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

Abstract

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)

INTRODUCTION

EVEN WITH PROPER selection of good quality embryos for transfer to the uterus, implantation rates remain low. The low frequency of implantation after embryo transfer has been hypothesized to result from impairment of hatching by non‐physiologic hardening of the zona pellucida (ZP). ¹ Assisted hatching, developed by Cohen et al. to promote the hatching process at the blastocyst stage, has improved implantation rates after embryo transfer. ¹ , ² Many studies have shown the clinical value of assisted hatching. ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ Mechanical zona dissection with glass pipettes ¹ or chemical zona opening using acidic Tyrode's solution ² are in wide use. However, these techniques require the use of micromanipulators and the meticulous performance of micro techniques by skilled laboratory staff. In addition, concerns exist that these techniques fail to reliably produce equally sized holes in the ZP, and that they might risk adverse effects in embryos, including direct mechanical injury or chemical damage.

These problems can be overcome by a recently introduced method of laser‐assisted hatching (LAH). ⁹ , ¹⁰ , ¹¹ , ¹² The main advantage of LAH is the absence of direct mechanical or chemical contact that could adversely affect embryos. Presently in Japan, few clinical investigators have examined the effectiveness of LAH. The aim of the present study was to make such an assessment of LAH using a 1.48‐µm diode laser in human embryos, comparing results between zona opening and zona thinning techniques.

MATERIALS AND METHODS

Embryos

BETWEEN JANUARY AND March 2004, we studied 56 embryos from 16 patients to investigate the efficacy of LAH procedures using a 1.48‐µm diode laser, including possibilities of embryo damage and disturbance of subsequent development. Informed consent for experimental use of remaining embryos not used for clinical purposes was obtained from all couples involved who attended the Yano maternity clinic IVF center. The present study was carried out according to the ethical guidelines of the Japan Society of Obstetrics and Gynecology regarding studies in which human gametes or embryos are used as materials for investigation.

Oocytes were obtained according to the regular procedure of our routine clinical in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) program. Controlled ovarian hyperstimulation (COH) was carried out using a long gonadotropin releasing hormone (GnRH) analog protocol. Buserelin acetate (Suprecur; Hoechst Japan, Tokyo, Japan) was given as a nasal spray in the midluteal phase of the cycle until the day of human chorionic gonadotropin (hCG; Mochida, Osaka, Japan) injection. Beginning on day 3 of the cycle, all patients were given gonadotropins at a dose of 150–300 IU daily in combination with follicular stimulating hormone (FSH, Fertinorm P; Serono Japan, Tokyo, Japan) and human menopausal gonadotropin (hMG, Humegon; Organon Japan, Tokyo, Japan). When two or more follicles reached 18 mm in diameter, oocytes were retrieved with transvaginal ultrasonographic guidance 35 h after injection of 10 000 IU of hCG.

In vitro fertilization procedure

After retrieval, all oocytes were incubated in Quinn's Advantage Fertilization Medium (SAGE In‐Vitro Fertilization, Pasadena, CA, USA) in an atmosphere of 5% CO₂, 5% O₂ and 90% N₂ at 37°C. For insemination, the oocytes were introduced into 1‐mL droplets containing 100 000 motile sperm under mineral oil (Squibb, Princeton, NJ, USA) in a 60‐mm plastic dish (Falcon 1007, Becton Dickinson, NJ, USA) and incubated overnight.

Intracytoplasmic sperm injection procedure

After being denuded of surrounding cumulus cells, oocytes were incubated in a 50‐mm plastic dish (Falcon 1006, Becton Dickinson, Franklin Lakes, NJ, USA) under mineral oil until the time of ICSI, which was carried out only with oocytes that had extruded their first polar bodies.

Embryo culture

After fertilization was detected, embryos were cultured in Quinn's Advantage Cleavage Medium (SAGE In‐Vitro Fertilization) in a four‐well multidish (Nunclon, Roskilde, Denmark) for 3 days. Culture media were refreshed daily. Day 3 embryos were assigned a numerical grade using the following scale: ¹³ grade 1, no fragmentation with equally sized homogenous blastomeres; grade 2, less than 20% fragmentation with equally sized homogenous blastomeres; grade 3, 20–50% fragmentation with unequally sized blastomeres; grade 4, over 50% fragmentation with unequally sized blastomeres. Up to three embryos of high quality (grade 1 or 2) were transferred to patients. Supernumerary embryos of high quality were cryopreserved. Embryos unsuitable for cryopreservation (grade 3 or 4) were discarded. Other remaining embryos of moderate quality (grade 2 or 3) were eligible for the present study. Embryos were assigned randomly to one of three groups: control, zona thinning, or zona opening.

Laser assisted hatching procedure

Laser assisted hatching was carried out at the eight‐cell stage on the morning of day 3. Zona dissection of embryos was carried out with a 1.48‐µm diode laser. The system used has been described in detail. ⁹ Briefly, a 1.48‐µm diode laser aiming beam and the collimated 1.48‐µm cw laser beam (OCTAX Laser Shot system, MTG Medical Technology, Altdorf, Germany) were passed into an inverted microscope (Olympus IX‐70; Tokyo, Japan), redirected by several mirrors and focused by the microscope's objective (×40) within the microscopic field. The power routinely available at the image plane of the objective was 47 mW, corresponding to a maximal power density of 94 kW/cm². Embryos were suspended in a 35‐mm plastic dish (Falcon 1008, Becton Dickinson) in 10‐µL droplets of Quinn's Advantage Medium with HEPES (SAGE In‐Vitro Fertilization) under mineral oil (Oil For Tissue Culture; SAGE In‐Vitro Fertilization). Each embryo was positioned by moving the microscope stage with X‐Y controls to bring a region of the ZP into the point of aim. No positioning of the embryo with a holding pipette was necessary.

In the zona thinning group, a maximum of eight ablations of 2.9 ms duration were carried out successively around the ZP to create a defect involving approximately 25% of the ZP circumference and 50–90% of the ZP thickness. The inner membrane of the ZP was not breached (Fig. 1). In the zona opening group, two to four ablations of 2.6 ms duration were carried out to ensure creation of a single hole 17 µm in diameter traversing the entire thickness of the ZP. The inner membrane of the ZP was broken to represent a full‐thickness opening (Fig. 2).

Figure 1.

Photomicrograph of human day‐3 embryo after zona thinning using the laser. The thinned area of the zona pellucida is indicated by an arrow (magnification: ×400).

Figure 2.

Photomicrograph of human day‐3 embryo after zona opening using the laser. The opening in the zona pellucida is indicated by an arrow (magnification: ×400).

Culture of embryos following laser assisted hatching

After the LAH procedure, embryos were cultured in a four‐well dish until they reached the hatched blastocyst stage. The culture of these embryos was extended further for 2–3 days in Quinn's Advantage Protein Plus Blastocyst Medium (SAGE In‐Vitro Fertilization). Embryo cleavage and morphology were evaluated every 24 h. The culture medium was refreshed daily. Blastocysts, arrested hatching blastocysts, or hatched blastocysts were evaluated and counted using an inverted microscope.

Statistical analysis

One‐factor anova was used to test differences in hatched blastocyst rate between zona thinning, zona opening and controls. The Mann–Whitney U‐test was used to test between‐group differences. A P‐value below 0.05 was considered indicative of statistical significance.

RESULTS

PROFILES AND OUTCOMES of 16 patients are described in Table 1. Mean grades of 56 embryos in control, zona thinning and zona opening groups were 2.7 ± 0.5 (n = 19), 2.6 ± 0.5 (n = 13), and 2.6 ± 0.5 (n = 24), respectively (Table 2). These were not statistically different. Numbers of embryos of grades 2 and 3 allocated to control, zona thinning and zona opening groups were 13 and 6, 8 and 5, and 15 and 9, respectively (Table 2).

Table 1.

Profiles and outcomes of patients supplying embryos

Patients (n)	16
Patient age (years)†	33.8 ± 3.8
Previous embryo transfers (n)†	1.6 ± 0.9
Duration of infertility (years)†	4.1 ± 2.4
No. oocytes retrieved (n)†	10.3 ± 3.4
2‐PN fertilization rate	76.0%
Cleavage rate	97.2%
No. embryos transferred (n)†	1.9 ± 0.7
Implantation rate	25.0% (8/32)
Clinical pregnancy rate	43.8% (7/16)
Multiple pregnancy rate	14.3% (1/7)
Abortion rate	28.6% (2/7)

^† Means are shown ± SD. PN, pro nucleus.

Table 2.

Characteristics of embryos

	Control	Zona thinning	Zona opening
Embryos (n)	19	13	24
Embryo grade†	2.7 ± 0.5	2.6 ± 0.5	2.6 ± 0.5
Grade‐2 embryos (n)	13	8	15
Grade‐3 embryos (n)	6	5	9

^† Means are shown ± SD.

Respective blastocyst development rates in control, zona thinning and zona opening groups were 42.1%, 46.2% and 37.5%, representing no significant difference between the three groups (Table 3). Rates of arrested hatching in blastocysts were 12.5%, 50.0% and 0%, respectively, whereas hatched blastocyst rates were 12.5%, 50.0% and 100%, respectively (Table 3). Thus, the zona opening group showed a significantly higher rate of hatched blastocysts than the control group (P ≤ 0.05). Furthermore, no blastocysts with arrested hatching were observed in the zona opening group. The hatching rate in the zona thinning group did not differ significantly from that of the control group.

Table 3.

Outcomes according to blastocyst developmental stage

Stage	Control	Zona thinning	Zona opening
Embryos (n)	19	13	24
Developed to blastocysts (n)	8	6	9
Arrested hatching (n)	1	3	0
Hatched blastocysts (n)	1	3	9
Blastocyst development rate (%)	8/19 (42.1)	6/13 (46.2)	9/24 (37.5)
Hatched blastocyst rate (%)	1/8 (12.5)* , **	3/6 (50.0)	9/9 (100)* , **

^* P = 0.0002 by one‐factor anova.

^** P = 0.009 by Mann–Whitney U‐tests.

DISCUSSION

THE FIRST REPORTED laser systems for ZP micromanipulation were the argon fluoride excimer (ArF) laser emitting at 193 nm ¹⁴ , ¹⁵ and the xenon chloride (XeCl) laser emitting at 308 nm ¹⁶ Later, the pulsed nitrogen laser (PALM UV) was introduced. ¹⁷ However, this type of laser posed a theoretical possibility of mutagenic effects because the wavelength approximated the absorption peak of DNA at 260 nm. More recently the yttrium‐aluminum‐garnet (YAG) laser has been applied to human embryos. ¹⁸ , ¹⁹ As the YAG laser is absorbed easily by water, the equipment must nearly touch the embryo, increasing the likelihood of embryo damage. The relatively high cost of the YAG laser also restricts its use.

Recently, the 1.48‐µm diode laser has become the standard choice for zona drilling and also for zona dissection of the embryo. ⁹ , ¹⁰ , ¹¹ , ¹² The OCTAX Laser Shot System used in the present study uses such a diode laser. The laser beam is focused through the microscope objective to allow rapid, easy and contact‐free microdrilling of the ZP. The size of the hole drilled in the ZP was dependent on irradiation time and the number of ablations induced by the laser. In the laser system used in the present study, one ablation of 2.6 ms or 2.9 ms duration was sufficient to create a hole 10 µm or 12 µm in diameter, respectively. This drilling can be carried out using conventional culture dishes and media. Furthermore, additional micromanipulation equipment is not required. However, 1.48‐µm diode lasers have not been available in Japan until recently. To our knowledge this is the first report to examine the effectiveness of LAH using human embryos in Japan.

The ZP of human embryos is bilayered; the outer layer is thick and easily dissolved, whereas the inner layer is more compact, resilient and difficult to dissolve. ²⁰ An earlier report suggested that complete breaching of the bilayered ZP was important for successful hatching in the human embryo. ²¹ Complete breaching or opening of the ZP using techniques such as mechanical zona dissection with glass pipettes ¹ or chemical zona opening using acidic Tyrode's solution ² has been long carried out as a classical assisted hatching method, and has shown clinical value. However, complete breaching of the ZP might increase the risk of bacterial infection of the embryo as well as other harmful interactions with the environment. Furthermore, blastomeres might be more likely to separate, as the embryo undergoes cleavage. ²¹ Finally, an experimental study concluded that a small breach or opening of the ZP inhibited completion of hatching. ²² With such treatment, embryos might also be at greater risk of becoming trapped, resulting in embryo damage or in splitting that has been described as a cause of monozygotic twinning. ²³ In contrast, thinning of the ZP with the inner layer kept intact and the outer layer dissolved or shaved away, appears unlikely to have a significant risk to the embryos. Recently, using a 1.48‐µm diode laser system, Mantoudis et al. ¹⁰ noted an improved clinical pregnancy rate using embryos treated by zona thinning as opposed to complete opening of the ZP. Thus, several investigators have recommended the practice of thinning of the ZP technique for assisted hatching. ³ , ¹⁰ , ¹¹ , ¹⁹ , ²⁰ However, in the present study the proportion of hatched blastocysts in the zona thinning group did not differ significantly from results in the control group. Opening of the ZP did not increase the likelihood of arrested hatching of blastocysts, rather, it resulted in a higher hatched blastocyst rate. No trapped hatching blastocysts were observed in our zona opening group (Table 3).

Thus, we found opening of the ZP to be more effective in aiding hatching of embryos than the thinning technique. These results are supported by two other recent studies. ²⁴ , ²⁵ However, neither these studies nor the present study showed an improvement of clinical end‐points, such as implantation or pregnancy rates. Further studies involving a larger number of embryos in a prospective randomized clinical trial design are needed to resolve these issues. A review of reports suggests that assisted hatching might be useful clinically and that individual ART programs should evaluate their own patient populations to determine which patient subgroups might benefit from the procedure. ²⁶ Routine or universal performance of assisted hatching in the treatment of all ART patients appears unwarranted at this point. ²⁶

In conclusion, assisted hatching using a 1.48‐µm diode laser in human embryos did not show an adverse effect upon subsequent embryo development. Among LAH procedures, full‐thickness opening of the ZP appeared to yield more frequent success in hatching embryos than the thinning technique.