植入前遗传学诊断/筛查技术指征进展

植入前遗传学诊断/筛查(PGD/PGS)技术发展多年,其指征始终存在争议。PGD指征较为明确,单基因遗传病、染色体异常人群、人类白细胞抗原(HLA)配型为其适用人群。PGS的指征争议较多,主要面向反复流产、反复植入失败、高龄人群,目的是提高妊娠率及活产率。然而第一代PGS技术[PGS#1,卵裂球活检及荧光原位杂交(FISH)-PGS]技术未显示明显效果,甚至降低了妊娠率及活产率。第二代PGS技术(PGS2.0)增加了严重男性因素不育为指征,其核心为囊胚活检及全染色体筛查(CCS),对上述人群的临床效果较为明显,降低了流产风险并提高了成功率及活产率。PGS2.0已极大地改变了辅助生殖技术(ART)面貌,可能成为未来生殖中心对所有患者的一个常规项目。目前仍然需要多中心前瞻性随机病例对照研究重新评估PGS。     

IVF-ET周期中1原核(PN)及0PN(2Pb)胚胎的染色体组成分析

目的:初步探寻体外受精-胚胎移植(IVF-ET)周期中高发育潜能的1原核(PN)及0PN(2Pb)胚胎的方法。方法:观察787个IVF周期患者的PN及卵裂情况,培养至第6日,观察其囊胚形成情况。取囊胚滋养层细胞活检,采用胚胎植入前遗传学筛查(PGS)技术检测0PN(2Pb)、1PN及2PN囊胚的染色体组成。结果:787个IVF周期共获卵8 352枚,2PN、0PN(2Pb)、1PN胚胎的形成率分别为64.35%、4.93%、3.65%,囊胚形成率分别为45.71%、30.03%、18.18%。PGS结果显示,1PN及0PN(2Pb)囊胚染色体正常的比例明显低于2PN囊胚,差异有统计学意义(P0.05)。0PN(2Pb)囊胚的染色体正常比例较1PN囊胚的略高,差异无统计学意义(P0.05)。此外,发育至第6日的0PN(2Pb)囊胚及1PN囊胚较第5日胚胎的正常染色体比例高。结论:对于本周期无可利用胚胎的患者,建议移植1PN及0PN(2Pb)来源的第6日评分较好的囊胚。     

滋养层细胞活检法分析人废弃胚胎源性囊胚发育与染色体异常

目的:探讨年龄、卵裂期胚胎碎片、囊胚生长速率与形态对染色体异常的影响。方法:收集226例患者授精培养后第3日不适于移植和冷冻的正常受精来源的废弃胚胎988枚,激光辅助打孔后,依年龄和胚胎碎片比率分组序贯培养至囊胚期。待第5～7日囊胚形成且滋养外胚层细胞脱出2～9个时活检,同时评估囊胚生长速率与形态;机械法分离活检后存活囊胚的内细胞团细胞,应用13q14.2,21q22.13特异位点探针行FISH检测,分析染色体异常发生情况。结果:FISH检测99枚囊胚,其中58枚(58.6%)存在染色体异常;当患者年龄≥35岁时,其囊胚染色体异常风险增高;尤其胚胎碎片比率>25%者,形成的囊胚形态较差且染色体异常程度复杂(P<0.05)。随囊胚生长速率的延缓与形态分级的降低,染色体异常发生率增高。滋养层细胞活检后囊胚均存活,在分离内细胞团的36枚囊胚中,滋养层与内细胞团细胞的FISH荧光信号吻合率为91.7%。结论:体外受精治疗周期部分废弃胚胎可发育至囊胚,但由于受年龄、胚胎碎片等因素影响,囊胚染色体异常率较高,可能导致胚胎发育延迟且囊胚形态较差;FISH检测囊胚滋养外胚层细胞法可评估胚胎的染色体组成。     

Ⅱ、Ⅲ级胚胎活检后体外发育能力的研究

目的:探讨形态学评价质量较差的胚胎有无活检价值,活检后能否正常发育。方法: 160个IVF和ICSI周期剩余的Ⅱ级、Ⅲ级胚胎,来自同一例患者分级相同的胚胎配对,随机分为实验组和对照组。实验组采用喷酸透明带打孔法取出1个卵裂球,对照组继续培养,对比两组囊胚的形成率、孵化率、囊胚细胞总数,并观察不同形态学特征的胚胎活检后的发育情况。结果:活检组胚胎囊胚形成率,囊胚总细胞数与对照组相比没有差别,但囊胚第6天孵出率活检组明显高于对照组;Ⅲ级胚胎活检后囊胚形成率、第6天孵化率、囊胚总细胞数均明显低于Ⅱ级胚胎;活检前细胞数少于7个的胚胎,活检后发育能力显著降低。结论:活检不影响胚胎体外继续发育的能力;Ⅱ级胚胎活检后具有较好的发育潜力,Ⅲ级胚胎活检后囊胚形成率较低,即使形成囊胚,细胞数也较少,不能正常发育;第3天活检时具有7个或7个以上卵裂球的胚胎具有较好的继续发育能力。     

GnRH-a联合低剂量HCG诱发卵母细胞成熟在行PGD/PGS助孕患者中的应用

目的:探讨促性腺激素释放激素拮抗剂(GnRH-ant)方案中促性腺激素释放激素激动剂(GnRH-a)联合低剂量绒促性素(HCG)扳机对行胚胎植入前遗传学诊断/筛查(PGD/PGS)助孕患者促排卵的效果。方法:回顾性分析2015年1月至2016年3月在我院因女方染色体异常行GnRH-ant方案中GnRH-a联合低剂量HCG双扳机诱导卵泡成熟的PGD/PGS助孕患者79例(A组),根据年龄、抗苗勒管激素(AMH)、基础卵泡刺激素(FSH)匹配选取行拮抗剂方案促排卵并单纯使用HCG扳机诱导卵泡成熟患者79例(B组)作对照,比较两组促排卵特点及促排卵结局。结果:两组促性腺激素总量、促排天数、HCG日雌二醇(E2)、HCG日孕酮(P)、HCG日黄体生成素(LH)、回收卵数、2个原核(2PN)数、第3天(D3)胚胎数、活检正常胚胎数、新发异常率差异均无统计学意义(P0.05)。与B组相比,A组获成熟卵数、D3优质胚胎数、形成囊胚数、优质囊胚数及优质囊胚率明显升高(P0.05),检测后正常的胚胎数虽然两组差异无统计学意义,但A组有升高趋势,两组OHSS发生率无明显差异(P0.05)。结论:GnRH-ant方案中GnRH-a联合HCG诱发卵母细胞成熟改善了行PGD/PGS助孕患者促排结局。     

废弃胚胎继续囊胚培养研究

目的:探讨体外受精治疗周期中废弃胚胎的体外发育潜能。方法:通过囊胚序贯培养法将无原核(0PN)、单个原核(1PN)、多个原核(≥3PN)和卵裂期发育延缓的2原核(2PN)废弃胚胎培养至囊胚期。比较不同来源胚胎囊胚形成情况和d3胚胎的卵裂球数、质量分级等;并利用废弃胚胎囊胚形成情况对体外受精妊娠结局进行预测。结果:共收集801个废弃胚胎,经序贯培养,形成209个囊胚(26.09%),其中58个为优质囊胚(27.75%)。1PN胚胎、0PN胚胎、d3卵裂球数为7-9-细胞胚胎、d3评分为Ⅰ-Ⅱ级胚胎以及卵裂球数为偶数的胚胎囊胚形成率较高(P均<0.05)。废弃胚胎中有囊胚形成者的临床妊娠率明显高于无囊胚形成者(P<0.05)。结论:废弃胚胎有不同程度的发育潜能,部分可发育为囊胚;特别是:①0PN和1PN胚胎;②d3的4-9-细胞胚胎,偶数卵裂球者更佳;③I级和Ⅱ级胚胎。     

微阵列技术在植入前遗传学筛查领域中的应用

胚胎植入前遗传学筛查(preimplantation genetic screening,PGS)是一种低风险的植入前遗传学诊断(preimplantation genetic diagnosis,PGD)。如今各种技术方法的不断涌现并应用于临床PGD中,大大增加了诊断的准确性,降低了误诊风险。而近几年微阵列技术如阵列比较基因组杂交(aCGH)和单核苷酸多态性阵列(SNP)已应用于临床PGS研究中,该项技术突破了以往经典遗传学检测技术如FISH等的诸多限制,能够在全基因组范围内同时检测多种因染色体失衡导致的疾病、微重复、微缺失等,检测结果更加精确、敏感,并能检测到≥10%水平的嵌合体。基于其各种优点,可见微阵列技术在胚胎PGS中具有重要的应用前景。     

微阵列比较基因组杂交胚胎植入前遗传学筛查技术在复发性流产患者中的应用研究

目的:探讨微阵列比较基因组杂交胚胎植入前遗传学筛查(aCGH-PGS)技术对复发性流产(RM)患者辅助生殖临床结局的影响。方法:54例RM患者分为aCGH-PGS组(n=25)与非PGS组(n=29)。aCGH-PGS组胚胎行aCGH筛查,选择染色体整倍性胚胎植入,其临床结局与非PGS组相比较。结果:aCGH-PGS组中染色体整倍性胚胎占26.93%(42/156);平均移植胚胎1.3±0.8个,低于非PGS组2.6±0.6个(P0.01);胚胎着床率(57.58%)与临床妊娠率(61.90%)高于非PGS组(27.03%,P0.01;46.43%,P0.05),早期流产率(7.69%)低于非PGS组(38.46%,P0.01)。结论:经aCGH-PGS,植入1~2个染色体整倍性胚胎,可使RM患者获得更高的胚胎着床率及较低的早期流产率,是RM患者临床治疗的有效途径。     

胚胎植入前遗传学诊断及筛查中7-细胞来源与9-细胞来源囊胚胚胎正常率和临床妊娠率的比较

目的研究在胚胎植入前遗传学诊断及筛查(PGD/PGS)中7-细胞来源与9-细胞来源囊胚的胚胎正常率和临床妊娠率,探讨胚胎体外培养第3日时的最适分裂速度。方法回顾性分析2014年4月1日—2015年12月31日期间207例PGD/PGS治疗周期的临床资料。比较7-细胞来源与9-细胞来源囊胚的胚胎正常率和临床妊娠率。结果 7-细胞来源与9-细胞来源囊胚的胚胎正常率分别为34.38%和52.63%,两者比较差异有统计学意义(c2=4.100,P=0.042 9)。7-细胞来源与9-细胞来源囊胚的临床妊娠率分别为55.56%和57.14%,两者比较差异无统计学意义(c2=0.004,P0.05)。结论在PGD/PGS中,9-细胞来源囊胚的胚胎正常率比7-细胞来源囊胚高,移植的两类胚胎能够获得类似的临床妊娠率。在体外培养第3日选择胚胎移植时,如果胚胎的分级相同,那么9-细胞胚胎优于7-细胞胚胎。     

植入前遗传学筛查技术争议与重新认识

植入前遗传学筛查（PGS）已开展多年,然而这一技术在适应证、临床效用、安全性和伦理管理等方面一直存在争议。随着胚胎实验室技术、单细胞分子遗传学技术和方法的不断发展与进步,胚胎活检创伤性减小,PGS检测时间缩短,准确性提高,为其临床应用的推广奠定了基础。因此,有必要对PGS进行重新认识和评估。     

A review of, and commentary on, the ongoing second clinical introduction of preimplantation genetic screening (PGS) to routine IVF practice

Purpose

Current re-introduction of “improved” preimplantation genetic screening (PGS#2) raises the question whether PGS#2 is ready for routine clinical application.

Methods

We assessed available evidence via review of published data for years 2005–2012, and review of currently ongoing registered clinical trials, based on searches under appropriate key words in PubMed, MEDLINE, Cochrane Database System Review and Google Scholar and http://www.ClinicalTrials.gov. In absence of prospective clinical trials, and due to limited available data, individual publications/ongoing studies are assessed.

Results

PGS#2 offers significant improvements in accuracy of aneuploidy diagnosis over PGS#1. By moving embryo biopsy from day-3 after fertilization (6–8 cell stage) to trophectoderm biopsy at blastocyst stage (day 5–6), PGS#2, however, adds additional co-variables to the analysis of efficacy of the procedure, which have special relevance for women with diminished ovarian reserve (DOR), who usually produce small egg and embryo numbers. Limited published data, claiming efficacy of PGS#2, as well as ongoing clinical trials, do not consider these additional co-variables, do not analyze outcomes by intent to treat and, therefore, have to be considered biased in patient selection.

Conclusions

Here reached conclusions are based on absence of adequate data rather than affirmative outcome assessments. They, therefore, are subject to change at any future date with generation of significant new data. Premature introduction of PGS#1 caused significant damage to patients. As currently no reliable PGS#2 data are available to suggest improvements in IVF outcomes, to avoid a repeat of the PGS#1 experience, PGS#2 should be considered experimental until data show otherwise.     

How can we improve current blastocyst grading systems?

PURPOSE OF REVIEW: To give an overview of the current status and future directions of blastocyst transfer and outcome with particular focus on markers of blastocyst quality and their relationship with implantation. RECENT FINDINGS: In addition to morphological markers, future embryo grading systems, in general, and blastocyst grading systems, in particular, will be based upon metabolic, genetic and epigenetic markers that will increase their efficiency. Metabolic markers such as soluble human leukocyte antigen-G, analysis of specific gene mutations in the trophectoderm by real-time multiplex polymerase chain reaction and analysis by microarray of differential gene expression will be operational in the near future for accurate blastocyst grading and selection. SUMMARY: Gamete and embryo quality as well as culture conditions affect blastocyst formation and quality. Characteristics of the zygote and the cleavage-stage embryo determine the developmental potential of the embryo up to the blastocyst stage. There appears to be a strong relationship between blastocyst quality and implantation. Accurate grading is important for selection of the most implantation-competent blastocyst. Similar to grading systems used in the cleavage-stage embryo, current blastocyst grading systems are mainly based upon morphological characteristics. Incorporation of metabolic, genetic and epigenetic markers will undoubtedly improve the selection process, making it possible to transfer a single blastocyst yielding high pregnancy rates.     

Developmental control of human preimplantation embryos: A comparative approach

The period for which oocyte-derived factors are engaged in the control of human embryonic development involves at least the first four cell cycles after fertilization. The maternal-embryonic transition in human 8-to 16-cell embryos is a relatively vulnerable process, the failure of which entails developmental arrest of the given blastomere. The very early cellular differentiative events in human embryos, including blastomere surface polarization and segregation of the inner cell mass and trophectoderm cell lineages, appear to be dependent largely on the maternal genetic program. However, the embryonic genome is required for the formation of the blastocyst cavity, which is necessary to allow further differentiation of the first two embryonic tissues. Blastomeres with major developmental defects are removed by fragmentation and their loss is compensated by proliferation of remaining normal blastomeres. This mechanism is also mainly responsible for the regulation of ploidy through elimination of aneuploid blastomeres. The data presented suggest that embryos of individual mammalian species may differ in the timing of relevant developmental changes at the cellular and molecular levels. This should be taken into account when findings obtained on embryos of one species are used to anticipate the behavior of embryos of another species under identical conditions.     

Should every embryo undergo preimplantation genetic testing for aneuploidy? A review of the modern approach to in vitro fertilization

Aneuploid conceptions constitute the majority of pregnancy failures in women of advanced maternal age. The best way to combat age-related decline in fertility is through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A allows for better embryo selection, which improves implantation rates with single embryo transfer and reduces miscarriage rates. Single embryo transfers decrease multiple gestations and adverse pregnancy outcomes such as preterm or low birth weight infants. Advancements in extended embryo culture, blastocyst biopsy techniques, and 24-chromosome aneuploidy screening platforms have made PGT-A safe and accessible for all patients who undergo in vitro fertilization. Improved genomic coverage of new sequencing platforms, such as next-generation sequencing, has increased the identification and diagnosis of mosaicism and partial aneuploidies in preimplantation embryos. Mosaic embryos have decreased viability compared to euploid embryos when transferred, but some mosaic embryos result in normal live births. Whole genome amplification artifacts may contribute to a misdiagnosis of mosaicism, or some mosaic embryos may self-correct to euploid after implantation. For this reason, patients without euploid embryos should be given the option of transferring mosaic embryos after genetic counseling. Further research is needed to characterize which mosaic embryos may be viable.     

Blastocyst quality criteria

Embryo culture to the blastocyst stage has progressed enormously with the new generation of acellular culture media, or sequential media. The main advantages of embryo transfer at the blastocyst stage are that it provides a natural embryo selection during culture, or a selection using preimplantation diagnosis, it provides better conditions for SET, avoiding multiple pregnancies and overcoming repeated IVF failures by improving embryo selection. The risks are failing to select a single blastocyst or obtaining a limited number of frozen blastocysts, with reduced survival after thawing. However, prolonged embryo culture seems to be good practice for day 3 embryos with delayed development, and blastocyst transfer. More effective at day 5 than at day 6.     

Prevalence,types and possible factors influencing mosaicism in IVF blastocysts: results from a single setting

Research questionAre intrinsic or extrinsic factors associated with embryo mosaicism prevalence in IVF cycles?DesignRetrospective cohort study of preimplantation genetic testing for aneuploidy (PGT-A) cycles carried out at a university-affiliated IVF clinic between October 2017 and October 2019. Trophectoderm biopsies were analysed by next generation sequencing. Mosaicism prevalence, type of anomaly and the chromosomes involved were analysed. Intrinsic and extrinsic factors potentially inducing mosaicism were studied: maternal and paternal age, antral follicle count, cumulus–oocyte complexes retrieved, female body mass index, PGT-A indication, sperm concentration, total dosage of gonadotrophins, embryo quality and day of blastocyst formation, single-step commercial media used and biopsy operator.ResultsOverall prevalence of mosaicism in our PGT-A setting was 13.9%. In segmental mosaicism, larger chromosomes tended to be more affected, which was not observed in whole-chromosome mosaicism. Additionally, segmental mosaicism was mostly observed in monosomy (69.6%; P < 0.01) compared with whole-chromosome mosaicism (49.7% monosomies versus 50.3% trisomies; P = 0.83). Although a high inter-patient variability was observed, only paternal age showed a positive association with mosaicism (adjusted OR 1.26, 95% CI 1.02 to 1.54) among the analysed variables.ConclusionsOur results suggest remarkable differences in the mechanisms generating segmental and whole-chromosome mosaicism, indicating that they may deserve different consideration when studying them and when prioritizing them for transfer. Male factor seems to be associated with mosaicism and may be worthy of specific assessment in future studies.     

Euploid rates among oocyte donors: is there an optimal age for donation?

To examine cycle blastocyst euploid rates among age subgroups of oocyte donors. Retrospective cohort analysis of ova donation in vitro fertilization cycles (OD-IVF) for which trophectoderm biopsy for preimplantation genetic testing for aneuploidy (PGT-A) by array comparative genomic hybridization (aCGH) or next generation gene sequencing (NGS) was employed between January 2015 and December 2018 in a single high-volume fertility center. Compared to oocyte donors age 26–30, oocyte donors age ≤ 25 had similar cycle blastocyst euploid rates (80 [66.7, 87.5]%, vs. 75 [62.5, 87.5]%, median [IQR], p = 0.07), blastocyst formation rates (66.7 [50, 75]%, vs. 62.5 [52, 75]%, p = 0.55), and number of retrieved oocytes (29 [23, 37] vs. 27 [20, 35], p = 0.18). Age of oocyte donor from 18 to 34 was not correlated with cycle blastocyst euploid rate. Oocyte donors age ≤ 25 had similar cycle blastocyst euploid rates, blastocyst formation rates, and number of retrieved oocytes compared to donors age 26–30. There was no correlation between cycle blastocyst euploid rates and age of the oocyte donor from 18 to 34 years. Given the lack of significant age-related change in cycle blastocyst euploid rates, our data support existing practices which do not favor a specific age subgroup of young oocyte donors.     

Superovulation fails to increase human blastocyst yield after uterine lavage.

Uterine lavage affords the potential for non-invasive human blastocyst recovery, with obvious potential for preimplantation genetic diagnosis. In an effort to duplicate in women the multiple blastocyst recovery per cycle that can be achieved in several other species, we initiated a programme in which fertile women underwent superovulation, followed by lavage and embryo collection. We superovulated 15 fertile women, aged 21-40, in 29 cycles using one of four regimens. Insemination was by either intercourse or artificial intracervical donor insemination with cryopreserved sperm from men of proven fertility. In 28 of 29 cycles, the uterus was lavaged daily for 1, 2, or 3 days between 5 and 10 days after human chorionic gonadotropin (hCG) administration or luteinizing hormone (LH) surge. Almost total fluid volume was recovered in every lavage. There were no retained pregnancies and no complications. Surprisingly, only two morulae, one blastocyst, and four unfertilized ova were recovered. Thus, alterations in ovulation induction, insemination timing, or lavage techniques must be contemplated in order to increase the blastocyst yield and thus fulfil the potential of uterine lavage for preimplantation diagnosis.     

First use of preimplantation genotyping in prevention of recurrent diandric complete hydatidiform mole

Complete hydatidiform moles have a diploid chromosome constitution, generally with only paternal genetic material present (diandry). Diandric complete moles are thought to arise either by fertilization of an anucleate oocyte by two spermatozoa or, more commonly, doubling of a single sperm genotype. Molar pregnancies are usually sporadic, and may be accompanied by malignant transformation; however, recurrence is associated with increased risk of further affected pregnancies and of persistent trophoblastic neoplasia or choriocarcinoma. This study presents the first use of preimplantation genotyping to ensure biparental inheritance in a woman presenting with recurrent diandric complete hydatidiform mole. Following an IVF cycle, a single cell from each of 11 embryos was tested by whole genome amplification and genotyping at 16 different simple tandem repeat loci. All embryos showed normal biparental inheritance; one blastocyst was transferred, resulting in the delivery of healthy monozygotic twin girls.