Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged

Objective: To examine whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres before embryo transfer.

Design: Prospective pilot study and observational clinical series.

Setting: Private hospital.

Patient(s): Two hundred thirty-five infertile couples undergoing frozen embryo transfer.

Intervention(s): Removal of necrotic blastomeres from frozen-thawed human embryos.

Main Outcome Measure(s): Pregnancy and implantation rates.

Result(s): Removal of necrotic blastomeres from partially damaged frozen-thawed embryos before transfer increased rates of pregnancy (45.7% vs. 17.1%), ongoing pregnancy (40.0% vs. 11.4%) and ongoing implantation (16.2% vs. 4.3%) compared with the control group, in which necrotic blastomeres were not removed. A similarly high implantation rate (16.7%) was seen a subsequent clinical series in which necrotic blastomeres were removed from all partially damaged embryos.

Conclusion(s): The viability of partially damaged frozen-thawed embryos can be improved by removal of necrotic blastomeres before embryo transfer.     

The impact of assisted hatching on live birth rates and outcomes of assisted conception: a systematic review

BACKGROUND: During the past decade in the UK, only one in six cycles of assisted conception has resulted successfully in a live birth. Assisted hatching (AH) has been proposed to improve outcome. This systematic review of randomized controlled trials addresses primary outcomes of live birth, clinical pregnancy and embryo implantation. METHODS: Trials on post-fertilization disruption of the zona pellucida were identified from the Cochrane Controlled Trials Register, MEDLINE, EMBASE and published bibliographies. Outcomes were analysed using random effects meta-analysis, sensitivity analysis, sub-grouping and meta-regression. RESULTS: Of 23 included trials recruiting 2572 women, only six reported live birth data. AH had no significant effect on live birth (OR 1.21, 95% CI 0.82-1.78). There was a significant benefit of AH on clinical pregnancy (OR 1.63, 95% CI 1.27-2.09), especially in the sub-group of women with previous failure of assisted conception (OR 2.33, 95% CI 1.63-3.34). Meta-regression suggested that AH might be more useful in older women. Implantation data were not considered valid for statistical analysis. The methodological quality of included trials was sub-optimal. CONCLUSIONS: AH probably enhances clinical pregnancy, especially in women with previous failure of assisted conception treatment and in older women; however, trials were of poor quality and so may be biased. Better quality trials reporting live birth are required to confirm any positive effects on the 'take-home-baby rate'.     

Randomized controlled study of human zona pellucida dissection using the zona infrared laser optical system: evaluation of blastomere damage, embryo development, and subsequent hatching

OBJECTIVE: To assess the effect of laser hatching on human embryo damage and subsequent development using the Zona Infrared Laser Optical System (ZILOS). DESIGN: Randomized controlled study. SETTING: Tertiary care fertility clinic. PATIENT(S): One hundred fourteen donated and discarded frozen human embryos. INTERVENTION(S): Embryos were thawed, cultured with cleavage and morphology evaluated periodically, and randomized into control, partial hatching, or complete hatching groups. The laser hatching procedure was performed by ZILOS. Zona thickness and embryo diameter were recorded. Complete hatching involved the production of a full-thickness defect in the zona and partial hatching, a defect in the outer half of the zona. No laser treatment was administered to the control group. MAIN OUTCOME MEASURE(S): Blastocyst development and completion of hatching process. RESULT(S): No significant difference was noted between the three study groups for their baseline characteristics. There was no significant difference in blastocyst development among the three groups. However, the complete hatching group showed a significant increase in hatching compared to the control group. CONCLUSION(S): Complete laser hatching of human embryos using the ZILOS does not have an adverse effect on subsequent development and increases the rate of completion of hatching.     

Hatching rate: An optimal discriminator for the assessment of single-blastomere biopsy

Purpose In order to determine an optimal marker to discriminate embryo injury following single-blastomere embryo biopsy, mouse embryos were examined for rates of blastocyst formation, hatching, implantation, and fetal development following single-blastomere biopsy.Results Early studies of single-blastomere biopsy (1–8 series) resulted in similar rates of blastocyst formation (P >0.05) but a lower rate of hatching of biopsied (n =140) versus control (nonbiopsied) (n =145) embryos (78.6 vs 95.2%; p <0.01). Subsequent experience (9–13 series) eliminated this difference between biopsied (n =145) and control embryos (n =133) (95.9 vs 94.0%; P >0.05). Embryo transfer of hatching blastocysts of biopsied (n =100) and nonbiopsied control (n =100) groups resulted in equivalent rates of implantation (96.0 vs 92.0%; p >0.05) and an equivalent rate of fetal development (70.0 vs 68.0%; p >0.05). Conclusions The hatching rate appeared to be a simple, sensitive, and reliable method to evaluate the singleblastomere biopsy technique.Presented at the Xth Annual Scientific Meeting of the Fertility Society of Australia, Lorne, Victoria, Australia, November 18–22, 1991.     

Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos

Objective: To verify the percentage of chromosomally abnormal preimplantation embryos in patients with a poor prognosis and possibly to increase the chance of implantation by selecting chromosomally normal embryos.

Design: A prospective, randomized, controlled study.

Setting: In vitro fertilization program at the Reproductive Medicine Unit of the Societá Italiana Studi Medicina della Riproduzione, Bologna, Italy.

Patient(s): In a total of 28 stimulated cycles, the maternal age was ≥38 years and/or the patient had ≥3 previous IVF failures, factors that indicated a poor prognosis. After consent, 11 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 17 controls underwent assisted zona hatching.

Intervention(s): Simultaneous analysis of chromosomes X, Y, 13, 18, and 21 in a blastomere biopsied from day-3 embryos. Chromosomal analysis was performed with fluorescence in situ hybridization. Assisted zona hatching was performed on day-3 embryos from the control-group patients.

Main Outcome Measure(s): Embryo morphology, results of fluorescence in situ hybridization, clinical pregnancies, and implantation.

Result(s): In the study group, a total of 61 embryos were analyzed by fluorescence in situ hybridization, and 55% were chromosomally abnormal. Embryo transfer with at least one normal embryo was performed in 10 cycles. Four clinical pregnancies resulted, with a 28.0% implantation rate. In the control group, 41 embryos were transferred in 17 cycles after the assisted zona hatching procedure, yielding four clinical pregnancies and an 11.9% implantation rate.

Conclusion(s): Infertile patients classified as having a poor prognosis have a high percentage of chromosomally abnormal embryos. The advantage of selecting and transferring embryos with normal fluorescence in situ hybridization results has an immediate impact on implantation.     

A randomized double-blind controlled study of the efficacy of laser-assisted hatching on implantation and pregnancy rates of frozen-thawed embryo transfer at the cleavage stage

BACKGROUND: Assisted hatching (AH) in fresh embryo transfer (ET) cycles increases the implantation and pregnancy rates, especially in women with a poor prognosis, repeated implantation failures and in older women. Little information exists in the literature regarding the role of AH in frozen-thawed embryo transfer (FET) cycles. METHODS: Embryos were cryopreserved at the cleavage stage. On the day of FET, 160 patients were randomized according to a computer-generated randomization list in sealed envelopes into the AH group and the control group. The patients and the clinicians were blinded to the group assigned. In the AH group, the outer half of the zona pellucida over a quarter of the diameter of zona was removed using a 1480 nm non-contact laser. RESULTS: The two groups were comparable in terms of demographic characteristics, ovarian response of the stimulated cycle and quality of fresh and frozen-thawed embryos. No differences in implantation, pregnancy and multiple pregnancy rates were found between the two groups. There was a non-significant trend of a higher implantation rate in the AH group when the zona thickness was > or = 16 mm. CONCLUSION: Laser AH did not improve the implantation rate of FET cycles and should not be performed routinely in all frozen-thawed embryos at the cleavage stage.     

Laser assisted zona hatching does not lead to immediate impairment in human embryo quality and metabolism

Laser assisted zona hatching (LAH) is a routinely used therapeutic intervention in assisted reproductive technology for patients with poor prognosis. However, results are not conclusive in demonstrating the benefits of zona hatching in improving the pregnancy rate. Recent observations on LAH induced genetic instability in animal embryos prompted us to look into the effects of laser assisted zona hatching on the human preimplantation embryo quality and metabolic uptake using high resolution nuclear magnetic resonance (NMR) technology. This experimental prospective study included fifty embryos from twenty-five patients undergoing intra cytoplasmic sperm injection. Embryo quality assessment followed by profiling of spent media for the non-invasive evaluation of metabolites was performed using NMR spectroscopy 24 hours after laser treatment and compared with that of non-treated sibling embryos. Both cell number and embryo quality on day 3 of development did not vary significantly between the two groups at 24 hours post laser treatment interval. Time lapse monitoring of the embryos for 24 hours did not reveal blastomere fragmentation adjacent to the point of laser treatment. Similarly, principal component analysis of metabolites did not demonstrate any variation across the groups. These results suggest that laser assisted zona hatching does not affect human preimplantation embryo morphology and metabolism at least until 24 hours post laser assisted zona hatching. However, studies are required to elucidate laser induced metabolic and developmental changes at extended time periods.

Abbreviations: AH: assisted hatching; ART: assisted reproductive technology; DNA: deoxy-ribo nucleic acid; LAH: laser assisted hatching; MHz: megahertz; NMR: nuclear magnetic resonance; PCA: principal component analysis; PGD: preimplantation genetic diagnosis; TLM: time lapse monitoring     

探讨辅助孵化在既往种植失败患者解冻周期中的合理化应用

目的:探讨在既往胚胎种植失败患者的解冻周期中如何合理化应用辅助孵化(AH)。方法:本研究共纳入了579名既往种植失败的患者,其中242例行激光AH(LAH组),337例未行AH(对照组)。比较组间临床妊娠率、种植率及活产率等;另根据患者的年龄、既往移植次数、胚胎评分及移植胚胎受精方式再进行分组,比较不同组别的妊娠结局。结果:妊娠率、种植率及活产率在LAH组与对照组组间无统计学差异(P0.05),但分组分析结果显示,既往移植次数2次的患者以及移植卵泡质内单精子显微注射(ICSI)胚胎的患者,LAH能显著提高活产率(25.5%vs 5.3%;37.0%vs 23.9%,P0.05)。结论:LAH并不适合作为解冻胚胎移植的常规移植策略,但对于既往移植次数2次的未孕者及移植ICSI胚胎的患者,LAH能明显改善妊娠结局。     

显微受精试管婴儿的基础及临床应用系列研究

目的:进行显微受精试管婴儿的基础及临床应用系列研究。方法:采用显微受精试管婴儿(卵胞浆内单精子显微注射,ICSI)治疗男性不孕症,同时进行了系列基础研究:显微激光薄化人胚胎透明带进行辅助孵出的效果研究;应用显微激光系统附带软件测量人胚胎透明带厚度(ZPT)及厚度变化率(ZPTV)与体外受精-胚胎移植妊娠结局的关系的研究。结果:显微受精试管婴儿应用于临床取得了可喜的成绩,我省首例显微受精试管婴儿于1999年11月在我中心妊娠成功并顺利分娩,填补了省内空白。截止2004年11月,共行显微受精试管婴儿533个周期,临床妊娠率达30%,已出生健康婴儿140个,系列基础研究为提高临床妊娠率提供了理论基础。结论:显微受精试管婴儿是目前治疗男性不孕症最有前景的辅助生殖技术。显微激光薄化人胚胎透明带可有效地增加胚胎种植潜力,提高临床妊娠率。ZPTV而非ZPT是评价胚胎质量的一个有效指标,对妊娠结局有很高的预测价值。     

Time-lapse observations to analyze the effects of assisted hatching

Purpose

Assisted hatching (AH) is an artificial disruption of the zona pellucida with the aim of facilitating embryo implantation. We used time-lapse observations of mouse embryos to examine the effect of AH in mouse blastocysts.

Methods

AH techniques were performed with acid Tyrode’s solution. We compared the rates of blastocyst formation and blastocyst attachment to Ishikawa cells between the control (n = 28) and the AH group (n = 24). To analyze the effects of AH, 8-cell mice embryos were cultured under time-lapse observations (every 15 min). The time required for hatching, the hatching rates, the frequency of contraction, and the contraction rates in the blastocysts were analyzed.

Results

There were no significant differences between the two groups in hatching rate or attachment rate. The times required for hatching were 286 ± 22 min in the AH group and 990 ± 437 min in the control group (P = 0.018). The contraction frequencies in blastocysts were 3.5 ± 0.7 times in the AH group and 7.5 ± 2.5 times in the control group (P = 0.020).

Conclusions

From the time-lapse observations we found that the time required for hatching and the frequency of contraction in blastocysts were both reduced by AH, although blastocyst formation and attachment were not affected.