robertsonian or reciprocal translocations was performed on all completed cycles of preimplantation genetic diagnosis at our center since the first reported case in 2004 until the end of 2010. Day 3 embryo biopsies were carried out, and the biopsied cell was checked by fluorescent in situ hybridization using relevant informative probes. Embryos with a normal or balanced translocation karyotype were transferred on Day 4.ResultsThirty-eight preimplantation genetic diagnosis cycles involving 17 couples were completed. A total of 450 (82.6%) of the total oocytes were MII oocytes, and 158 (60.0%) of the two-pronuclei embryos were biopsied. In 41.4% of the fluorescent in situ hybridization analyses, the results were either normal or balanced. Embryos were transferred back after 21 cycles. Three babies were born from Robertsonian translocation carriers and another two from reciprocal translocation carriers. The miscarriage rate was 0%. Among the reciprocal translocation group, the live delivery rate was 8.3% per ovum pick-up cycle and 18.2% per embryo transfer cycle. Among the Robertsonian translocation group, the live delivery rate was 14.3% per ovum pick-up cycle and 20.0% per embryo transfer cycle.ConclusionThere is a trend whereby the outcome for Robertsonian translocation group carriers is better than that for reciprocal translocation group carriers. Aneuploidy screening may possibly be added in order to improve the outcome, especially for individuals with an advanced maternal age. The emergence of an array-based technology should help improve this type of analysis.     

Conversion and non-conversion approach to preimplantation diagnosis for chromosomal rearrangements in 475 cycles

Due to the limitations of preimplantation genetic diagnosis (PGD) for chromosomal rearrangements by interphase fluorescent in-situ hybridization (FISH) analysis, a method for obtaining chromosomes from single blastomeres was introduced by their fusion with enucleated or intact mouse zygotes, followed by FISH analysis of the resulting heterokaryons. Although this allowed a significant improvement in the accuracy of testing of both maternally and paternally derived translocations, it is still labour intensive and requires the availability of fertilized mouse oocytes, also creating ethical issues related to the formation of interspecies heterokaryons. This method was modified with a chemical conversion procedure that has now been clinically applied for the first time on 877 embryos from PGD cycles for chromosomal rearrangements and has become the method of choice for performing PGD for structural rearrangements. This is presented within the context of overall experience of 475 PGD cycles for translocations with pre-selection and transfer of balanced or normal embryos in 342 (72%) of these cycles, which resulted in 131 clinical pregnancies (38%), with healthy deliveries of 113 unaffected children. The spontaneous abortion rate in these cycles was as low as 17%, which confirms an almost five-fold reduction of spontaneous abortion rate following PGD for chromosomal rearrangements.