体外受精中3原核胚胎的发育及可利用价值的探讨

目的:探讨体外受精(IVF)中异常的3原核(PN)胚胎的发育及可利用价值。方法:收集IVF治疗周期中废弃的3PN受精卵204个进行体外培养,观察其发育能力,并与同周期的1 138个2PN受精卵进行比较;采用胚胎植入前遗传学筛查(PGS)技术对由3PN发育成的19枚囊胚进行非整倍体分析。结果:3PN组和2PN组的卵裂率无统计学差异(P0.05);但3PN组囊胚形成率显著低于2PN组[9.6%(19/97)vs 37.9%(204/342),P0.01]。整倍体分析显示,10.5%(2/19)的3PN来源的囊胚为正常二倍体核型。结论:3PN受精卵有继续发育能力;囊胚培养和高通量测序可作为有效筛选异常PN受精卵中正常核型胚胎的一种方法。     

PGT周期中0PN和1PN来源胚胎的整倍性结果分析

目的:通过array-SNP技术分析1PN和0PN(2pb)来源的囊胚染色体的整倍性,探讨其移植价值。方法:回顾分析2013年至2018年于北京大学第三医院生殖医学中心行ICSI-PGT助孕,且1pn和0pn(2pb)来源的胚胎有囊胚形成的患者,共92个PGT取卵周期,。结合PGT检测,比较1pn和0pn(2pb)胚胎来源与2PN胚胎来源活检囊胚的染色体整倍性及临床妊娠率等。结果:2PN来源、1PN来源、0PN来源的活检囊胚的染色体整倍体率分别为46.53%(161/346)、63.33%(19/30)、28.21%(22/78)。1PN来源和2PN来源的活检囊的胚染色体整倍体率均高于0PN来源的活检囊胚,差异有统计学意义(P0.05);1PN来源的活检囊胚的染色体整倍体率高于2PN来源的活检囊胚,但差异无统计学意义(P0.05)。0PN与1PN来源囊胚移植的临床妊娠率比较,差异无统计学意义。结论:对于PGT周期患者,行ICSI后的0PN(2pb)以及1PN来源的胚胎培养后形成囊胚,通过芯片技术检测其染色体正常的囊胚仍占一定比例,可作为可移植囊胚进行移植。     

人类常规体外受精中3原核(3PN)受精卵及胚胎的染色体组成

目的:分析人类常规体外受精(in vitro fertilization,IVF)中3原核(tripronuclear,3PN)受精卵及胚胎的染色体组成。方法:收集IVF来源的3PN受精卵及胚胎,与秋水仙素共培养14～20 h后制备染色体。结果:共收集到46枚3PN受精卵,277枚3PN来源的胚胎,核型分析显示:受精卵多为三倍体,胚胎可为近单倍体、近二倍体、近三倍体、多倍体等。结论:人类IVF来源3PN受精卵多为三倍体,3PN胚胎染色体紊乱。     

体外受精周期中不同原核数发育囊胚使用价值的探讨

目的:比较3种受精卵来源胚胎的囊胚形成率和冻胚移植周期的临床妊娠率,初步探讨异常受精来源囊胚的使用价值。方法:体外受精(IVF)和卵胞浆内单精子注射(ICSI)周期中,比较2PN、1PN和0PN来源胚胎的囊胚形成率、优质囊胚形成率和在冻融胚胎移植(CET)周期中的临床妊娠率。结果:IVF周期中,2PN来源胚胎的囊胚形成率与0PN比较,无显著差异(P0.05),1PN来源胚胎的囊胚形成率显著低于0PN、2PN来源(P0.01)。ICSI周期中,1PN、0PN来源胚胎的囊胚形成率均显著低于2PN来源(P0.05);3种受精卵来源的优质囊胚在CET周期的临床妊娠率无显著差异(P0.05)。结论:1PN、0PN来源的囊胚可视为与2PN来源的囊胚同等价值,可在患者接受产前检查的基础上用于移植。     

移植无原核胚胎出生健康子代

目的:观察无原核(PN)胚胎的发生率、影响因素及移植0PN胚胎周期的临床治疗结局。方法:回顾性分析4 424个IVF-ET周期移植0PN来源胚胎的结局。结果:0PN胚胎的发生率为11.3%。女方的年龄、获卵数及优质胚胎率与0PN的发生率相关。70个周期移植了112个0PN来源的卵裂期胚胎,获得的临床妊娠率为24.3%(17/70),植入率为17.0%(19/112),流产率为11.8%(2/17)。1例死产。13个周期活产13个健康子代,未见畸形的发生。对0PN来源的囊胚进行复苏周期移植,可获得与2PN囊胚相似的临床妊娠率及植入率,但流产率较高,差异有统计学意义。结论:IVF周期中,含有0PN优质胚胎的周期可能有较好的临床治疗结局。形态正常的0PN胚胎有较好的胚胎发育潜能,在无2PN胚胎的情况下,可考虑移植0PN来源胚胎。     

受精方式、女方年龄、生育史和取卵数对体外异常受精的影响

目的:探讨人卵子体外受精后单原核(PN)和多原核胚胎形成的影响因素,为提高正常受精率探寻可行的方法。方法:回顾性分析1 004个IVF周期和250个ICSI周期,共计15 364个卵细胞资料,研究胚胎原核形成与体外受精方式、女方年龄、生育史、获卵数的关系。结果:①ICSI周期的1PN率明显高于IVF周期,而3PN和4PN形成率则明显低于IVF(P<0.05)。②当女方年龄>35岁时,1PN、3PN和4PN形成率均显著高于年龄≤35岁者(P<0.05);当女方年龄在28￣35岁时2PN(正常受精率)生成率最高(P<0.05),此年龄段的患者施行ICSI时3PN生成率也最高(P<0.05)。③女方无生育史的1PN形成率上明显高于有生育史患者(P<0.05)。④获取6￣10枚卵组,2PN形成率明显高于1￣5枚卵组和>20个卵组(P<0.05);而对于1PN、3PN、4PN形成率,获11￣15枚卵组明显高于其他获卵数组(P<0.05);而在获卵数>20枚时ICSI组2PN(正常受精率)生成率显著降低。结论:人体外受精正常与否受到受精方式、女方年龄、生育史和取卵数目等因素的影响,因此,在进行辅助生殖技术(ART)时应综合考虑这些因素,有助于提高正常受精率,降低异常受精率。     

101例IVF-ET新鲜周期移植未见原核来源胚胎临床结局分析

目的评估体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)新鲜周期移植完全来源未见原核(non-pronuclear,0PN)胚胎的临床结局。方法回顾分析了2009年1月至2012年9月,在北京大学第三医院生殖中心接受IVF-ET治疗(包括常规IVF和常规ICSI),移植完全来源0PN的胚胎的新鲜移植周期共计101例。结果 101例新鲜移植完全来源于0PN胚胎中,99例移植分裂期胚胎,临床妊娠率为:9.09%(9/99),着床率:7.09%(9/127);截止到2013年1月,妊娠结局:1例胎停育,2例继续,6个健康婴儿出生。移植囊胚2例,没有妊娠。结论 IVF-ET新鲜周期中,在没有正常2PN受精来源的胚胎时,0PN来源的胚胎可用于移植。     

异常受精胚胎临床利用价值探讨

目的:探讨常规体外受精(IVF)和卵胞浆内单精子注射(ICSI)周期中异常受精胚胎的临床利用价值。方法:回顾性分析2013年4月至2015年6月在白求恩国际和平医院生殖中心接受体外受精-胚胎移植(IVF-ET)治疗(包括常规IVF和ICSI)的955个周期的实验室和临床数据。结果:ICSI周期双原核(2 PN)受精率显著高于IVF周期2 PN受精率(88.20%vs 72.04%,P0.01),而ICSI周期的未见原核(0 PN)、单原核(1 PN)及多原核受精率均低于IVF周期(P0.01),差异有统计学意义。第5天(D5)0 PN的囊胚形成率和可利用囊胚率显著高于2 PN和1 PN组(P0.01)。新鲜移植周期和冻融卵裂胚移植周期,0 PN组种植率和临床妊娠率显著低于2 PN组(P0.01),但是冻融囊胚移植周期,0 PN组的种植率和临床妊娠率与2 PN组相比,差异均无统计学意义(P0.05)。结论:IVF/ICSI周期中没有2 PN胚胎可供选择移植时,可选择0 PN胚胎,囊胚培养是合理利用异常受精胚胎的有效手段。     

低质量人类胚胎体外发育至囊胚能力的研究

目的:探讨临床上D3丢弃的低质量胚胎培养至囊胚的发育潜能,为从低质量胚胎获得胚胎干细胞提供依据。方法:收集IVF/ICSI35例,184枚低质量胚胎(包括异常受精胚胎),用序贯法微滴培养,D3～D9连续观察囊胚形成情况。结果:(1)184枚低质量胚胎于D4～D6形成23枚囊胚,囊胚形成率12.5%;(2)胚胎卵裂球数越多,囊胚形成率越高,胚胎级别越低,囊胚形成率越低;(3)3PN胚胎囊胚形成率低于2PN囊胚形成率(14.8%vs15.9%),3PN来源的胚胎具有一定的发育潜能。结论:低质量胚胎具有发育至囊胚的潜能,有可能成为分离胚胎干细胞的材料来源。     

输卵管积水对人早期胚胎体外发育的影响

目的:探讨人输卵管积水(HSF)对人早期胚胎体外发育的影响。方法:因输卵管积水、阻塞行IVF/ICSI-ET患者取卯时穿刺抽吸HSF30例,送细菌培养及成分分析等。同期收集161例行IVF/ICSI-ET患者受精第1日的261枚异常受精卵(1PN,≥3PN),随机分成3组,分别培养于含不同体积分数的HSF培养液中:A组(50%HSF,n=91)、B组(100%HSF,n=89)和C组(对照组,0%HSF,n=81),观察胚胎发育情况,计算卵裂率、优质胚胎率、囊胚形成及孵出率、优质囊胚率等。结果:4例HSF细菌培养阳性(13.33%);HSF中总蛋白及葡萄糖显著低于该患者同目的血清水平。与C组比,未放入卵子的A、B组培养液平衡后,pH值升高,B组渗透压降低(P<0.05)。3组卵裂率、优质胚胎率无统计学差异(P>0.05);囊胚形成率及优质囊胚率A组(15.38%,21.43%)、B组(11.24%,10%)明显低于C组(28.40%,82.61%)(P<0.05)。结论:HSF影响人囊胚形成及囊胚质量,可能与HSF中低葡萄糖、低蛋白、低渗透压、高pH有关,自然状态下可能还与存在一定比例的细菌有关。     

Triploidy after in vitro fertilization: Cytogenetic analysis of human zygotes and embryos

Tripronuclear zygotes obtained from a clinical IVF program were studied cytogenetically. Successful analysis was possible of 42 specimens at the zygote stage and 21 embryos after the first or second cleavage division. In the majority of zygotes (88%) the expected triploidy was confirmed, whereas only 14% of embryos had solely triploid cells. Therefore it is concluded that after tripolar cleavage division, many different types of mosaicism may originate from irregular chromosome distributions. Since the findings in individual blastomeres in embryos resulting from multipronuclear zygotes do not reflect the genetic content of the whole embryo, these embryos are less suitable in a model system for preimplantion diagnosis. The distribution of the sex chromosomal types (XXX, XXY, and XYY) confirmed theoretical expectations. Since in abortion material or in liveborn triploidy cases, the XYY karyotype is hardly ever observed, this indicates that most likely the 69,XYY karyotype has a very high embryonic mortality.     

卵胞质内单精子显微注射后异常受精的影响因素分析

目的:探讨卵胞质内单精子显微注射(ICSI)后异常受精发生的影响因素。方法:回顾性分析299个ICSI周期,按照是否有异常受精分为异常受精组(n=118)和正常对照组(n=181),异常受精组至少发生1个非2原核(2PN)的受精卵。比较分析异常受精组和对照组临床和实验室资料。结果:异常受精组hCG注射日E2水平(14 097±3 066 pmol/L)高于对照组(1 2461±6 836 pmol/L),差异有统计学意义(P<0.05);异常受精组获卵数及成熟卵数分别为17.8±7.2个、15.3±6.1个,多于对照组的13.6±7.0个、10.2±5.3个,差异均有统计学意义(P<0.01);卵子成熟率(81.7%vs76.4%)、2PN受精率(78.4%vs 86.9%),组间比较差异均有统计学意义(P<0.01);而患者年龄、基础激素水平、促性腺激素(Gn)使用总量、精子来源及质量、着床率、临床妊娠率组间比较,差异均无统计学意义(P>0.05)。结论:卵巢对Gn刺激的高反应性可能与ICSI后异常受精的发生有关。异常受精卵的发生虽对临床结局无明显影响,但减少异常受精发生率,增加卵子利用率,可能会提高累积妊娠率。     

电针对重复周期患者卵细胞质量的影响

目的观察控制性促排卵期间电针对重复周期患者卵细胞质量以及胚胎情况的影响。方法将重复控制性卵巢刺激(COS)的患者94例,分为观察组(48例)和对照组(46例)。两组患者再分别按女方年龄进行分组,分为3个亚组:A组(35岁)、B组(35~39岁)和C组(≥40岁)。所有对象均采用改良方案进行COS,观察组在促排卵第1日开始进行电针治疗,直到h CG注射日,选用穴位为子宫、关元、中极、三阴交、太溪;对照组不加用电针干预。结果观察组A组h CG注射日E2峰值、临床取卵数和MⅡ卵子数与自身前一周期和同年龄组对照组相比差异具有统计学意义(P0.05),Gn剂量及使用时间、双原核(2PN)数、获胚数和优质胚胎率则差异无统计学意义(P0.05)。B组和C组与自身前一周期和同年龄组对照组相比则差异无统计学意义。结论电针可改善重复周期患者卵细胞质量,提高卵巢反应性,其效果可能与女方年龄有关。     

Recent cadmium exposure among male partners may affect oocyte fertilization during in vitro fertilization (IVF)

Purpose  

We recently reported evidence suggesting associations between urine cadmium concentrations, reflecting long-term exposure, measured in 25 female patients (relative risk = 1.41, P = 0.412) and 15 of their male partners (relative risk = 0.19, P = 0.097) and oocyte fertilization in vitro. Blood cadmium concentrations reflect more recent exposure.     

IVF和ICSI中多原核受精卵的发育及遗传多态性分析

目的:了解IVF和ICSI治疗周期中多原核受精卵的发育情况及其基因座遗传多态性。方法:分别收集IVF和ICSI治疗中的废弃多原核(≥3PN)受精卵315个和67个,进行体外培养,观察比较其发育能力,并复合扩增多原核来源的1株胚胎干细胞和2个囊胚细胞DNA的15个短串联重复序列(STR)基因座,利用3100遗传分析仪对其进行STR基因座多态性检测。结果:IVF组和ICSI组的多原核受精卵卵裂率无显著性差异(P>0.05),但IVF组胚胎停育率和囊胚形成率显著低于ICSI组(P<0.01)。STR基因座多态性检测显示,IVF组3PN受精卵来源的胚胎干细胞为典型的三倍体特征,但ICSI组4PN受精卵来源的2个囊胚未表现多倍体特征。结论:多原核受精卵有继续发育能力,其中IVF组多原核来源胚胎干细胞表现遗传物质倍性改变,而ICSI组多原核来源囊胚无遗传物质倍性变化。     

The relationship of semen parameters to fertilization in patients participating in a program of in vitro fertilization

Approximately 80% of the patients in a program of in vitro fertilization (IVF) will fertilize an oocyte. The purpose of this study was to determine which parameters of the semen analysis influence fertilization in vitro. Of 120 patients participating in an in vitro fertilization program, 98 achieved fertilization of at least one mature oocyte and 22 did not. Ovulation induction was standardized and patients whose sperm was exposed to at least one mature oocyte (by light microscopy) were included in the study. Semen washing was accomplished using a swim-up technique. Semen parameters were assessed both before (raw) and after washing. Following insemination with 100,000 motile sperm, fertilization was determined by the presence of pronuclei or cleavage. Mean sperm count and motility were higher in patients who fertilized. However, morphology was similar. Fertilization was more likely to occur with a raw density>104 million/ml and a motility>64%, as well as with a density>18 million/ml and a motilkity>86% following washing. Furthermore, washing lowered sperm counts by 75% and increased motility by 25% but had no effect on morphology. This study demonstrates that sperm count and motility, but not morphology, influence fertilization in a program of in vitro fertilization and that patients with higher counts and motility have and greater probability of fertilization.Presented at the Third Annual Congress of Andrology, April 27–May 2, 1985, Boston, Massachusetts.     

Triploid and diploid embryonic stem cell lines derived from tripronuclear human zygotes

Purpose

Human embryonic stem cells (hESCs) are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. The need for new hESC lines motivated our attempts to find a new resource for the derivation of hESC lines. The aim of this work was to establish more hESC lines from abnormal fertilized zygotes and to meet the emerging requirements for their use in cell replacement therapies, disease modeling, and basic research.

Methods

A total of 130 tripronuclear human zygotes was collected 18–20 h post-insemination and cultured in a modified culture medium. The inner cell mass of 12 blastocysts were isolated by a mechanical method in order to establish embryonic stem cell lines.

Results

We established four hESC lines derived from 130 trinuclear zygotes, one of which was triploid and the others were diploid. The efficiency of deriving hESC lines is 3.08 %. The ratio of deriving triploid and diploid hESC lines is 1:3. All of these hESC lines exhibited similar markers of undifferentiated hESCs and had the typical morphology of hESCs, a capacity for long-term proliferation, and pluripotent differentiation potential both in vivo and in vitro.

Conclusions

These abnormal zygotes, which otherwise would have been discarded, can serve as an alternative source for normal euploid hESC lines.     

Changes in the freezing protocol for human zygotes alter embryonic development and pregnancy rates

OBJECTIVE: To determine the effect of various freezing protocols on postthaw development and pregnancy rates resulting from transfer of human zygotes. DESIGN: Prospective study. SETTING: Tertiary care center. PATIENT(S): Couples undergoing assisted reproductive technology (ART) procedures who wished to have their excess zygotes cryopreserved. INTERVENTION(S): We cryopreserved zygotes with one of three protocols. MAIN OUTCOME MEASURE(S): Post-thaw survival and development of the zygotes as well as pregnancy rate after transfer of these zygotes. RESULT(S): A 3-minute hold time after seeding, followed by a final preplunging temperature of -180 degrees C, resulted in a clinical pregnancy rate of 28.6%. In contrast, a 15-minute postseed hold time and a -30 degrees C final chamber temperature resulted in a 37.3% clinical pregnancy rate. When we combined the protocols to provide a 15-minute postseed holding time and a -180 degrees C before plunging into liquid nitrogen, we achieved a 69.6% clinical pregnancy rate. CONCLUSION(S): By increasing the postseeding hold time and decreasing the temperature of the freezing chamber before plunging the zygotes into liquid nitrogen, significant improvements can be made in postthaw development and pregnancy rates.     

Associations between toxic metals in follicular fluid and in vitro fertilization (IVF) outcomes

Purpose

We previously reported associations between trace concentrations of Hg, Cd and Pb in blood and urine and reproductive outcomes for women undergoing in-vitro fertilization (IVF). Here we assess measurements in single follicular fluid (FF) specimens from 46 women as a presumably more relevant marker of dose for reproductive toxicity.

Methods

FF specimens were analyzed for Hg, Cd and Pb using sector field-inductively coupled plasma-mass spectrometry (SF-ICP-MS). Variability sources were assessed by nested ANOVA. Multivariable regression was used to evaluate associations for square root transformed metals with IVF outcomes, adjusting for confounders.

Results

An inverse association is detected for FF Pb and fertilization (relative risk (RR) = 0.68, P = 0.026), although positive for Cd (RR = 9.05, P = 0.025). While no other statistically significant associations are detected, odds ratios (OR) are increased for embryo cleavage with Hg (OR = 3.83, P = 0.264) and Cd (OR = 3.18, P = 0.644), and for embryo fragmentation with Cd (OR = 4.08, P = 0.586) and Pb (OR = 2.22, P = 0.220). Positive estimates are observed for Cd with biochemical (RR = 19.02, P = 0.286) and clinical pregnancies (RR = 38.80, P = 0.212), yet with very low precision.

Conclusions

We have identified associations between trace amounts of Pb and Cd in FF from a single follicle, and oocyte fertilization. Yet, the likelihood of biological variation in trace element concentrations within and between follicles, coupled with levels that are near the limits of detection suggest that future work should examine multiple follicles using a ‘one follicle-one oocyte/embryo’ approach. A larger study is merited to assess more definitively the role that these environmental factors could play with respect to egg quality in IVF programs.     

Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure

Purpose: Fertilization of an egg by injection of a single spermatozoon into the cytoplasm has been shown to be an effective procedure to obtain a pregnancy in human in vitro fertilization. This, as one of the most invasive micromanipulation techniques, has generated concern about inducing embryo abnormalities. The objective of this study was to obtain insight into the chromosomal constitution of zygotes proceeded after the intracytoplasmic sperm injection procedure. Methods: For this purpose the first cleavage division of 33 single- and 16 multipronucleated zygotes proceeded after the intracytoplasmic sperm injection procedure was cytogenetically analyzed. Results: Chromosome spreading permitted adequate karyotyping in 28 (84.8%) single-pronucleated zygotes. Among these, 19 (67.9%) were haploid, 5 (17.9%) hypohaploid (n=20–22), and 4 (14.3%) hyperhaploid (n=24–25). The overall rate of aneuploidy found here for single-pronucleated zygotes was 32.1%. In the 23 (82.1%) analyzed single-pronucleated zygotes, besides single mitotic complements, an intact sperm head or sperm nuclei structure has been found, indicating the maternal origin of these chromosomes. Conclusions: Ten digynic zygotes with three pronuclei and six zygotes with more than three pronuclei were identified after injecting a single spermatozoon, representing 3.2% of all the fertilized oocytes. Aneuploid chromosome complements were found in three of seven tripronuclear zygotes and each one exhibited a hypo-, hyper-, and haploid complement (23,X, 21,X,–A,–B, 25,X,+A.+B; 23,Y, 22,X,–D, 24,X,+D; 23,X, 22,X,–G, 24,X,+G). The absolute difference in the number of chromosomes between each of these two imbalanced corresponding haplotypes was the same and this difference was caused by the chromosomes belonging to the same groups of karyotype. Of the remaining four tripronuclear zygotes, three had triploid and one had diploid numbers of chromosomes. Furthermore, five zygotes having more than three pronuclei at the first cleavage division displayed severely depleted chromosome complements. The majority of imbalanced multipronuclear zygotes was found after delay of the microinjection, suggesting that aging of oocytes might be the reason for their abnormal chromosomal arrangements.