Introduction
Surplus embryos from in vitro fertilization (IVF) cycles can be cryopreserved for future use. A successful cryopreservation program improves cumulative rates of conception from IVF. Nevertheless, it is widely recognized that cryopreserved embryos have a lower implantation potential than fresh embryos.
Aside from intrinsic embryo abnormalities or poor uterine receptivity, failure of the embryo to hatch could partially explain the low rates of implantation observed in IVF [1]. The freezing and thawing process, along with the prolonged in vitro culture of frozen-thawed embryos, may cause changes in the glycoprotein matrix, resulting in the hardening of the zona pellucida (ZP) [2,3].
The elasticity and thinning of the ZP are essential for successful embryo hatching. If the ZP is excessively thick or hard, hatching and implantation of the embryo may be impaired. Given the importance of hatching for implantation, various assisted hatching (AH) methods have emerged to address hatching failures resulting from ZP hardening.
Several AH techniques have been developed to facilitate embryo hatching. These include mechanical ZP dissection, chemical ZP drilling, and laser-assisted thinning or breaching of the ZP. Each of these methods involves disrupting the ZP to aid in the hatching process [4-6].
In laser-assisted hatching (LAH), two different techniques are commonly used: thinning and breaching [7]. Thinning reduces the thickness of the ZP, whereas breaching entails creating an opening in the ZP.
Several studies have demonstrated that LAH employing the breaching method is more effective than mechanical or chemical AH in improving clinical outcomes [7-9]. Accordingly, LAH has become the most widely used technology, favored for its straightforward, easy, and rapid operation [7,10,11]. In addition to the absence of thermal or mutagenic side effects, the simplicity, ease of use, and time-effectiveness of LAH confer benefits such as convenient handling, technical precision, and time savings in embryo manipulation.
In one study, with careful avoidance of damage to the microvilli on the blastocyst ooplasmic membranes, no instances of failed embryo development were observed [1]. A subsequent study supported these findings, suggesting that LAH may be a safe and effective technique that does not adversely affect later development [12]. The impacts of thinning and breaching on pregnancy outcomes from frozen embryo transfer cycles have been compared with varying results, as illustrated in Table 1.
Three studies reported that thinning was superior to breaching [1,13,14], but one study reported the opposite [10]. Another study described similar IVF outcomes between the thinning and breaching methods [15].
In a retrospective study, Mantoudis et al. [1] found that the thinning method yielded a higher implantation rate (IR) and clinical pregnancy rate (PR) than the breaching technique in frozen cleavage-stage embryo transfer cycles. In a double-blind randomized trial conducted by Ng et al. [13], thinning resulted in higher IR and ongoing PR values compared to breaching in frozen cleavage-stage embryo transfer cycles.
In a retrospective study performed by Wang et al. [14], thinning appeared superior to breaching in terms of IR and clinical PR in frozen day-4 cleavage-stage embryo transfer cycles.
However, in a retrospective study, Liu et al. [10] suggested that breaching resulted in a higher IR, but not clinical PR or ongoing PR, compared to thinning in single frozen blastocyst transfer cycles.
Contradicting these findings, in a randomized trial, Le et al. [15] found similar IR and clinical PR between thinning and breaching techniques in frozen cleavage-stage embryo transfer cycles, irrespective of the woman’s age or ZP thickness.
Although the Practice Committee of the American Society for Reproductive Medicine (ASRM) has noted that insufficient evidence is available supporting the benefit of LAH in patients undergoing frozen embryo transfer cycles [11], consensus has yet to be reached regarding the most effective technique for improving IR or clinical PR with LAH. Furthermore, whether a combined approach is superior to a single method remains largely unknown. To date, no studies have specifically addressed the efficacy of combined thinning and breaching. Initially, our center adopted a breaching-only approach; however, a combination of thinning and breaching was later introduced, and both methods have been used interchangeably since. Here, we present our experience comparing the clinical efficacy of 'thinning and breaching' versus 'breaching only' in vitrified-warmed cleavage-stage embryo or blastocyst transfer cycles.
Methods
1. Study population
We selected 110 vitrified-warmed day-3 embryo transfer cycles and 50 vitrified-warmed day-5 blastocyst transfer cycles from a total of 72 infertile couples. These embryo/blastocyst transfers were performed between March 2021 and November 2022 at Seoul National University Bundang Hospital. In all transfer cycles, at least one embryo/blastocyst survived after warming. We received approval from the Institutional Review Board of Seoul National University Bundang Hospital for the use of patients' medical records (IRB No. B-2310-856-103). Written informed consent by the patients was waived due to a retrospective nature of our study. The indications for IVF included female factor infertility in 37 couples, male factor infertility in nine couples, unexplained infertility in 12 couples, and combined infertility factors in 14 couples.
2. Vitrification procedure
Embryos or blastocysts were vitrified using a CryoTop device (Kitazato BioPharma) and a Kitazato vitrification kit (Kitazato BioPharma), following the manufacturer’s instructions and referencing a previous report by Moulavi et al. [16]. The procedure entailed incubating the embryos or blastocysts in an equilibration solution (7.5% ethylene glycol+7.5% dimethyl sulfoxide [DMSO]+20% synthetic serum substitute [SSS]) for 10 minutes. Subsequently, they were transferred to a vitrification solution (VS; 15% ethylene glycol+15% DMSO+0.5 M trehalose+20% SSS) at room temperature. After removing excess VS, the embryos or blastocysts were placed on the thin polypropylene strip of the CryoTop and immediately submerged vertically in liquid nitrogen within 1 minute to achieve vitrification.
3. Endometrial preparation
Two methods were employed for endometrial preparation: a natural cycle and a hormone replacement cycle. In the natural cycle approach, the growth of a dominant follicle was monitored. Once the follicle grew to 19–20 mm, a trigger of recombinant human chorionic gonadotropin (hCG) (Ovidrel; Merck-Serono) was administered. Vitrified-warmed day-3 embryo transfers were carried out 5 days after the hCG trigger (hCG+5 days), while vitrified-warmed day-5 blastocyst transfers were conducted 7 days after hCG administration (hCG+7 days).
In the hormone replacement cycle, patients were administered either 6 or 8 mg of estradiol valerate (Progynova; Bayer) once daily, starting on the 3rd or 4th day of the menstrual cycle. Luteal phase support commenced once the endometrial thickness reached 7.0 mm or greater, using one or a combination of the following medications: subcutaneous progesterone 25 mg once daily (Prolutex; IBSA Institut Biochimique SA), micronized vaginal progesterone 100 mg three times daily (Lutinus; Ferring Pharmaceuticals), and vaginal progesterone gel 8%/90 mg once daily (Crinone; Merck-Serono). Occasionally, the vaginal form was supplemented with oral progesterone 10 mg taken three times daily (Duphaston; Abbott Biologicals BV). Transfers of vitrified-warmed day-3 embryos were conducted on the 3rd day after progesterone initiation (P+3 days), while vitrified-warmed day-5 blastocyst transfers took place on day P+5.
4. Warming procedure
The CryoTop was submerged in 1 mL of pre-warmed (37 °C) warming solution (1.0 M trehalose in tissue culture medium+20% SSS) for 1 minute. Then, the embryos or blastocysts were gently drawn from the capillary tip and transferred to a diluent solution (0.5 M sucrose) for 3 minutes at room temperature. Following this, they underwent two washes in wash solution droplets, first for 5 minutes and then for 1 minute. Finally, they were incubated in global medium supplemented with 20% human serum albumin (LifeGlobal) at 37 °C with an atmosphere of 6% CO2 and 5% O2, in air, for further development.
5. LAH
Embryos or blastocysts underwent LAH immediately after warming. The embryologist selected one of the two methods: thinning combined with breaching or breaching alone. In brief, the embryos or blastocysts were positioned on the stage of an inverted microscope (Eclipse TE2000; Nikon) equipped with a 1.48-μm (infrared) diode laser (Saturn 5 Active; Research Instruments Limited). The laser was positioned within the field, and the laser beam was subsequently activated.
In the breaching-only method, a laser was used to create a hole, 40 μm in diameter, in the ZP. For combined thinning and breaching, either one-quarter or one-sixth of the ZP’s circumference was thinned using an appropriately intense laser, immediately followed by breaching. Throughout the entire process, the laser did not directly contact the embryo or blastocyst. After the procedure, the culture was maintained at 37 °C with 6% CO2/5% O2 and saturated humidity until embryo transfer. Representative photographs of the LAH procedure are presented in Figure 1.
6. Embryos or blastocyst transfer and identification of pregnancy
For all cycles, the embryos or blastocysts were warmed between 3:00 PM and 6:00 PM and transferred the following morning between 8:00 AM and 10:00 AM. The embryo/blastocyst score was assessed at the time of transfer. For cleavage-stage embryos, the score was calculated as the product of cell number and grading score, with A=4, B=3, C=2, and D=1 [17]. When multiple embryos were present, their scores were summed. Compacted embryos were treated as a superior stage to 8-cell embryos, receiving a score of 40 (8×5 points). A morula, considered an advanced-stage 12-cell embryo, was assigned a score of 72 (12×6 points). The scoring system for blastocysts involved multiplying the development score, the inner cell mass score, and the trophectoderm score together [18]. The development score was assigned as follows: early blastocyst=1, middle expanding blastocyst=2, expanded or fully expanded blastocyst=3.5, expanded blastocyst with partial hatching=5, and fully hatched=6. The inner cell mass and trophectoderm were each graded separately, with A=3, B=2, and C=1. For instance, an expanded blastocyst with partial hatching and a grade of BB received a score of 20 (5×2×2). When multiple blastocysts were present, their scores were summed.
A clinical pregnancy was indicated by the presence of one or more intrauterine gestation sacs on transvaginal ultrasound at 6 to 7 weeks of gestation or later. Miscarriage was defined as the loss of an intrauterine pregnancy before 20 weeks of gestation, following the identification of clinical pregnancy. A live birth was defined as the delivery of a neonate at 24 weeks of gestation or later, with gestational age determined based on the date of embryo transfer. The clinical PR was calculated per embryo transfer, while the miscarriage rate was calculated per clinical pregnancy.
7. Statistical analysis
Statistical analyses were conducted using SPSS ver. 26.0 (IBM Corp.). Continuous variables were expressed as median and interquartile range. The Mann-Whitney U test, chi-square test, and Fisher exact test were performed as appropriate. A p-value less than 0.05 was considered to indicate statistical significance.
Results
Table 2 presents the baseline clinical and laboratory characteristics, as well as pregnancy outcomes, for the 'thinning and breaching' and 'breaching only' groups among women who underwent vitrified cleavage-stage embryo transfer cycles. Hormonal endometrial preparation was the primary preparation method in both groups. In the group subjected to combined thinning and breaching, a combination of subcutaneous and vaginal luteal support was predominantly utilized, whereas those treated with breaching alone mainly received vaginal and oral luteal support. Median data regarding endometrial thickness at the initiation of luteal support, the number of embryos transferred, and the embryo score at the time of transfer were comparable between the two groups. Similarly, the IR, clinical PR, miscarriage rate, and live birth rate did not differ significantly (Table 2).
Table 3 presents the baseline clinical and laboratory characteristics, as well as pregnancy outcomes, for the 'thinning and breaching' and 'breaching only' groups among women who underwent vitrified blastocyst transfer cycles. Hormonal endometrial preparation was the only preparation method used in either group. For both groups, three types of luteal support were employed, with the usage proportions being comparable between them. Median data regarding endometrial thickness at the initiation of luteal support, the number of embryos transferred, and the blastocyst score at the time of transfer were similar between the groups. The IR, clinical PR, miscarriage rate, and live birth rate also did not differ significantly (Table 3).
Discussion
To our knowledge, this is the first study to compare pregnancy outcomes following two different LAH techniques: combined thinning and breaching versus breaching alone. Our findings indicate similar pregnancy outcomes for both methods, suggesting that breaching alone may be sufficient for achieving favorable pregnancy results.
As stated in the Introduction, no consensus has yet formed regarding the preferred LAH method for optimizing pregnancy outcomes. In this study, we investigated whether a combination of the thinning and breaching techniques yields superior results. Our analysis indicated that thinning did not have an additive effect when the breaching method was employed. Since hatching already occurs through the opening created by breaching the ZP, thinning does not appear to contribute additional benefits. Consequently, based on our findings, we recommend employing breaching, rather than a combined method, in LAH. This approach minimizes the time required for manipulation.
Two main factors contribute to hatching: lysis and mechanical pressure. Lysis is triggered by substances originating from either the embryo or the female reproductive tract. During in vitro culture, cleaving embryos may secrete Lysine proteases, which play a crucial role in thinning the ZP during hatching [19]. Perona and Wassarman [20] identified a substance known as strypsin, a trypsin-like protease derived from the mural trophectoderm, which is present immediately before hatching. This finding indicates that strypsin is closely involved in the hatching process. Furthermore, Yamazaki et al. [21] found that treating the culture medium with a protease inhibitor prevented hatching, further indicating that lysine proteases are essential for this process. However, in vitro-cultured frozen-thawed embryos often exhibit impaired hatching due to zona hardening. For frozen-thawed embryos, impaired hatching may result from the extended duration of culture in an artificial environment, which affects the physicochemical properties of the ZP and leads to hardening [22]. Moreover, the freeze-thaw process itself might exacerbate the hardening of the ZP [23].
Given the pivotal role of hatching in successful implantation, various AH techniques have been employed to overcome hatching failure due to ZP hardening. In recent years, LAH has emerged as a prominent method. However, AH has been associated with an increased risk of monozygotic twinning [24,25]. This risk is attributed to the narrow opening of the ZP, which can trap the hatching blastocyst in a figure-eight configuration, potentially leading to its subdivision and the development of monozygotic twins [24]. Additionally, blastomere loss can occur through a larger hole before the formation of tight junctions, also raising the likelihood of monozygotic twins [22]. Of the two LAH techniques—thinning and breaching—breaching is theoretically more likely to result in monozygotic twins, although this has not yet been clearly demonstrated. In the present study, two twin pregnancies were observed, one involving a set of monozygotic twins and the other dizygotic twins. Both pregnancies occurred when the breaching method alone was applied to vitrified-warmed day-3 cleavage-stage embryo transfer cycles. Due to the limited sample size, we were unable to determine whether breaching is associated with an elevated incidence of monozygotic twins.
The limitations of this study include its retrospective, observational design and the fact that it was conducted at a single center, potentially limiting the generalizability of the results. Additionally, our study lacked a control group that did not undergo LAH. The Practice Committee of the ASRM has noted that insufficient evidence is available to support the benefit of LAH in patients undergoing frozen embryo transfer cycles [11]. Similarly, the Good Practice Recommendations on Add-ons in Reproductive Medicine, published by the European Society of Human Reproduction and Embryology Add-ons working group, do not advise using AH for IVF [26]. Consequently, it is essential to clarify the effectiveness of LAH by comparing PRs between individuals who undergo LAH and those who do not. Researchers must also assess whether LAH is particularly beneficial for specific groups of patients. Moreover, as described in the Introduction, thinning is generally slightly preferred over breaching; thus, further investigation is required to directly compare the efficacy of these two techniques.
In conclusion, our findings indicate that the pregnancy outcomes of combined thinning and breaching are comparable to those of breaching alone. Therefore, a breaching-only technique appears sufficient to achieve favorable pregnancy results.
PDF Links
PubReader
ePub Link
XML Download
Full text via DOI
Download Citation
