Controlled hatching at the prescribed site using femtosecond laser for zona pellucida drilling at the early blastocyst stage

PurposeTo study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability.MethodsMouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5–8.5 μm. Additional longitudinal incisions (5–7-μm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated.ResultsFemtosecond laser–assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening’s small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3–94.7% vs. 83.3–85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups.ConclusionsBlastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.     

新型定量集卵直检技术现场诊断血吸虫病的效果评价

目的　建立一种简便、经济、快速、准确、敏感性高于 Kato- Katz法的新型诊断血吸虫病的方法 ,以便在现场推广应用。　方法　对血吸虫病流行区的 15 9名居民的同一份粪便 ,用新型定量集卵直检法、Kato- Katz法和集卵孵化法同时进行检测。　结果　 3种方法血吸虫卵检出率依次为 2 5 .16 % (40 /15 9)、6 .2 9% (10 /15 9)和 2 5 .79% (41/15 9)。经配对计数资料的卡方检验 ,定量集卵直检法与集卵孵化法之间差异无显著性 (χ2 =0 ,P>0 .0 5 ) ;而定量集卵直检法与 Kato-Katz法之间差异有极显著性 (χ2 =2 8.0 3,P<0 .0 1)。定量集卵直检法镜下检出的虫卵形态不变 ,卵内毛蚴结构清晰 ,很容易辨认。　结论　定量集卵直检法不仅简便、快速、经济 ,而且诊断准确 ,敏感性高 ,明显优于 Kato- Katz法和集卵孵化法 ,值得进一步研究、推广。     

Asymmetrical hatching behaviors: the development of postnatal motor laterality in three precocial bird species

The effects of asymmetrical hatching behaviors on the development of turning bias and footedness in domestic chicks, bobwhite quail, and Japanese quail chicks were examined. Control tests with incubator reared domestic chicks and bobwhite quail revealed significant individual and population left-side turning bias and right footedness. When late stage hatching behaviors were disrupted, population laterality was not evident and individual laterality was reduced. By contrast, Japanese quail chicks demonstrated no population turning bias or footedness and only weak individual biases. Disruption of hatch behaviors further decreased laterality. Examination of discarded eggshells showed significant differences in the degree of rotation made to cut out of the egg by Japanese quail versus domestic chicks and bobwhite quail. Taken together these findings suggest that the counterclockwise hatching behaviors that are characteristic of many precocial bird species serve to facilitate the development of motor laterality at both the individual and population level.     

Ovicidal activity of three insect growth regulators against Aedes and Culex mosquitoes

Interspecific variations in the susceptibility of freshly and embryonated eggs of Aedes albopictus, Ae. aegypti, Ae. atropalpus and Culex pipiens were tested against three classes of insect growth regulators (IGRs) including ecdysone agonist (azadirachtin), chitin synthesis inhibitor (diflubenzuron) and juvenile hormone analog (pyriproxyfen) at 0.001, 0.01, 0.1 and 1.0 ppm concentrations. Egg hatching inhibition was dose dependent, the highest being at 1.0 ppm concentration for freshly laid eggs of Ae. albopictus (pyriproxyfen: 80.6%, azadirachtin: 42.9% and diflubenzuron: 35.8%). Aedes aegypti showed lower egg hatching inhibition when exposed to pyriproxyfen (47.3%), azadirachtin (15.7%) and diflubenzuron (25.5%). Freshly laid eggs of Cx. pipiens were most susceptible to diflubenzuron. Aedes atropalpus eggs were tolerant to all three classes of IGRs. Embryonated eggs of Ae. albopictus, Ae. aegypti, Ae. atropalpus and Cx. pipiens were resistant to pyriproxyfen, azadirachtin and diflubenzuron than freshly laid eggs. The median desiccation time (DT₅₀) of Ae. atropalpus eggs was maximum (5.1 h) as compared to Ae. aegypti (4.9 h), Ae. albopictus (3.9 h) or Cx. pipiens (1.7 h) eggs. Insignificant relationship between the rates of desiccation and egg hatching inhibition suggests other factors than physical providing eggs the ability to tolerate exposures to various IGRs. Egg hatching inhibition was due to the alteration in embryonic development caused by IGRs. Changes in the egg shell morphology and abnormal egg hatching from the side of the egg wall instead of operculum, was observed at higher concentrations of diflubenzuron. Morphological and physiological variations in eggs may be the key factor to influence the ovicidal efficacy of IGRs. The present data provide a base line for the improvement of the ovicidal efficacy of the insecticide and its formulation.     

Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in-vitro fertilization patients

Older patients and those who consistently return for embryotransfer but without implnatation were studied to see if a combinationof day 3 assisted hatching and co-culture (AH +CC) might bebeneficial compared to assisted hatching alone (AH-alone). Femalepatients of 38 years and couples who had previously failedto implant embryos three times or more were prospectively andrandomly assigned to either an experimental or a control group.In the experimental group all embryos were co-cultured on partialmonolayers of bovine oviductal epithelial cells for 2 days followedby assisted hatching by zona drilling (AH +CC). All controlembryos were cultured by standard procedures until day 3 whenthey also underwent zona drilling prior to uterine transfer(AH -alone). With 50 cycles in each group there was unfortunatelya marginal bias against the AH + CC group in that these patientshad undergone a higher number of previous transfer cycles. Therewas a marginally lower percentage of fragmentation and a signficantlyhigher degree of zona thickness variablity in the AH + CC embryogroup. Embryonic implantation was significantly increased (P< 0.05) in the AH xCC group (18%) when compared to the AH-alone group (10%). This difference was reflected in a significantlyhigher (P < 0.05) initial pregnancy rate (52 versus 32%)in the AH +CC group, and a higher (not significant) viable pregnancyrate (38 versus 22%).     

改良激光辅助孵化技术在解冻移植（FET）周期中的应用

目的 探讨改良激光辅助孵化技术（LAH）对冻融胚胎（D3、D5、D6）临床结局的影响。方法 回顾性分析2019 年1 月至2020 年12 月在本生殖中心实施玻璃化冻融胚胎移植的患者共计786 例,其中进行改良激光辅助孵化LAH 338 例,传统激光辅助孵化（LAH）448 例。观察两组受精胚胎的复苏率、胚胎植入率、临床妊娠率、早期流产率、异位妊娠率等临床指标。结果 两组胚胎复苏率、胚胎植入率、临床妊娠率、早期流产率、异位妊娠率分别为100.00%和99.32%、49.35%和42.34%、62.43%和57.81%、9.48%和17.76%、1.42%和2.70%;改良LAH 组的胚胎植入率、临床妊娠率、早期流产率、异位妊娠率和传统LAH 组相比较,差异有统计学意义（P＜0.05）,胚胎复苏率比较,差异无统计学意义（P＞0.05）;改良LAH 组中的囊胚（D5、D6）组临床妊娠率、胚胎植入率与D3 胚胎组比较,差异有统计学意义（P＜0.05）;囊胚（D5、D6）早期流产率、异位妊娠率与D3 胚胎组相比较,差异有统计学意义（P＜0.05）;两组的胚胎复苏率比较,差异无统计学意义（P＞0.05）。结论 改良激光辅助孵化技术比传统激光辅助孵化技术对冻融胚胎（D3、D5、D6）在临床结局上有一定的优化作用。     

江苏省血吸虫病查病质控体系的构建与应用 Ⅱ 县级人员病原学检测能力评估

目的 评价江苏省县级血防机构开展血吸虫病病原学检测水平,提高专业技术人员血吸虫病病原学检测能力,为构建血吸虫病现场查病质控体系提供技术支撑。方法 人工获取家兔血吸虫肝卵,制备成4个不同浓度的虫卵悬液,采用单盲法开展虫卵孵化检测,比较县级工作员检测结果与标准结果的符合率、误检率和漏检率。结果 江苏省28个县（市、区）共检测虫卵悬液560份,检出阳性283份,阴性203份,总符合率为86.79%,总误检率为9.38%,总漏检率为15.77%,检测结果与标准结果差异有统计学意义（[χ2] = 12.99,P < 0.01）。28个县（市、区）中有20个县（市、区）出现了漏检,占71.43%;13个县（市、区）出现了误检,占46.43%。江滩、山丘、水网和湖滩4类地区病原学检测误检率为4.55%～43.75%,差异有统计学意义（[χ2] =30.34, P < 0.01）;漏检率为4.17%～20.45%,差异无统计学意义（[χ2] = 5.09,P = 0.17）。传播控制和传播阻断2类流行区病原学检测误检与漏检率分别为7.50%、13.33%与10.42%、17.13%,差异无统计学意义（[χ2] = 0.229、0.575,P 均> 0.05）。达到血吸虫病传播控制10年及以上和不足10年地区误检与漏检率分别为11.81%、5.00%与16.67%、14.17%,差异无统计学意义（[χ2] = 2.804、2.848,P均 > 0.05）。达到血吸虫病传播阻断10年及以上与不足10年地区误检率分别为11.54%与10.00%,差异无统计学意义（[χ2] = 0.069,P = 0.792）;漏检率分别为10.90%与35.00%,差异有统计学意义（[χ2] = 17.364,P < 0.01）。结论 江苏省县级水平血吸虫病查病工作存在漏检、误检现象,现场病原学检测能力有待进一步提高。     

囊胚玻璃化冻存前激光打孔对冻融后移植效果的影响

背景：一系列研究均表明人工皱缩囊胚腔能明显提高囊胚的玻璃化冻融效果,但在皱缩过程中采用的物理方法和使用材料的不同可能会造成囊胚细胞的机械性损伤。 目的：探讨囊胚玻璃化冻存前激光打孔对冻融后移植效果的影响。 方法：3例不孕患者,平均年龄30岁,于囊胚玻璃化冻存前行激光打孔使囊胚皱缩,平均冻存时间为1年,确定移植前给予解冻。 结果与结论：3例患者中有2例取得了满意的妊娠结局,1例未妊娠。说明采用激光辅助打孔的方法使囊胚皱缩,结合玻璃化冻融后行囊胚移植安全有效。