Abstract
Objective
To investigate the impact of laser-assisted hatching (LAH) on clinical outcomes in patients undergoing frozen-thawed embryo transfer (FET) cycles with embryos at different developmental stages.
Methods
We conducted a retrospective analysis of 2208 patients who underwent vitrification-thawed FET at our reproductive center between October 2018 and June 2024, with some patients contributing multiple cycles. Patients were stratified by embryonic developmental stage into cleavage-stage FET (n = 793) and blastocyst-stage FET (n = 1415) groups. Each group was further subdivided based on post-thawing LAH implementation: Non-LAH and LAH subgroups (cleavage-stage: Non-LAH (n = 363) vs. LAH (n = 430); blastocyst-stage: Non-LAH (n = 532) vs. LAH (n = 883)). Embryological parameters and clinical outcomes were compared across subgroups. Binary logistic regression analysis was performed to assess the impact of LAH on live birth rates after adjusting for confounding factors.
Results
In cleavage-stage FET, LAH group had higher clinical pregnancy rate (39.3% vs 27.5%), implantation rate (25.2% vs 17.7%) and live birth rate (25.8% vs 19.8%) than Non-LAH group (P < 0.05), but lower multiple pregnancy rate (5.9% vs 15.0%, P < 0.05). In blastocyst-stage FET, the LAH group had a higher clinical pregnancy rate (67.6% vs 58.5%) and implantation rate (65.0% vs 52.3%) than the Non-LAH group (P < 0.001). There were no statistically significant differences in the miscarriage rate, birth defect rate, and singleton birth weight between the LAH group and the Non-LAH group of different embryo ages (P > 0.05). Binary logistic regression analysis showed that after adjusting for confounding factors, LAH could increase the live birth rate of patients with cleavage FET (OR=1.529, 95% CI=1.081–2.162, P = 0.016). However, it had no effect on the live birth rate of FET patients at the blastocyst stage (OR=0.988, 95% CI=0.794–1.230, P = 0.914).
Conclusion
In the FET cycles, LAH can improve the live birth rate of patients undergoing cleavage-stage FET, especially for women aged ≥ 35 years or those with ≤ 2 previous transfers, but has no significant effect on the live birth rate of patients undergoing blastocyst-stage FET. LAH did not increase the risk of perinatal outcomes in either the cleavage-stage or blastocyst-stage embryo transfer.
Keywords: Laser-assisted hatching, Frozen-thawed embryo transfer, Clinical outcomes, Cleavage stage, Blastocyst stage
1. Introduction
The rapid advancement of assisted reproductive technology (ART) has provided diverse treatment options for infertile patients. Among these, FET is one of the ART solutions, and it involves the cryopreservation of embryos formed after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), followed by thawing and transferring them into the female uterus at an appropriate time. Compared with fresh embryo transfer, FET can effectively mitigate the risk of ovarian hyperstimulation syndrome (OHSS) via a whole embryo freezing strategy. Additionally, it enables the selection of an appropriate endometrial window period for transplantation based on the patient's condition, thereby significantly enhancing embryo implantation efficiency [1]. However, cryopreservation and thawing may cause physical or biochemical damage to the zona pellucida (ZP), thereby reducing its hatching ability. This has become one of the key factors limiting the success rate of FET [2].
To address the issue of reduced embryo hatching ability caused by freezing, LAH technology has emerged, which creates tiny openings on the zona pellucida by precisely focusing lasers, weakening its mechanical strength. This process mimics the natural hatching process and is theoretically capable of improving embryo implantation potential [3]. Nevertheless, there is still controversy over whether LAH can improve patient clinical outcomes. Some studies have indicated that LAH can enhance live birth rate and clinical pregnancy rate during blastocyst-stage FET cycles [4]. By contrast, other studies have suggested that LAH has no effect on the live birth rate among patients with repeated implantation failures [5]. Moreover, existing literature predominantly focuses on specific developmental stages of embryos and lacks a systematic comparison of the effects of LAH on embryos at different developmental stages.
In light of these findings, this study aims to investigate whether the use of LAH at different embryo stages during FET cycles could improve clinical outcomes for patients. It is anticipated that the results of this study will provide an evidence-based foundation for the rational clinical application of LAH and promote the optimization of individualized embryo transfer strategies.
2. Materials and methods
2.1. Patient selection and ethics approval
In this retrospective analysis, we included 2208 patients undergoing vitrified-warmed FET cycles at our reproductive center between October 2018 and June 2024, including repeated cycles. Our center began using LAH technology post-thaw in June 2022. Accordingly, patients undergoing vitrified-warmed FET between October 2018 and May 2022 without post-thaw LAH were assigned to the Non-LAH group (n = 895), while those undergoing vitrified-warmed FET between June 2022 and June 2024 with routine post-thaw LAH were assigned to the LAH group (n = 1313). Based on embryonic developmental stage at thawing and LAH application, these groups were further stratified: the cleavage-stage (Day 3, D3) FET Non-LAH subgroup (n = 363) and LAH subgroup (n = 430), and the blastocyst-stage (Day 5/6, D5/D6) FET Non-LAH subgroup (n = 532) and LAH subgroup (n = 883). The inclusion criteria are: ① maternal age ≤ 42 years; ②fertilization by IVF/ICSI; ③underwent vitrified FET. Exclusion criteria: ① women aged > 42 years; ② endometrial thickness < 7 mm on the day of transplantation; ③ incomplete clinical data; ④ patients who underwent rescue ICSI due to IVF failure; ⑤ patients with endocrine dysfunction (e.g., thyroid or adrenal dysfunction).
This retrospective study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Affiliated Hospital of Youjiang Medical University for Nationalities (Ethics Review No.: YYFY-LL-2024–285). Informed consent was obtained from all participants.
2.2. Methods
2.2.1. Research design and group comparison
In this study, the LAH grouping was based on the date when the technology was routinely introduced in the reproductive laboratory. The study design and grouping details are provided in Table S1. The primary objectives and the corresponding multiple-comparison schemes are summarized in Table S2. All analyses were performed according to these predefined groups; the primary comparisons were clinical outcomes between LAH and non-LAH groups at the same embryonic stage, and factors influencing live birth rates.
2.2.2. Ovulation induction and embryo culture
Ovarian stimulation protocols included the long-acting GnRH agonist protocol, the GnRH antagonist protocol, and the minimal stimulation protocol. Gonadotropin (Gn) dosage was determined based on patient age, baseline hormone levels, and ovarian reserve. Follicular development was monitored via serial transvaginal ultrasound. Ovulation was triggered with human chorionic gonadotropin (hCG) when the leading follicle diameter reached ≥ 18 mm. Following oocyte retrieval, fertilization was achieved via conventional IVF or ICSI, with subsequent embryo culture in incubators. Fertilization status was assessed 16–18 h post-insemination/injection. Embryos were cultured until Day 3, at which point morphological evaluation was performed according to the following criteria: High-quality D3 embryos were defined as those derived from normally fertilized oocytes (2 pronuclei, 2PN) with 7–9 cells and a grade 1–2. viable D3 embryos encompassed those derived from oocytes fertilized with 2PN, 0PN, or 1PN, meeting either ≥ 6 cells with grade ≥ 3, or 4–5 cells with grade ≥ 2. The decision to extend culture to the blastocyst stage (Day 5/6) was based on clinical indications and embryo quality. Extended culture was typically recommended for patients with ≥ 3 viable D3 embryos and was strongly considered for those who are elderly or have a history of implantation failure. The final decision was made jointly by the embryologist and clinician. The blastocysts are classified according to the Gardner grade, and the high-quality blastocysts on the 5th or 6th day and the available blastocysts are transferred or cryopreserved.
2.2.3. Vitrification and thawing of embryos
Embryo vitrification was performed using the KITAZATO Vitrification Kit (Japan) with the following protocol: Following dual-operator verification at room temperature, embryos were washed in equilibration solution (ES). Cleavage-stage embryos underwent ES equilibration for 8 min, while blastocysts were equilibrated for 10 min; well-expanded blastocysts received pre-equilibration-assisted LAH, with laser-collapsed blastocysts equilibrated for 10 min and non-collapsed counterparts for 12 min. Embryos were then serially transferred to the central surface of Vitrification Solution 1 (VS1) and subsequently to the periphery of a VS2 droplet. Using minimal fluid volume, embryos were loaded onto the designated area (approximately 3 mm from the tip) of the cryotop carrier. Within ≤ 60 s of VS2 exposure, the loaded cryotop was immediately plunged into liquid nitrogen with gentle agitation. After securing the protective cap and final verification, cryotops were stored in labeled goblets within cryocanes under liquid nitrogen vapor phase storage.
The thawing process is as follows: Thawing solution (TS) should be pre-warmed at 37℃ for over 60 min. After double verification, take out the carrier rod from liquid nitrogen and quickly immerse it in TS solution to shake gently to make the embryo fall off; the embryo is transferred to diluent (DS) solution and allowed to stand for 3 min, washing solution (WS1) and washing solution (WS2) surface for 5 min in turn. The residual liquid content is controlled during the whole process and finally transferred to the G2 culture dish.
2.2.4. Laser-assisted hatching
LAH was systematically performed on all frozen-thawed embryos during the defined LAH period, using laser pulses of 2–3 ms. For cleavage-stage embryos, laser application targeted areas with a thicker zona pellucida or significant fragmentation, resulting in a thinning of approximately 30% of the zona without perforation. For blastocysts, continuous laser ablation perforated and removed 20–25% of the zona. Post-procedure embryos were returned to the incubator for at least 30 min before transfer.
2.2.5. Endometrial preparation and embryo transfer
The choice of endometrial preparation and embryo transfer strategy was based on the patient's ovulation function and ovarian reserve, with options including artificial cycles, natural cycles, hormone replacement cycles, or ovulation induction cycles. Artificial Cycle: Starting on the 3rd day of menstruation, patients took oral estradiol valerate (4–8 mg/day) for 14 days. When ultrasound indicated an endometrial thickness of 7 mm or greater, intramuscular progesterone (60 mg/day) was administered for luteal phase support. FET was performed on the 5th day after progesterone initiation. Natural Cycle: From the 10th day of menstruation, ultrasound monitoring was conducted until the dominant follicle reached ≥ 18 mm and a positive urinary LH surge was detected. Cleavage-stage embryo transfer occurred 3 days post-ovulation, accompanied by vaginal progesterone gel (90 mg/day). Hormone Replacement Cycle: Patients took oral estradiol valerate (10 mg/day) for 21 days. Once the endometrium met the criteria, progesterone was administered as in the artificial cycle protocol. Ovulation Induction Cycle: Starting on the 3rd day of menstruation, patients took letrozole (5 mg/day for 5 days). When follicles reached ≥ 14 mm, estradiol valerate (2 mg/day) was added. Blastocyst transfer was scheduled 5 days after hCG triggering.
2.2.6. Pregnancy determination and observation indicators
Transvaginal ultrasound was performed at 4–5 weeks post-transfer, corresponding to gestational weeks 6–7. Clinical pregnancy was confirmed by visualization of an intrauterine gestational sac containing a yolk sac or embryonic pole with cardiac activity. Live birth was defined as the delivery of a viable neonate at ≥ 28 weeks' gestation. The relevant indicators of this study are as follows in the Table 1.
Table 1.
The relevant indicators of this study.
| Outcome | Definition |
|---|---|
| Primary | |
| Live birth rate | Number of live birth cycles / Number of initiated treatment cycles × 100% |
| Secondary | |
| Clinical pregnancy rate | Number of clinical pregnancy cycles / Total number of transfer cycles × 100% |
| Implantation rate | Number of gestational sacs / Total number of embryos transferred × 100% |
| Multiple pregnancy rate | Number of multiple pregnancies / Number of clinical pregnancies × 100% |
| Miscarriage rate | Number of pregnancy losses / Number of clinical pregnancies × 100% |
| Birth defect rate | Number of birth defects / Total number of live births × 100% |
| Embryo recovery rate | Number of survived embryos after thawing / Total number of thawed embryos × 100% |
| High-quality embryo transfer (%） | Number of high-quality embryos transfer / Total number of embryos transfer× 100% |
| Singleton birth weight | Birth weight (g) of single live birth infants |
2.2.7. Statistical methods
Analysis in this study was performed using SPSS （version 27.0）. For non-normally distributed continuous variables, data were presented as medians with interquartile ranges [M (P25, P75)], and the Mann-Whitney U test was used to compare differences between two groups. Categorical variables were expressed as frequencies (%), with comparisons between groups conducted using the chi-square (χ²) test. To evaluate the effect of LAH on live birth rate, binary logistic regression analysis was applied. The model was adjusted for potential confounding variables, including maternal age, body mass index（BMI）, duration of infertility, endometrial thickness, and number of previous embryo transfer attempts. To account for potential within-patient correlation arising from the inclusion of multiple cycles of some individuals, we conducted a sensitivity analysis using generalized estimating equations (GEE). Subgroup analyses were performed using stratified logistic regression models, adjusting for maternal age, BMI, duration of infertility, endometrial thickness, and number of embryo transfers. Additionally, post-hoc power analysis for live-birth rate was performed with G*Power software, employing Fisher’s exact test (two-tailed, α = 0.05) to assess the statistical power of the study under the observed effect size.
3. Results
3.1. Comparison of basic patient data
A total of 2208 patients were included in this study, of whom 793 were treated with vitrified FET at cleavage stage, divided into Non-LAH group (n = 363) and LAH group (n = 430), and 1415 were treated with FET at blastocyst stage, divided into Non-LAH group (n = 532) and LAH group (n = 883). There was no statistical difference in general basic data between the Non-LAH group and the LAH group of cleavage-stage FET and blastocyst-stage FET(P > 0.05). Detailed results are shown in Table 2.
Table 2.
Comparison of basic data in cleavage-stage FET / blastocyst-stage FET [M(P25, P75)].
| Characteristics | Cleavage stage |
Blastocyst stage |
||||||
|---|---|---|---|---|---|---|---|---|
| Non-LAH（n = 363） | LAH（n = 430） | Z/X2 | P-value | Non-LAH（n = 532） | LAH（n = 883） | Z/X2 | P-value | |
| Maternal age（years） | 35.0(31.0,39.0) | 36.0(32.0,39.0) | -1.235 | 0.217 | 33.0(30.0,36.0) | 33.0 (30.0,36.8) | -0.487 | 0.627 |
| Male age（years） | 37.0 (32.5,42.0) | 37.0(34.0,41.5) | -0.899 | 0.369 | 35(31,39) | 35(31,38) | -0.575 | 0.565 |
| Duration of infertility (years) | 3.0(2.0,5.0) | 4.0(2.0,6.0) | -1.114 | 0.265 | 3.0(2.0,5.0) | 3.0(2.0,5.0) | -0.296 | 0.767 |
| Endometrial thickness(mm） | 9.0(8.0,9.5) | 9.0(8.0,10.0) | -0.097 | 0.923 | 8.5(8.0,9.5) | 8.8(8.0,9.5) | -0.283 | 0.777 |
| bFSH（U/L） | 6.8(5.4,8.2) | 7.0(5.5,9.0) | -1.780 | 0.075 | 6.0(4.9,7.2) | 6.0(4.7,7.2) | -1.513 | 0.130 |
| BMI（kg/m2） | 22.2(20.6,24.3) | 22.8(20.7,25.2) | -1.718 | 0.086 | 22.2(20.5,24.4) | 22.6(20.6,25.0) | -1.941 | 0.052 |
| Number of prior transfers | 2.0(1.0,2.0) | 2.0(1.0,2.0) | -0.447 | 0.655 | 2.0(1.0,2.0) | 1.0(1.0,2.0) | -0.706 | 0.480 |
| Fertilization method | 0.546 | 0.460 | 0.871 | 0.351 | ||||
| IVF | 46.5% (280/363) | 43.5% (322/430) | 84.0% (447/532) | 85.8% (758/883) | ||||
| ICSI | 53.5% (83/363) | 56.5% (108/430) | 16.0% (85/532) | 14.2% (125/883) | ||||
* Differences between the two groups were compared by X2 test (reported as X2 value) and Mann-Whitney U test (reported as Z value)
3.2. Comparison of FET cycle embryos at different embryonic ages
There was no statistical difference in the number of frozen embryos, the number of transferred embryos, and the embryo recovery rate between the two groups of cleavage-stage FET(P > 0.05). In blastocyst-stage FET, the median number of transferred blastocysts in the Non-LAH group (1.0(1.0,2.0)) was significantly higher than that in the LAH group (1.0(1.0,1.0)) (P < 0.001), while there were no statistically significant comparisons of the other parameters (P > 0.05). Detailed results are shown in Table 3.
Table 3.
Comparison of embryo status in cleavage-stage FET / blastocyst-stage FET [M(P25, P75)].
| Characteristics | Cleavage stage |
Blastocyst stage |
||||||
|---|---|---|---|---|---|---|---|---|
| Non-LAH（n = 363） | LAH（n = 430） | Z/X2 | P-value | Non-LAH（n = 532） | LAH（n = 883） | Z/X2 | P-value | |
| Frozen embryos | 2.0(2.0,2.0) | 2.0(1.0,2.0) | -0.821 | 0.412 | 3.0(2.0，6.0) | 6.0(2.0，6.0) | -0.456 | 0.649 |
| Embryo transfer | 2.0(2.0,2.0) | 2.0(2.0,2.0) | -1.511 | 0.131 | 1.0(1.0,2.0) | 1.0(1.0,1.0) | -5.115 | ＜0.001 |
| Embryo recovery（%） | 98.3%（643/654） | 98.2%（743/757） | 0.057 | 0.812 | 99.3%（669/674） | 99.5%（1022/1027） | 0.122 | 0.727 |
| High-quality embryo transfer rate（%） | 50.4%（1050/1999） | 49.6%（1035/1967） | 0.003 | 0.954 | 52.4%（1108/2113） | 52.7%（1182/2243） | 0.029 | 0.864 |
* Differences between the two groups were compared by X2 test (reported as X2 value) and Mann-Whitney U test (reported as Z value)
3.3. Comparison of clinical outcomes of FET cycles at different embryonic ages
In cleavage-stage FET, the clinical pregnancy rate, implantation rate, and live birth rate in the LAH group were higher than those in the Non-LAH group (P < 0.05), while the multiple pregnancy rate in the Non-LAH group was higher than that in the LAH group (P < 0.05).In blastocyst-stage FET, the LAH group had significantly higher clinical pregnancy rate (67.6% vs 58.5%) and implantation rate (65.0% vs 52.3%) than the Non-LAH group (P < 0.001). There was no statistically significant difference in miscarriage rate, birth defect rate, and singleton birth weight between the LAH group and the Non-LAH group in FET at the cleavage stage and FET at the blastocyst stage (P > 0.05). Detailed results are shown in Table 4.
Table 4.
Comparison of clinical outcomes in cleavage-stage FET / blastocyst-stage FET [M(P25, P75)].
| Outcomes | Cleavage stage |
Blastocyst stage |
||||||
|---|---|---|---|---|---|---|---|---|
| Non-LAH（n = 363） | LAH（n = 430） | Z/X2 | P-value | Non-LAH（n = 532） | LAH（n = 883） | Z/X2 | P-value | |
| Clinical pregnancy rate（%） | 27.5%（100/363) | 39.3%（169/430) | 12.132 | ＜0.001 | 58.5%（311/532) | 67.6%（597/883) | 12.093 | ＜0.001 |
| Implantation rate（%） | 17.7%（116/654） | 25.2%（191/757） | 11.576 | ＜0.001 | 52.3%（348/665） | 65.0%（654/1006） | 26.809 | ＜0.001 |
| Live birth rate（%） | 19.8%（72/363） | 25.8%（111/430） | 3.964 | 0.046 | 46.4%（247/532） | 45.5%（402/883） | 0.109 | 0.742 |
| Multiple pregnancy rate（%） | 15.0%（15/100） | 5.9%（10/169） | 6.148 | 0.013 | 9.3%（29/311） | 6.7%（40/597） | 2.006 | 0.157 |
| Miscarriage rate（%） | 22.0%（22/100） | 15.4%（26/169） | 1.875 | 0.171 | 11.9%（37/311） | 8.2%（49/597） | 3.246 | 0.072 |
| Newborn(male/female) | 53/34 | 64/57 | 1.325 | 0.250 | 160/116 | 267/175 | 0.418 | 0.518 |
| Gestation age (weeks) | 39.0 (38.0，41.0） | 39.0 (38.0，41.0） | -0.113 | 0.910 | 39.0（38.0，41.0） | 40.0 (38.0，41.0） | -0.879 | 0.380 |
| Birth defect rate（%） | 1.4%（1/72） | 0.0%（0/111） | 0.048 | 0.827 | 1.2%（3/247） | 0.0%（0/402） | 2.621 | 0.105 |
| Singleton birth weight（g） | 3.1（2.8，3.5） | 3.2（2.9，3.4） | -1.729 | 0.084 | 3.2（2.8，3.5） | 3.2（2.9，3.5） | -1.331 | 0.183 |
* Differences between the two groups were compared by X2 test (reported as X2 value) and Mann-Whitney U test (reported as Z value)
3.4. Binary regression analysis of factors affecting live birth rate in FET cycles at different embryonic ages
Covariates including maternal age, BMI, duration of infertility, endometrial thickness, and number of prior embryo transfers, were incorporated into logistic regression models to adjust for potential confounding. After adjustment, LAH demonstrated a significant association with live birth rate in cleavage-stage FET cycles (OR=1.529, 95% CI=1.081–2.162, P = 0.016). Maternal age remained independently associated with live birth outcomes (OR=0.908, 95% CI=0.875–0.942, P < 0.001). For blastocyst-stage FET, LAH showed no significant effect on live birth rate (P > 0.05). However, maternal age, endometrial thickness, and number of prior transfers independently influenced live birth outcomes (P < 0.05). Detailed results are shown in Table 5.
Table 5.
Binary logistic regression analysis of LAH and other factors affecting live birth rate.
| Cleavage stage |
Blastocyst stage |
||||
|---|---|---|---|---|---|
| OR (95%CI) | P-value | OR (95%CI) | P-value | ||
| Maternal age | 0.908（0.875–0.942） | < 0.001 | 0.952（0.930–0.975） | < 0.001 | |
| BMI | 0.991（0.939–1.045） | 0.732 | 0.986（0.953–1.019） | 0.399 | |
| Duration of infertility | 1.009（0.953–1.069） | 0.752 | 1.001（0.965–1.040） | 0.943 | |
| Endometrial thickness | 1.097（0.968–1.244） | 0.146 | 1.095（1.009–1.188） | 0.030 | |
| Number of prior transfers | 0.987（0.832–1.171） | 0.881 | 0.841（0.744–0.952） | 0.006 | |
| LAH | 1.529（1.081–2.162） | 0.016 | 0.988（0.794–1.230） | 0.914 | |
*Binary logistic regression analysis was used to adjust for confounding factors such as maternal age, BMI, duration of infertility, endometrial thickness and number of transplants
3.5. Comparison of live birth rates among FET subgroups of different embryonic ages
In subgroup analyses of cleavage-stage FET stratified by maternal age, covariates including maternal age, BMI, duration of infertility, endometrial thickness, and number of prior transfers were adjusted via logistic regression. After adjustment for confounding factors, LAH was significantly associated with higher live birth rates in patients with advanced maternal age (≥35 years) (OR=2.238, 95% CI=1.334–3.729, P = 0.002) and those with ≤ 2 prior embryo transfers (OR=1.725, 95% CI=1.166–2.554, P = 0.006). For blastocyst-stage FET, LAH demonstrated no significant improvement in live birth rates across all subgroups (P > 0.05). Detailed results are shown in Table 6.
Table 6.
Adjusted live birth rates in cleavage-stage FET / blastocyst-stage FET subgroups.
| Subgroup | Cleavage stage |
Blastocyst stage |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Non-LAH (n = 363) | LAH(n = 430) | OR（95%CI） | P-value | Non-LAH (n = 532) | LAH(n = 883) | OR（95%CI） | P-value | ||
| Maternal age（years） | |||||||||
| ＜35 | 30.5%（47/154） | 31.3%（46/147） | 1.083（0.655–1.790） | 0.756 | 50.0%（167/334） | 51.8%（274/529） | 1.093（0.829–1.441） | 0.529 | |
| ≥ 35 | 12.0%（25/209） | 22.9%（65/284） | 2.238（1.334–3.729） | 0.002 | 40.4%（80/198） | 36.2%（128/354） | 0.837（0.580–1.208） | 0.342 | |
| Endometrial thickness(mm) | |||||||||
| ＜9 | 18.1%（32/177） | 24.1%（51/212） | 1.618（0.963–2.716） | 0.069 | 41.6%（117/281） | 44.0%（196/445） | 1.116（0.822–1.517） | 0.481 | |
| ≥ 9 | 21.5%（40/186） | 27.4%（60/219） | 1.465（0.917–2.341） | 0.110 | 51.8%（130/251） | 47.0%（206/438） | 0.853（0.622–1.169） | 0.321 | |
| Number of prior transfers | |||||||||
| ≤ 2 | 23.0%（32/139） | 29.7%（51/172） | 1.725（1.166–2.554） | 0.006 | 50.4%（125/248） | 49.2%（222/451） | 0.949（0.753–1.197） | 0.659 | |
| ＞2 | 17.9%（40/224） | 23.2%（60/259） | 0.914（0.415–2.011） | 0.823 | 43.0%（122/284） | 41.7%（180/432） | 1.370（0.667–2.817） | 0.392 | |
*Subgroup analysis was performed by stratified logistic regression to adjust for confounding factors such as maternal age, BMI, duration of infertility, endometrial thickness, and number of transplants
3.6. Sensitivity analysis and post-hoc power assessment
To control for potential intra-patient correlation introduced by multiple cycles of some individuals, we performed a sensitivity analysis of live-birth rate using generalized estimating equations (GEE). The results were consistent with those of binary logistic regression (Supplementary Table S3): LAH was significantly associated with improved live-birth rate in cleavage-stage FET (OR=1.527, 95% CI=1.075–2.168, P = 0.018), whereas no significant effect was observed in blastocyst-stage FET (OR=0.988, 95% CI=0.791–1.233, P = 0.912). Post-hoc power analysis for live-birth rate indicated that statistical power was approximately 50% for cleavage-stage FET, but only 5.9% for blastocyst-stage FET, suggesting that the latter may have been underpowered to detect small between-group differences (Supplementary Table S4).
4. Discussion
Zona pellucida (ZP) is a tough, primarily glycoprotein-based protective layer surrounding oocytes and early embryos [6]. Its presence prevents polyspermy and serves as a crucial barrier, protecting the developing embryo [7]. For successful implantation into the endometrium, the embryo must escape this "shell" through a process termed hatching [3]. However, cryopreservation can damage the ZP, primarily through ice crystal-induced mechanical stress, osmotic shock, and cryoprotectant effects, often manifesting as zona hardening or thickening. This significantly impedes the embryo's ability to hatch [8]. With the continuous advancement of ART, assisted hatching (AH) has emerged as a key laboratory intervention to overcome these zona-related hatching barriers. AH techniques include mechanical, chemical, and laser methods. LAH, owing to its precision, safety, and efficacy, has become the most widely applied AH technique in clinical practice [9], [10]. This study evaluated LAH efficacy in enhancing clinical outcomes following FET at the cleavage and blastocyst stages. Given significant inter-stage variations in embryonic morphology, ZP properties, and developmental competence, we designed and incorporated distinct LAH strategies for cleavage and blastocyst embryos, respectively.
The results showed that there was no significant difference in the rate of embryo recovery between the Non-LAH group and LAH group (P > 0.05), suggesting that LAH had no significant harm to embryo recovery. In addition, the clinical pregnancy rate and implantation rate in the LAH group were significantly higher than those in the Non-LAH group (P < 0.05), and the multiple pregnancy rate was significantly lower (P < 0.05). This is consistent with previous studies showing that LAH in freeze-thaw embryo transfer improves clinical pregnancy and implantation rates [11]. The potential underlying mechanisms are as follows [12], [13]: (1) Cryostress-induced glycoprotein cross-linking hardens the ZP, but localized precision thinning restores physiological elasticity, directly reducing mechanical resistance to embryo hatching. (2) By decreasing ATP consumption required for spontaneous embryo hatching, LAH diverts metabolic resources toward blastomere repair and mitotic progression, thereby enhancing developmental potential. (3) LAH intrinsically selects embryos with intact cytoskeletal structures and genomic stability while eliminating cryodamaged, low-quality embryos. Notably, the observed reduction in multiple pregnancy rate within the LAH group directly correlates with improved single-embryo implantation efficiency. This enables clinical reduction in the number of embryos transferred, rather than indicating that the intervention itself alters the mechanisms of multiple gestation.
Previous studies indicate that the size of the zona pellucida opening during assisted hatching in blastocyst-stage FET significantly impacts blastocyst hatching [14]. In this study, precise ablation of 20–25% of the blastocyst's zona pellucida facilitated directional hatching of trophectoderm cells. Concurrent blastocyst retraction created a protective cavity between the embryo and zona, mitigating potential laser-induced damage to the inner cell mass and ensuring procedural safety. In blastocyst-stage FET, the Non-LAH group received significantly more transferred blastocysts than the LAH group (P < 0.001), reflecting an evolution in practice towards stricter SET policies over time. Nevertheless, the LAH group demonstrated superior clinical pregnancy (P < 0.001) and implantation rates (P < 0.001). Despite fewer embryos transferred, the LAH group achieved higher implantation efficiency per embryo (P < 0.001), and the multiple pregnancy rate was lower, although the P value was not significant（P = 0.157）, strongly supporting LAH's intrinsic role in enhancing implantation potential. This result is consistent with previous studies [4]. LAH precisely punctures and penetrates the zona pellucida, reducing the difficulty of embryo hatching and thereby enhancing the implantation potential. Furthermore, our study detected no increased risks of miscarriage, birth defects, or abnormal neonatal birth weights associated with LAH (P > 0.05), consistent with existing evidence regarding the safety of laser-assisted hatching in vitrified-warmed embryo transfers [15], [16].
In this study, live birth rate served as the primary outcome measure. In cleavage-stage FET, both univariate and multivariable logistic regression analyses demonstrated significantly higher live birth rate in the LAH group than the Non-LAH group (OR=1.529, 95% CI=1.081–2.162, P = 0.016), consistent with prior findings [17]. This effect may be attributed to the artificial gap created by LAH serving as a conduit for metabolite and growth factor exchange between the embryo and endometrium, thereby facilitating implantation. Such microstructural modification potentially enhances embryo-endometrial synchrony during the receptive window, creating favorable conditions for gestation. However, the precise protective mechanisms remain unclear and warrant further investigation. A prospective study on vitrified blastocyst cryopreservation and thawing reported that LAH significantly improved the live birth rate [18]. Interestingly, in this study, LAH increased the implantation rate and clinical pregnancy rate in patients undergoing blastocyst-stage transfer (P < 0.001), while LAH itself showed no independent effect on the live birth rate at the blastocyst stage (OR = 0.988, 95% CI = 0.794–1.230, P = 0.914). The observed discrepancy may be attributed to several plausible explanations: (1) Patient heterogeneity across studies, encompassing variations in maternal age, ovarian reserve, endometrial receptivity, and prior cycle failure history, likely influenced live birth outcomes.(2) Despite lower per‑embryo transfer numbers in the LAH group (P < 0.001), reflecting stricter SET policies, the Non‑LAH group’s higher embryo number per transfer partially offset its lower implantation efficiency, yielding comparable cumulative live birth rates per cycle; (3) Although pregnancy loss after implantation was numerically lower in the LAH group (8.2% vs. 11.9%, P = 0.072), this difference did not reach statistical significance or confer a live birth advantage; (4) Embryonic aneuploidy, an unmeasured confounder, may limit the benefit of LAH, which aids hatching but cannot overcome developmental impairments from chromosomal abnormalities [19].
Subgroup analyses in this study revealed significant heterogeneity in LAH effects. For cleavage-stage FET, LAH substantially improved live birth rates in specific groups. Among advanced maternal age (≥35 years) patients, LAH significantly increased live birth rate (OR=2.238, 95% CI 1.334–3.729), potentially attributable to age-related zona pellucida hardening [20]. Cryopreservation stress compounded by aging may further impair intrinsic hatching capacity, which LAH mitigates through targeted zona thinning. Patients with ≤ 2 prior embryo transfers also demonstrated improved live birth rate with LAH (OR=1.725, 95% CI 1.166–2.554). These patients likely exhibit preserved endometrial receptivity [21], where LAH optimizes embryo-endometrial synchrony by reducing hatching resistance. Conversely, in blastocyst-stage FET, we found that no LAH-associated live birth improvement was observed across any subgroup (P > 0.05). Blastocysts possess innate hatching competence, and natural zona thinning during expansion may diminish LAH's mechanical advantage. These findings reinforce that blastocyst live birth outcomes depend more critically on maternal factors (e.g., age, endometrial thickness) than LAH intervention, warranting greater clinical attention to maternal parameters during treatment planning.
Several limitations should be considered when interpreting the results of this study. (1) The study’s non‑randomized, period‑based retrospective design carries a risk of temporal confounding due to evolving laboratory techniques, embryo selection criteria, and clinical practices over time. This represents the primary limitation of this study. (2) Detailed embryo morphology scores were not consistently available for analysis owing to incomplete records. Although embryo recovery rates and the proportion of high‑quality embryos transferred were comparable between groups (P > 0.05), residual confounding by embryo quality cannot be excluded. (3) The choice between cleavage‑stage and blastocyst‑stage transfer was based on clinical factors such as embryo number, developmental potential, maternal age, and prior transfer history. This clinical judgment may have introduced unmeasured confounding and affected group comparability. (4) Information on endometrial preparation protocols was not available for adjustment. Given that endometrial receptivity is a key determinant of success, unmeasured differences in protocol may have influenced outcomes. (5) Post‑hoc power analysis revealed moderate power for cleavage‑stage transfers (about 50%), but extremely low power for blastocyst‑stage transfers (about 5.9%). The study was therefore underpowered to detect small between‑group differences in blastocyst transfers, and the true effect of LAH in this context awaits confirmation in larger prospective studies.
In conclusion, this study confirms the safety profile of LAH in vitrified-warmed embryo transfer cycles. For cleavage-stage FET, LAH significantly improves clinical pregnancy rates, implantation rates, and live birth rates. Subgroup and multivariable analyses collectively demonstrate population-specific enhancement of live birth rates by LAH in cleavage-stage transfers. In blastocyst-stage FET, although LAH increases clinical pregnancy and implantation rates, its effect on live birth rates remains limited, highlighting the predominant influence of maternal factors and embryonic genetic competence at this stage. Future prospective randomized controlled trials should validate the independent effect of LAH on live birth outcomes across distinct embryonic stages (cleavage versus blastocyst) and defined subpopulations, particularly women with advanced maternal age or recurrent implantation failure, to establish optimal application strategies.
List of abbreviations
- LAH
Laser-assisted hatching
- AH
Assisted hatching
- FET
Frozen-thawed embryo transfer
- ZP
Zona pellucida
- AFC
Antral follicle count
- ART
Assisted reproductive technology
- ICM
Inner cell mass
- VS
Vitrification solution
- bFSH
Baseline follicle-stimulating hormone
- BMI
Body mass index
- ICSI
Intracytoplasmic sperm injection
- IVF
In vitro fertilization
- OHSS
Ovarian hyperstimulation syndrome
- PN
Pronuclei
- hCG
Human chorionic gonadotropin
- ES
Equilibration solution
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Affiliated Hospital of Youjiang Medical University for Nationalities (Ethics Review No.: YYFY-LL-2024–285). All procedures followed the Declaration of Helsinki. Informed consent was obtained from all participants.
Funding
This research was supported by the Guangxi Natural Science Foundation Project (2024JJA140002) and the Guangxi Medical and Health Appropriate Technology Development and Promotion Project (S2023110).
CRediT authorship contribution statement
Xing-Hong Chen: Data curation, Conceptualization. Yu-Lan Lu: Supervision, Methodology. Lin-Lin Hu: Visualization, Conceptualization. Jun-Li Wang: Investigation. Hai-Mei Qin: Methodology. Yu-Xia Wei: Writing – review & editing, Conceptualization. Bi-Yun Liao: Writing – review & editing, Writing – original draft, Data curation. Min-Min Ou: Writing – original draft, Validation, Data curation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank the participants who participated in this study, as well as all staff at the Reproductive Medicine Center, Affiliated Hospital of Youjiang Medical College for Nationalities, China, for their support in collecting the data.
Consent for publication
All the authors read and approved the final manuscript for publication
Footnotes
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.eurox.2026.100448.
Contributor Information
Min-Min Ou, Email: 1259606709@qq.com.
Bi-Yun Liao, Email: 1985795213@qq.com.
Xing-Hong Chen, Email: 1398953347@qq.com.
Lin-Lin Hu, Email: 751745135@qq.com.
Yu-Lan Lu, Email: 747475146@qq.com.
Hai-Mei Qin, Email: 903910211@qq.com.
Jun-Li Wang, Email: baiscwangjunli@163.com.
Yu-Xia Wei, Email: 359627186@qq.com.
Appendix A. Supplementary material
Supplementary material
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