Assisted reproductive techniques using frozen embryo transfer (FET) are becoming more common. Cryopreservation of embryos helps increase the cumulative pregnancy rate in in vitro fertilization (IVF) cycles [1]. However, embryo cryopreservation can cause zona pellucida (ZP) hardening, which leads to impaired embryo hatching and a lower implantation rate [2,3]. Scanning electron microscopy data have shown that the number and size of holes in the ZP of vitrified embryos were smaller than in fresh ZPs [4]. Other studies have shown that enzymatic digestion of the ZP with pronase in cryopreserved embryos required more time than in fresh embryos [5].

The implantation potential of a frozen/thawed embryo is lower than that of a fresh embryo. One of the reasons for this is that vitrification may impair hatching of the embryo. Theoretically, assisted hatching (AH) may facilitate the hatching process and subsequently increase the implantation rate [2,3,6]. Many authors have suggested that perforating or thinning the ZP of frozen-thawed embryos by AH facilitates embryonic implantation by making it easier for the embryo to exit the surrounding ZP [7,8].

AH has been used in humans since the 1990s [9]. There are four main techniques for AH: mechanical partial ZP dissection with glass pipettes, chemical ZP opening using acidic Tyrode solution, enzymatic ZP thinning using pronase, and diode laser-assisted hatching (LAH) [7]. The first three methods require manual or motorized micromanipulators and skilled embryologists, and carry a risk of direct mechanical injury or toxicity. Research has shown LAH to be more effective, simpler, and faster than mechanical and chemical methods [6]. Non-contact laser systems using infrared wavelengths (1.4 µm) are commonly used in LAH today. This enables easy automated operation and avoids adverse effects, as the thermal effects on the embryo are negligible. LAH is performed by two methods: drilling or thinning. In drilling LAH (D-LAH), a laser beam is used to create a hole 20–40 µm in diameter in the ZP, putting the embryo in direct contact with the culture medium. Embryos can easily hatch after they develop [10]. However, this method has disadvantages, including the risk of losing blastomeres through the opening and the direct contact of an embryo with high concentrations of nutrients and antibiotics in the environment [11]. In thinning LAH (T-LAH), a quarter to half the ZP is thinned from the outside, causing blastomeres to be less affected by heating. The size of the ZP thinning area in LAH affects the outcomes of vitrified-warmed embryo transfer (ET) at the cleavage stage [12]. Drilling a large enough hole in the ZP makes a clear path for the embryo to hatch. Thinning removes only 50% of the ZP thickness, and the embryo must break down the rest. Complete hatching through a thinned ZP occurs more slowly than through a drilled ZP, and sometimes the embryo can be trapped when trying to hatch via two routes simultaneously [13]. Incomplete hatching of an embryo significantly affects the implantation process and subsequent development of the fetus [14]. Given the advantages and disadvantages of the D-LAH and T-LAH methods for AH, we wished to study whether either of these techniques was advantageous for FET. No studies have yet compared the efficacy of these two methods of LAH on the results of FET.

Data were collected from 171 IVF/ICSI cycles randomly divided into D-LAH (85 cycles with 223 embryos) and T-LAH (86 cycles with 243 embryos) groups. The groups were not significantly different regarding the age of the female partner, body mass index, or the number of embryos transferred. In the T-LAH group, 243 embryos were transferred and 40 (16.5%) implanted. In the D-LAH group, 223 embryos were transferred and 32 (14.4%) implanted. As shown in Table 1, no difference in the rate of β-hCG positivity was observed between the two groups. The results were similar in the T-LAH and D-LAH groups in terms of implantation and clinical pregnancy rate.

The median thickness of the ZP in our study group was 16.9 µm. We analyzed the differences between two subgroups defined based on the ZP thickness (greater than or less than 17 µm). We observed no differences in the β-hCG positivity rate, clinical pregnancy rate, ongoing pregnancy rate, or multiple pregnancy rate between the two subgroups defined based on ZP thickness (Table 2).

Table 3 shows the outcomes when subgroups of ≤34-year-old (median, 31 years) and ≥35-year-old (median, 38 years) were compared. The differences were not statistically significant. However, there was a trend for better results to be found for the clinical pregnancy rate in younger patients using thinning (p=0.097).

Previous studies have used a wide variety of AH techniques, and a meta-analysis of those studies produced few meaningful results due to the heterogeneity of the studies [19]. AH procedures vary in terms of time and mode of application, thereby resulting in differences in hatching dynamics. In our study, we compared two main methods of LAH: thinning and drilling. The effectiveness of these two methods has not been directly compared in frozen and thawed embryos, and varying results for their efficacy have been reported [2,10,20]. In our study, we focused on the effect of drilling and thinning on FET results.

Studies similar to our own have shown that the choice of the method of LAH (i.e., D-LAH vs. T-LAH) had no effect on the pregnancy rate [14,21]. In a previous study, the implantation rate was found to be similar (p=0.44) in the T-LAH group (n=19, 34.5%) and the D-LAH group (n=111, 29.4%) when fresh embryos were used [20]. In our study group, in which AH was performed on day 2 frozen and thawed embryos, the differences were not statistically significant between the thinning and drilling group in terms of the biochemical pregnancy rate (38.4% vs. 29.4%) or the clinical pregnancy rate (36.0% vs. 25.9%) (p>0.2 for both). However, a trend for thinning to be more effective, similar to the results of Padula et al. [20], was observed.

The thickness of the ZP can affect the embryo's hatching. The thicker the ZP, the harder it is for the embryo to hatch [11]. Several studies have shown that a ZP thickness of more than 15 µm had a negative effect on embryo hatching and pregnancy outcomes, meaning that such cases need AH [22]. In our study group, the median ZP thickness was 16.9 µm, so we used 17 µm as a cut-point for a subgroup analysis. The rate of success was similar in the T-LAH and D-LAH groups in both groups defined by ZP thickness (greater than or less than 17 µm). The early miscarriage rate appeared to be higher for patients in the <17 µm group who underwent T-LAH (13.2%, 5/38 for T-LAH vs. 2.1%, 1/48 for D-LAH; p=0.046). However, we believe that this is attributable to chance due to the low number of cases involved.

According to many studies, maternal age has a significant influence on the genetics and viability of the embryo. Unsuccessful hatching may affect the outcomes of ICSI-ET. AH methods are thought to be effective in improving the treatment outcomes for older patients [23]. In this study, we compared the efficacy of two AH methods in subgroups of patients ≤34 and ≥35 years of age. The differences were not statistically significant. However, a trend (p=0.097) for a higher clinical pregnancy rate was demonstrated for T-LAH cycles in the younger age group, which represented more than half (56.1%, 96/171) of the entire study cohort (Table 3). This difference was primarily due to the lower pregnancy rate in the D-LAH group.

The clinical pregnancy rate in our study is comparable to published results for the 25- to 35-year age group (35%–36%) [24]. The clinical pregnancy rate in the thinning group did not differ between age groups (36.7% in those ≤34 years of age vs. 35.1% in those ≥35 years of age). It is possible that embryo hatching is one of the steps responsible for this difference and that AH eliminates it. Other factors acting later in pregnancy development reduce the success rate further for older patients, as can be seen from the lower ongoing pregnancy rates in the older group (32.7% in those ≤34 years of age vs. 27.0% in those ≥35 years of age) although the difference was not significant. Other factors involved may be related to ethnicity or lifestyle.

In early studies of LAH, thinning was reported to be superior to drilling. Comparing those results to ours, it can be seen that this difference arose from very low rates of success in the drilling group (clinical pregnancy rate: D-LAH, 5.2% vs. T-LAH, 22.1%) [25]. Later reports confirmed that drilling is more technically demanding, requires greater operator skill, and can be damaging to the embryo [20]. In our study, the success rate was higher than the early results in both groups (clinical pregnancy rate: D-LAH, 25.9% vs. T-LAH, 36.0%); the difference was not significant although T-LAH showed slightly better outcomes.

In conclusion, the present study demonstrated that there were no significant differences in the implantation and pregnancy rates when transferring thawed embryos after T-LAH or D-LAH procedures. ZP thickness and the women's age had no effect on the implantation and clinical pregnancy rates in women who underwent ICSI-FET and AH treatment by these two methods. Although there were no statistically significant differences between the two groups, the T-LAH group in which one-fourth of the ZP was thinned showed a tendency for better ET results than the D-LAH group. Thinning is also considered to be safer for the embryo and easier to perform [20]. However, the small sample size may have affected the results of this study, so studies of larger groups or pooling similar studies for meta-analysis may provide data that would reach statistical significance.