Improved implantation rate after chemical removal of the zona pellucida

OBJECTIVE: To evaluate the implantation rate achieved after chemical removal of the zona pellucida from day 5 human in vitro-derived embryos. DESIGN: Prospective, randomized, controlled study. SETTING: A tertiary care infertility clinic. PATIENT(S): Two hundred fifty-seven patients undergoing IVF with transfer of morulas or blastocysts on day 5 after oocyte retrieval. All patients had had at least two previous implantation failures. INTERVENTION(S): Chemical removal of zona pellucida by using acidic Tyrode's solution vs. no removal (controls). MAIN OUTCOME MEASURES: Clinical pregnancy rate and implantation rate per transfer. RESULT(S): Embryos without zona pellucida implanted at nearly twice the rate of control embryos (15.7% vs. 27.5%). The pregnancy rate was also significantly higher in the zona pellucida-free group than the control group (31.0% vs. 46.1%). Removal of zona pellucida was most effective in embryos with delayed development, which reached the morula or early cavitating stage on day 5 of in vitro culture (implantation rate, 12.1% vs. 25.7%). CONCLUSION(S): Chemical removal of zona pellucida from day 5 in vitro cultured human embryos is an effective and safe method of significantly improving the implantation rate, especially of embryos with delayed development.     

Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate

Two hundred eighteen consenting patients entered a randomized study of the application of chemical zona pellucida thinning on their day 3 embryos, prior to uterine transfer. Of those control patients (n =108), whose embryos remained unmanipulated, 40 (37.0%) have ongoing/delivered pregnancies, while in the experimental group (n =110), whose embryos had their zonae pellucidae chemically thinned, there are 49 patients (44.6%) who have ongoing/delivered pregnancies. Although this difference is not significant, clearly the application of this micromanipulative intervention has not been detrimental, and this bodes well for routine application of embryonic micromanipulation procedures in general. Certain patient subgroups were studied including older women, those with elevated basal follicle stimulating hormone levels, patients with embryos of differing zona thickness, and patients with embryos of differing uniformity of zona thickness. No significant influence of chemical removal of the outside of the zona on the implantation rate of embryos in any of these subgroups was observed other than a marginally significant (P =0.095) improvement of implantation of embryos with less than 4.0 µm variation in zona thickness when chemical zona thinning was applied. Failure of chemical zona thinning to enhance human embryo implantation significantly, compared to assisted hatching by complete zona drilling, strongly suggests that the bilayered human zona pellucida needs to be fully breached, unlike that of the mouse.     

Mechanical zona pellucida removal of vitrified-warmed human blastocysts does not affect the clinical outcome

Research QuestionDoes complete mechanical removal of the zona pellucida modify the outcome of transfer of vitrified–warmed human blastocysts?DesignIn a prospective randomized controlled study, 419 couples were allocated to either zona pellucida-free (n = 209) or zona intact (n = 210) vitrified–warmed embryo transfer. Main outcome measures included clinical pregnancy, implantation and ongoing pregnancy rates.ResultsTransfer of zona pellucida-free blastocysts resulted in clinical pregnancy, implantation and ongoing pregnancy rates (35,9%, 33,9% and 32,1%, respectively), similar to those achieved with zona intact control embryos (39%, 36,4% and 33,1%, respectively).ConclusionTotal mechanical removal of the zona pellucida did not affect the tested parameters of clinical outcomes.     

Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system

Purpose A noncontact holmium:yttrium scandium gallium garnet (Ho:YSGG) laser system has been designed and tested for the micromanipulation of mammalian embryos. The purpose of this preliminary investigation was to determine the effectiveness of this laser for assisted hatching and evaluate its impact on embryo viability. The Ho:YSGG system, utilizing 250-sec pulses at a wave-length of 2.1 m and 4 Hz, was used to remove a portion of the zona pellucida (ZP) of two-to four-cell FVB mouse embryos.Results In the first experiment there was no difference in blastocyst production or hatching rates following laser or conventional assisted hatching (LAH or AH, respectively) in contrast to control embryos cultured in a 5% CO₂ humidified air incubator at 37°C. In the second experiment a blastocyst antihatching culture model was employed and LAH-treated embryos were cultured in a serum-free HTF medium (HTF-o). Blastocyst formation was not influenced by LAH treatment and hatching was increased (P < 0.01) from 4 to 60% compared to HTF-o control group.Conclusions These preliminary data demonstrate the utility and nontoxic properties of the Ho:YSGG laser system for quick and precise ZP drilling.     

Infertility and Immunocontraception based on zona pellucida

The zona pellucida (ZP) is an extracellular matrix surrounding ovarian oocytes, ovulated eggs and preimplantation embryos. It plays several important roles at different stages of reproduction. Its constituent glycoproteins are expressed specifically in the ovary. It is thus possible to produce autoantibodies to ZP proteins that interfere with reproductive functions including folliculogenesis, fertilization and implantation. First, this article describes the history of anti-ZP antibodies detected in women with idiopathic infertility. Second, the current relationship between anti-ZP antibodies and infertility is discussed in relation to assisted reproductive medicine. Third, we introduce the latest studies of animal experiments involving the ZP. Finally, immunocontraceptive vaccine development using various ZP antigens is reviewed.     

Assisted hatching using a 1.48-μm diode laser: Evaluation of zona opening and zona thinning techniques in human embryos

Aim:  We assessed the effectiveness of assisted hatching using a 1.48-µm diode laser in human embryos, comparing zona opening and zona thinning techniques.
Methods:  A total of 56 day 3 embryos were assigned randomly to a zona opening group, a zona thinning group, or a control group. We then carried out assisted hatching using the OCTAX Laser Shot system (MTG Medical Technology, Altdorf, Germany) in the first two groups. In the zona opening group, the inner membrane of the zona pellucida was broken to create a full-thickness opening. In the zona thinning group, the inner membrane of the zona pellucida was not breached. After the laser procedure, embryos were cultured to the hatched blastocyst stage.
Results:  Blastocyst development rates did not differ significantly between the three groups. In the zona opening group, blastocysts were significantly more likely to hatch than those in the control group ( P  ≤ 0.05) and no arrested hatching of blastocysts was observed.
Conclusions:  Assisted hatching using a 1.48-µm diode laser in the zona opening technique increases the likelihood of blastocyst hatching in human embryos and does not adversely effect subsequent embryo development. (Reprod Med Biol 2006; 5 : 221–226)     

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.     

Analysis of the human zona pellucida during culture: Correlation with diagnosis and the preovulatory hormonal environment

Purpose: The objective of this study was to analyze sequentially the human zona pellucida changes in an in vitro fertilization program as it relates to several variables. Methods: The zona pellucida thickness was measured daily in zygotes and cleavage-stage embryos on a Nikon inverted microscope equipped with Hoffman modulation contrast optics, using an ocular micrometer. A total of 512 embryos from 96 patients was evaluated. Results: There was a highly significant direct correlation between zona thickness and preovulatory estradiol and basal day 3 FSH levels (P < 0.02 andP < 0.0006, respectively). This relationship showed a rapid reversal following 48 hr of culture; embryos from patients with the highest FSH levels had thinner zonae prior to transfer (P < 0.0007). The zonae from patients with unexplained infertility were thicker (19.4 ± 2.7 μm) than those from patients with endometriosis (17.7 ± 2.2 μm), tubal (17.5 ± 2.4 μm), or male-factor infertility (16.4 ± 2.7 μm) (P < 0.0001) on the first day of culture. Conclusions: We hypothesize that the thickness of the human zona pellucida is influenced by the preovulatory hormonal environment and diagnosis. These factors should be considered as part of the embryo quality evaluation prior to transfer or when assessing the possibility of using assisted hatching. More studies are needed to understand the factors regulating the thickness of the human zona pellucida.     

辅助生殖技术子代体格和精神健康

辅助生殖技术（ART）的安全性,尤其是对子代身心健康的影响,是近年来生殖医学研究的重点。多数研究表明,ART子代和自然妊娠子代的健康状况相似,辅助生殖技术是相对安全的;然而当前研究多集中于ART子代出生后早期的随访,尚无权威证据揭示其对子代成年后的远期影响,也无法明确已有的研究结论是否与遗传起源、不孕背景、体外受精（IVF）技术本身（如超促排卵用药、体外操作与培养等）和IVF相关的不良围产结局等有关。     

A follow-up study of children born after diode laser assisted hatching

This follow-up study of children born after laser assisted hatching (LAH) with a non-contact laser aims to compile data on karyotypes, deliveries, congenital malformations and growth parameters to evaluate the safety of this new technique. The study design consisted of karyotyping and completion of a standardized questionnaire. Data were analyzed for the first 134 children born after LAH.

Indication for LAH were the age (over 35 years) (group I), previously minimum three unsuccessful IVF-ET treatment cycles (group II) and age >35 years with at least three unsuccessful IVF cycles (group III).

In our study group of 134 babies we found no increase in the major congenital malformation rate (2.2%) which is comparable with the major congenital malformation rate (3%) among all deliveries at our hospital. No increase in chromosomal aberrations could be observed. Neither could we observe a difference in minor congenital malformations between the LAH treated group and all deliveries at our hospital (10.4% versus 11.1%).

On this limited number of children born after LAH there was neither evidence of an increased incidence of chromosomal aberrations nor of congenital malformations.     

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.     

In vitro fertilization in women age 40 and older: The impact of assisted hatching

Purpose To assess the impact of assisted hatching on in vitro fertilization (IVF) outcome in women age 40 and older.Methods A retrospective analysis was performed to compare 28 cycles of IVF without assisted hatching to 38 cycles of IVF with assisted hatching. All patients in both groups were age 40 or older and the mean age was similar.Results The delivery rate per oocyte retrieval was significantly higher in the assisted hatching group (18/38; 48%) compared to the nonhatched controls (3/28; 11%, P=0.0003). The implantation rate of hatched embryos (40/175; 22%) was clearly enhanced, compared to the non-hatched embryos (7/126; 6%, P<0.001). The fertilization rate, number of oocytes and the number of embryos per patient were comparable in the two groups.Conclusions Assisted hatching dramatically improves embryonic implantation and term pregnancy rates in women age 40 and older undergoing IVF.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, April 3–7, 1995, Vienna, Austria.     

阴道微生态环境对辅助生殖技术安全性影响

尽管辅助生殖技术是当代治疗不孕症的核心技术,女性的生殖能力仍然主要取决于卵母细胞质量以及支持胚胎种植和发育的母体环境。阴道微生态环境是衡量女性生殖健康重要的评估指标,与不孕症及妊娠结局具有一定的相关性。在辅助生殖技术的实施过程中,阴道微生态环境随着控制性超促排卵、手术操作、内膜准备、妊娠状态等变化而改变,对助孕结局产生一定影响。重视阴道微生态环境,增强辅助生殖技术的安全性,为不孕症患者获得良好的助孕结局提供了新的思考方向。     

辅助生殖技术妊娠丢失的相关因素分析

辅助生殖技术(ART)后妊娠丢失病因复杂,困扰着大多数不孕患者与临床工作者,明确其相关因素对改善ART妊娠结局至关重要。经过文献综述,夫妻双方年龄、胚胎染色体异常影响ART妊娠结局,而ART方法、多胎妊娠是否影响其妊娠结局,及ART妊娠丢失率是否高于自然妊娠仍需进一步探讨,这为今后的研究提示了相应的研究方向。     

Ethical application of Shared Risk programs in assisted reproductive technology

Shared Risk programs require adherence to core principles: transparency, patient autonomy, and appropriate medical care. These programs improve utilization of and perseverance with fertility treatment, receiving strong patient endorsements.