Laser‐assisted hatching on clinical and neonatal outcomes in patients undergoing single vitrified Blastocyst transfer: A propensity score–matched study

Abstract Purpose This study determined the effect of laser‐assisted hatching on the clinical and neonatal outcomes of single vitrified blastocyst transfer. Methods From June 2014 to March 2018, 289 matched pairs after propensity score matching were analyzed. During the blastocyst warming procedure, a small section of the zona pellucida area in the empty perivitelline space was sliced off using multiple laser beams. The clinical and neonatal outcomes of the laser‐treated group and non‐treatment control were analyzed. Results In the laser‐assisted hatching group, significantly higher rates of clinical pregnancy (40.8% vs 29.4%, P < .01) and live delivery (34.3% vs 22.5%, P < .01) were observed compared to the control group. Other variables such as the average gestational weeks, the sex of the baby, birthweight, or congenital malformations were found to have no significant differences in neonatal outcomes. Moreover, all babies were singleton live births. Conclusions Single vitrified blastocyst transfer treated with laser‐assisted hatching increases the live birth rate and has no adverse effects on neonatal outcomes.

but the researchers did not find a significant difference in the LBR between the AH treatment group and a control group. 4,5 A US multicenter analysis from the National ART Surveillance System focused on AH procedures from 35,518 cycles and found that AH was not associated with improved pregnancy outcomes. 6 Also, the Society for Assisted Reproductive Technology Clinic Outcome Reporting System database found that AH slightly decreased the LBR in 151,533 first-cycle, autologous frozen embryo transfers (FETs). 7 However, data accuracy on this topic is still under examination.
Many meta-analyses did not evaluate the different techniques and protocols of AH, the different stages of the embryo, the type of cycle, or the indications for the procedure. 8 This heterogeneity was not well discussed in previous publications; thus, there are conflicting views on the effectiveness of AH among researchers.
Alteri et al 8 evaluated current AH protocols and found studies that support the benefit of ZP breaching in thawed blastocysts.
The integration of a laser application system and frozen blastocyst transfer is an effective treatment that improves the IVF success rate, due primarily to the following advantages: (a) AH might overcome ZP hardening, which is caused by freezing-thawing procedures and the prolonged embryo culture in vitro 9,10 ; (b) blastocyst transfer results in a more significant implantation potential than cleavage-stage embryos owing to the improvement in embryo selection and embryo-endometrium synchrony 11,12 ; and (c) frozen single blastocyst transfer results in a higher singleton LBR than a fresh single blastocyst transfer. 13 The laser system has become widely used in AH because it is simple and less damaging to the embryos. However, the potential effects of using laser for frozen blastocyst have not been proven, and reports regarding the LBR are limited. Because the clinical findings on the LBR and neonatal outcomes are insufficient, we conducted a retrospective cohort study using propensity score analysis to investigate the references of single vitrified blastocyst transfer with laser treatment.
This study determined the effect of laser-AH (LAH) on the clinical and neonatal outcomes of single vitrified blastocyst transfers among IVF patients.
F I G U R E 1 Patient inclusion flowchart 2 | MATERIAL S AND ME THODS

| Experimental data and patient selection
This study was a retrospective, observational study based on data

| Embryo culture
In the oocyte retrieval cycle, ovarian stimulation was achieved using standard gonadotropin-releasing hormone agonist/folliclestimulating hormone (FSH) protocols or an antagonist/FSH protocol. A vaginal ultrasound-guided follicle puncture was conducted 36 hours after injection of human chorionic gonadotropin. The retrieved oocytes were inseminated by conventional IVF or intracytoplasmic sperm injection in accordance with a previously reported method. 14,15 The oocytes with two pronuclei and a second polar body at 17-19 hours following insemination were defined as normally fertilized. They were cultured for 5 days in a medium (global ® ; Life Global) and supplemented with recombinant human albumin (G-MM; Vitrolife) at 37°C in 6% CO 2 , 5% O 2 , and 89% N 2 .

| Blastocyst vitrification and warming
All blastocysts were vitrified and warmed using Kitazato vitrification and warming media (Kitazato Company), as described previously. 16 On day 5, some blastocysts were transferred in fresh cycles, and surplus viable blastocysts were vitrified using open-system Cryotop ® (Kitazato Company). The blastocyst with a diameter greater than 150 µm was treated in 0.2 M hyperosmotic sucrose solution and mechanically punctured using a micro-needle following a published report. 17 The inner cell mass of the blastocyst was located at the 12 o'clock position, and the injection pipette punctured the blastocyst at the 3 o'clock position. The blastocoel fluid was completely aspirated with negative pressure, and then it was moved into the equilibration solution. The blastocyst was immersed in the equilibration solution within 15 minutes and sequentially exposed in the vitrification solution for 1.5 minutes. The blastocyst was immediately transferred to the Cryotop and submerged into the liquid nitrogen. After being stored in the storage tank for several months, the blastocyst was warmed in the thawing solution for 2 minutes and sequentially transferred to the dilution solution for 3 minutes.
They were then transferred into the washing solution for 5 minutes.
All were cultured overnight (≧16 hours), and the blastocyst that reexpanded and having greater than 50% of the morphologically intact cells was considered as survival.

| LAH procedure
Laser treatment (Saturn 5; Research Instruments Ltd.) was performed during the blastocyst warming procedure in the dilution solution. A small section of the ZP area, where the empty perivitelline space was found, was sliced off using 10-15 laser beams ( Figure 2).
The laser pulse length was 999 µs, and a hole size was 18 µm.

| Blastocyst scoring and single vitrified blastocyst transfer
Before the vitrified blastocyst transfer, the blastocyst quality was graded according to a previous report. 18

| Follow-up and evaluation index
Implantation was confirmed when a gestational sac was visualized via an ultrasound examination, and clinical pregnancy was confirmed by the detection of a fetal heartbeat. LBR was calculated by dividing the live birth delivery cycles by the transfer cycles. The evaluated neonatal outcomes included the delivery method, weeks of gestational age, sex, birthweight, and neonatal malformations.

| Study outcomes
The primary outcome was LBR. The secondary outcomes were the rate of complete hatching, CPR, and neonatal outcomes, including average gestational weeks, birthweight, malformation rate, and twin birth rate.

| Statistical analysis
All statistical analyses were conducted using EZR (Saitama Medical Center, Jichi Medical University). 19 Continuous variables are represented as means ± SD. The data were evaluated using chi-square test, chi-square test for contingency tables, and Mann-Whitney U test to compare the LAH treatment group and the control. P < .05 was considered statistically significant. The patient characteristics including the woman's age at oocyte pickup and FET, previous failed cycles, a type of infertility, insemination methods, type of endometrial preparation and infertile cause were analyzed using propensity score matching conducted on adjusting for potential confounders in this study. It was judged to be balanced if the absolute value of the standardized mean difference (SMD) is <0.1.

| Sample size estimation
It is difficult to define the sample size based on the published studies because there were no statistically significant differences in the effect of LAH on LBR using vitrified blastocysts. 8 One study found a 13% difference in the LBR between the LAH treatment group and the control group. 20 Based on that report, 558 transfer cycles (279 per group) are required to obtain an 90% chance of detecting (at a 5% significance level) an increase in the primary outcome measure from 28% in the control group to 41% in the LAH treatment group.

| Patient characteristics
A total of 1180 FET cycles were performed during the study period.
Of these, 482 cycles were excluded because of the exclusion criteria shown in Figure 1. The remaining 698 cycles, including 340 cycles in the LAH group and 358 cycles in control, were available for analysis and slight differences in women's age at FET (P = .14) and infertility cause (P = .08). All covariates were in good balance after propensity score matching. The 289 matched pairs were analyzed for differences in patient characteristics: 201 women who underwent the LAH treatment and 196 women in the control group; 42 women out of all experienced both treatments in different FET cycles (Table 1).

| Comparison of clinical outcomes
The clinical outcomes for the two groups are presented in Table 2.

| Comparison of neonatal outcomes
The neonatal outcomes of 153 children born after transfer are presented in Table 3. Live birth data were collected from 93 babies in the LAH treatment group and 60 babies in the control group. There were no differences between the LAH treatment and control groups in terms of delivery style, average gestational age weeks, the sex of the baby, or birthweight. One baby with muscular dystrophy was born in the LAH and a baby with a cleft lip in control. The rate of malformations did not differ between the groups.

| D ISCUSS I ON
The main question of the current study is whether the use of LAH However, in studies focusing on LAH, a surprising benefit was found when LAH was used along with a vitrified blastocyst, in which the ZP was widely opened (around a one-quarter to one-half opening of the ZP) with multiple laser shots, 20,24 or was removed entirely. 21 Alternatively, LAH was not effective on ZP hole opening, drilling, or thinning when the laser was applied on cleavage-stage embryos or blastocysts in the FET cycle. 8 Also, no benefits were found for L AH with a ZP opening of any size in fresh embryos, including those in the blastocyst stage. 8 In this study, LAH was performed during the warming procedure in a dilution solution. The procedure was completed within 2 minutes, with still enough time to finish the work during blastocyst warming. Blastocyst warming in a dilution solution resulted in a large perivitelline space, which allowed a small section of the ZP to be sliced easily and safely using a noncontact laser beam.
Hiraoka et al 23 were the first to report that ZP removal using multiple laser shots and mechanical pipetting improved the clinical outcomes of vitrified blastocysts compared with a single-hole ZP opening and the control group. After LAH treatment, the ZP-free blastocyst increases the expression of integrin, which is the fibronectin receptor and is considered the critical outgrowth for blastocyst attachment and adhesion to the endometrial epithelium. 25 The current study suggests that 83.4% of LAH blastocysts completely escaped from the ZP after overnight culture and improved the CPR and LBR compared with the control group.
Extending the culture of vitrified blastocysts would be helpful for measuring embryonic development and improving CPR and LBR. 26 Also, these did not affect either the clinical or neonatal outcomes in the current study.
After escaping the ZP, the blastocyst is sticky, which may provide a definite advantage in terms of increasing the chance of cell attachment to the endometrium of the uterus after transfer.
Simultaneously, this stickiness is considered a significant disadvantage in the laboratory, where it tends to adhere tenaciously to the surface of the plastic culture dish, handling pipette, and embryo transfer catheter. 9 Therefore, some embryologists prefer not to handle hatched blastocysts and avoid the potential risks involved with handling one. To resolve this issue, the use of inorganic or organic macromolecules, such as polyvinyl alcohol or albumin, is useful. In this study, all naked blastocysts were handled using PVP-coated pipette and enriched albumin medium; therefore, we did not have an issue with adhesion to the equipment. We consider reliable embryo handling to be an essential key for a successful transfer, which can result in high CPR and LBR.
The follow-up studies have been conducted to evaluate the effect of AH, including fresh cycles of cleavage-stage embryos treated with acidic Tyrode's solution 27,28 and laser for ZP drilling, 29 and frozen cycles on cleavage-stage embryos treated with laser ZP thinning 30,31 and on blastocyst stage treated with laser ZP thinning or bleaching. 32 AH would be considered a safe method of ART because it has no adverse effects on obstetrical and neonatal outcomes, including congenital malformation. 30,32,33 We evaluated neonatal outcome data of vitrified blastocysts with LAH from 93 babies and from 60 babies in the control group. No differences in the average gestational weeks, sex of the child, birthweight, or malformations of the newborn were found that are consistent with a previous report. 34 In addition, no monozygotic twins occurred in the current study, which used a single blastocyst transfer in FET cycles. The risk of monozygotic twinning would not be increased in widely ZP opening but in ZP drilling and thinning. 33,35,36 However, the overall number of reports is small.
This study was analyzed the propensity score-matched data, and the potential biases were removed. However, difficulty in removing the bias could also be a limitation. blastocysts, which were graded "Good" after overnight culture ( One may have a concern about comparing the clinical outcomes between the LAH and control groups, because the LAH group was

ACK N OWLED G EM ENTS
The authors would like to thank Enago (www.enago.jp) for the English-language review.

E TH I C A L A PPROVA L
The study design was approved by the appropriate ethics committee of Kurashiki Medical Clinic, Okayama, Japan.