Controlled hatching at the prescribed site using femtosecond laser for zona pellucida drilling at the early blastocyst stage.

PURPOSE: To study whether the application of femtosecond laser pulses for zona pellucida (ZP) drilling of blastocysts at the embryonic or abembryonic poles can promote hatching to start immediately through the hole formed and ensure high hatching rates and embryo viability. METHODS: Mouse blastocyst (E3.5) ZP were microdissected with femtosecond laser pulses (514-nm wavelength, 280-fs pulse duration, 2.5-kHz repetition rate) close to the trophoblast or inner cell mass (ICM). The sizes of the holes formed were in the range of 4.5-8.5 µm. Additional longitudinal incisions (5-7-µm long) on either side of the hole were created to determine whether hatching had started at the correct position. Embryos post-laser-assisted ZP drilling and intact embryos were cultured under standard conditions for 2 days; embryo quality was assessed twice daily. The hatching rates and in vitro and in vivo implantation rates (only for embryos with ZP dissected close to the ICM) were estimated. RESULTS: Femtosecond laser-assisted ZP drilling at the early blastocyst stage facilitated embryo hatching to start at the artificial opening with probability approaching 100%. Despite the artificial opening's small size, no embryo trapping during hatching was observed. Both experimental groups had higher hatching rates than the control groups (93.3-94.7% vs. 83.3-85.7%, respectively). The in vitro implantation rate was comparable with that of the control group (92.3% vs. 95.4%). No statistically significant differences were obtained in the in vivo implantation rates between the experimental and control groups. CONCLUSIONS: Blastocyst-stage femtosecond laser microsurgery of ZP is fast and delicate and enables the hatching process to be initiated in a controlled manner through a relatively small opening, with no embryo trapping.

Higher implantation and live birth rates with laser zona pellucida breaching than thinning in single frozen-thawed blastocyst transfer.

While zona pellucida (ZP) breaching of day-3 frozen blastocysts embryos can increase the blastocyst hatching rate, compared with ZP thinning, the pregnancy and implantation rates are similar. The aim of this study was to compare pregnancy outcomes and the risks associated with frozen-thawed blastocysts between laser ZP breaching and laser ZP thinning. For the thinning group, ZP of thawed blastocyst was thinned to a length of 30-40 µm using laser between January 2013 and October 2015. On the other hand, for the breaching group, thawed blastocysts were breached with a 60-80 µm hole in the ZP using laser between November 2015 and April 2018. The implantation rate of ZP breaching (72.7%) was higher than that of ZP thinning (61.8%). In single frozen blastocyst transfer, the implantation rate, clinical pregnancy rate, and live birth rate of ZP breaching (73.9%, 73.9%, 61.8%, respectively) were significantly higher than those of ZP thinning (60.9%, 60.9%, 46.7%, respectively). The abortion rate, preterm birth rate, congenital malformation, birth defects, and birth weight did not significantly differ between the two groups. In conclusion, laser assisted hatching during single frozen blastocyst transfer using ZP breaching exhibit higher implantation, pregnancy, and live birth rates compared with ZP thinning. No significant differences were observed between the two assisted hatching methods in terms of adverse effects on pregnancy and newborns.

The impacts of laser zona thinning on hatching and implantation of vitrified-warmed mouse embryos.

Embryo vitrification has advantages in assisted reproduction yet it also induces zona hardening. Laser zona thinning (LZT) is considered as a solution yet its efficacy and security have not been well studied. In this study, we used vitrified-warmed morulae from 2-month-old and 10-month-old ICR female mice as model to investigate the impacts that LZT treatment brings to the in vitro hatching process and implantation by analyzing hatching rate, implantation rate, and blastocyst quality. The results showed that the fully hatched rate was significantly higher after LZT treatment for both young (25.7% vs. 16.2%, P < 0.05) and aged (36.6% vs. 13.2%, P < 0.01) mice. For zona-thinned morulae in young mice, its onset of hatching occurred earlier (28.6% vs. 8.8%, P < 0.01) at D4 and with a greater percentage of U-shaped hatching at D5 (48.3% vs. 33.0%, P < 0.05). LZT treatment did not induce expression change of apoptosis-related genes in all groups (P > 0.05), but for young mice, the total cell number of day 5 blastocyst in zona-thinned group was significantly less than that of the control group (40.6 ± 5.1 vs. 59.9 ± 14.5, P < 0.01). At last, there was an increasing implantation rate in zona-thinned compared to the control group for young (63.8% vs. 52.5%, P > 0.05) and aged (55.6% vs. 47.2%; P > 0.05) mice after embryos were bilaterally transferred in the same recipient. In conclusion, the significant increase of fully hatched rate after LZT treatment is related to the advanced onset of hatching as well as the enhancement of superior hatching structure, and LZT also lead to a better implantation after embryo transfer.

Effect of laser-assisted zona thinning, during assisted reproduction, on pregnancy outcome in women with endometriosis: randomized controlled trial.

OBJECTIVE: To compare the ICSI-ET outcomes in patients with endometriosis with or without laser-assisted zona pellucida thinning. DESIGN: Randomized controlled trial. SETTING: The study was conducted in the Obstetrics & Gynecology Department, Cairo University hospital, and two private IVF centers in Cairo & Beni-Suif from July 2015 to January 2017 upon infertile and known endometriosis patients who planned to do ICSI-ET. INTERVENTIONS: Before randomization, all patients received the same ovarian stimulation preparation, oocyte retrieval procedures, and the same intracytoplasmic sperm injection procedures. After randomization, laser-assisted hatching was performed only for embryos of 158 patients, while the other group (n = 150) no laser-assisted hatching was made. The verification of pregnancy was achieved by the serum hCG concentration 14 days after the embryo transfer, and the clinical pregnancy was confirmed 2 weeks later by the presence of gestational sac with pulsating fetal pole on vaginal ultrasonography. MEASUREMENTS: The main outcome measures were the clinical pregnancy rate and the clinical implantation rate. MAIN RESULTS: Both groups were comparable with regard their baseline characteristics, baseline hormonal profile, the ovarian stimulation characteristics, and the ovulation characteristics. The mean number of embryos developed per patient and the mean transferred number of embryos per patient were comparable between groups (p value > 0.05). The implantation rate was significantly higher (p value 0.002) in the study group than the control group with an odds ratio of 1.86 (CI 95% 1.24-2.80) and NNT 13.81 (CI 95% 8.35-39.94). The clinical pregnancy rate, was significantly (p value 0.022) higher in the study group than in the control group with an odds ratio of 1.79 (CI 95% 1.05-3.06) and NNT 9.57 (CI 95% 5.03-98.99). CONCLUSION: That laser-assisted hatching by thinning of the zona pellucida may be a suitable method to improve the ICSI-ET outcomes, in term of the implantation and the pregnancy rates, in cases of endometriosis. CLINICAL TRIAL REGISTRATION: Pan African Clinical Trials Registry (PACTR), http://www.pactr.org/ATMWeb/appmanager/atm/atmregistry?dar=true&tNo=PACTR201502001022393 , PACTR201602001467322.

A Prospective Randomized Controlled Study of Laser-Assisted Hatching on the Outcome of First Fresh IVF-ET Cycle in Advanced Age Women.

There is no sufficient data to conclude the benefit of assisted hatching (AH) for advanced age patients. However, AH is routinely performed for advanced age patients undergoing in vitro fertilization (IVF) in China based on some retrospective evidence. It is important to assess the benefit of AH procedure for advanced age patients, especially by analyzing the data from China. This is a prospective randomized controlled trial to evaluate the effect of laser AH in the advanced age patients undergoing IVF. A total of 256 patients conformed to the inclusion criteria, and 78 were excluded by exclusion criteria. A total of 178 patients were eligible and randomized to 2 groups (82 AH group and 96 control group). Laser AH (zona thinning) was performed in the AH group. There were no statistical significance in basic clinical parameters between the 2 groups. No difference was found in implantation rate (AH vs control, 32.45% vs 39.29%) and clinical pregnancy rate (AH vs control, 48.78% vs 59.38%). Our data did not find any benefit of laser AH in improving implantation or pregnancy rates in advanced age women. Due to the potential risk and increasing financial burden, AH should not be routinely performed in first fresh IVF embryo transfer cycle for advanced age women.

Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.

Multi-Pulse laser ablation modeling with applications to automated zona removal.

Laser zona drilling (LZD), the ablation of a portion of the zona pellucida (ZP) in embryos with the use of a laser, is a required step in many embryonic surgical procedures such as assisted hatching and preimplantation genetic diagnosis. The objective of LZD is to remove specific locations of the ZP while minimizing potential harmful thermal effects to important structures of the embryo, namely the blastomeres. Current thermal analyzes of lasers used in LZD only encompass the use of a single pulse, whereas LZD is typically performed using multiple pulses. In this paper we analyze the effect of multipulse LZD and introduce a linear approximation method for multi-pulse LZD. Furthermore, we describe a novel method of measuring the thermal effect of a single laser pulse using the thermosensitive fluorescent dye Rhodamine B and a high speed camera.

Effect of red light on the development and quality of mammalian embryos.

PURPOSE: To assess irradiance and total energy dose from different microscopes during the in-vitro embryonic developmental cycle in mouse and pig and to evaluate its effect on embryonic development and quality in pig. METHOD: Spectral scalar irradiance (380-1050 nm) was measured by a fiber-optic microsensor in the focal plane of a dissection microscope, an inverted microscope and a time-lapse incubation system. Furthermore, the effect of three different red light levels was tested in the time-lapse system on mouse zygotes for 5 days, and on porcine zona-intact and zona-free parthenogenetically activated (PA) embryos for 6 days. RESULTS: The time-lapse system used red light centered at 625 nm and with a lower irradiance level as compared to the white light irradiance levels on the dissection and inverted microscopes, which included more energetic radiation <550 nm. Even after 1000 times higher total energy dose of red light exposure in the time-lapse system, no significant difference was found neither in blastocyst development of mouse zygotes nor in blastocyst rates and total cell number of blastocysts of porcine PA embryos. CONCLUSIONS: Our results indicate that red light (625 nm, 0.34 W/m(2)) used in the time-lapse incubation system does not decrease the development and quality of blastocysts in both mouse zygotes and porcine PA embryos (both zona-intact and zona-free).

A study comparing three different laser-assisted hatching techniques.

PURPOSE OF INVESTIGATION: Laser-assisted hatching (LAH) is recognized as a useful technology to improve clinical pregnancy rates and implantation rates. This study reports the differences between a new LAH method and two conventional LAH techniques. MATERIALS AND METHODS: The authors studied 151 patients with repeated implantation failure, who were divided into three groups. RESULTS: In group 1, the zona pellucida (ZP) was opened using LAH (n = 52). In group 2, laser-assisted thinning was performed to dissolve the outer layer of the ZP (n = 49). In group 3, laser-assisted thinning was performed to dissolve the inner layer of the ZP (n = 50). The clinical pregnancy rates and implantation rates among the groups were compared. The results demonstrate that there are significant differences in the clinical pregnancy rates and implantation rates between group 3 and the other two groups. CONCLUSION: Performing laser-assisted thinning to dissolve the inner layer of the ZP markedly increases the pregnancy rates and implantation rates of patients with repeated implantation failure.

The effect of modified quarter laser-assisted zona thinning on the implantation rate per embryo in frozen/vitrified-thawed/warmed embryo transfer cycles: a prospective randomized controlled trial.

BACKGROUND: Freezing/vitrifying and thawing/warming of embryos may impair the successful hatching process of the embryo out of its zona pellucida (ZP) and its following implantation into the uterus. Theoretically, assisted hatching (AH) may facilitate the hatching process and subsequently increase implantation rates (IRs). METHODS: In this prospective randomized controlled trial (RCT), the hypothesis was tested that the IR per embryo transferred is higher after transfer (ET) of frozen/vitrified-thawed/warmed embryos with thinned ZP after AH by modified quarter laser-assisted zona thinning (mQLAZT) when compared with ET of frozen/vitrified-thawed/warmed embryos without mQLAZT. Patients with frozen/vitrified embryos were randomized at the time of thawing/warming to a study group (with mQLAZT) or a control group (without mQLAZT). After thawing/warming, embryos were kept in culture for 24h, and mQLAZT was performed prior to ET. RESULTS: A total of 647 thawing cycles were randomized to either the mQLAZT group (n = 324) or the control group (n = 323). Reproductive outcome data were available for 302 cycles in the mQLAZT group and 317 cycles in the control group. Transfer could be performed in 73.5% and in 71.9% of the thawing/warming cycles in the mQLAZT group and the control group (P = 0.78), respectively. No significant differences were observed between the mQLAZT group and the control group for the IR [13.3%; 15.6%; rate ratio 0.85; 95% confidence interval (CI), 0.596-1.224], the ongoing IR (10.5 and 13.5%, P = 0.25) and the live birth rate [10.5%;13.3%; rate ratio 0.79; (95% CI), 0.530-1.189] per embryo transferred. CONCLUSIONS: In this RCT, mQLAZT did not improve the IR per embryo transferred in frozen/vitrified-thawed/warmed embryo transfer cycles. ClinicalTrials.govID NCT00593775.

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa.

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 µm² vs. 18.8 ± 1.4 µm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Impact of the size of zona pellucida thinning area on vitrified-warmed cleavage-stage embryo transfers: a prospective, randomized study.

PURPOSE: The aim of this study was to determine if the size of zona pellucida thinning area by laser assisted hatching could affect the rates of pregnancy and implantation for vitrified-warmed embryo transfers at the cleavage-stage. METHODS: A total of 120 vitrified-warmed cleavage-stage embryo transfers were randomly assigned to either quarter or half of zona pellucida thinning group. RESULTS: The rates of clinical pregnancy (46.7 versus 25.0%) and implantation (32.0 versus 16.2%) were significantly greater in the half thinning group than in the quarter thinning group (P = 0.0218 and P = 0.0090, respectively). CONCLUSIONS: The results of this investigation show that, in vitrified-warmed embryo transfers at the cleavage-stage, the size of zona pellucida thinning area by laser assisted hatching impacts the rate of clinical pregnancy and implantation and that half of zona pellucida thinning significantly increases both of these results compared with quarter of zona pellucida thinning.

Impact of assisted hatching on fresh and frozen-thawed embryo transfer cycles: a prospective, randomized study.

The aim of this study was to determine if assisted hatching (AH) could improve the rates of pregnancy and implantation for both fresh and frozen-thawed embryo transfer cycles. A total of 760 fresh embryo transfer cycles and 200 frozen-thawed embryo transfer cycles were randomly assigned to either the treatment group (AH) or the control group (no AH). Zona thinning by laser was performed just before embryo transfer. In fresh embryo transfer cycles, the AH group and control group results were comparable. There were no significant differences in the rates of positive human chorionic gonadotrophin (HCG; 47.5 versus 48.8%), clinical pregnancy (42.4 versus 42.6%), or implantation (26.3 versus 25.2%) between the two groups. However, in frozen-thawed embryo transfer cycles, the rates of positive HCG (32.0 versus 17.0%), clinical pregnancy (25.0 versus 14.0%) and implantation (16.7 versus 7.3%) were significantly greater in the AH group than in the control group (P <: 0.05). The results of this investigation show that in the fresh embryo transfer cycles, laser-assisted hatching by zona thinning has no impact on the rates of positive HCG, clinical pregnancy and implantation, whereas in frozen-thawed cycles, assisted hatching by zona thinning significantly increases all three of these rates.

Effect of laser zona pellucida opening on clinical outcome of assisted reproduction technology in patients with advanced female age, recurrent implantation failure, or frozen-thawed embryos.

OBJECTIVE: To determine whether laser-assisted hatching can improve clinical outcome of assisted reproductive techniques in patients with advanced female age, with recurrent implantation failure, or who are using frozen-thawed embryos. DESIGN: A prospective randomized study. SETTING: The infertility and IVF unit at a research facility in Iran. PATIENT(S): Four hundred ten patients with advanced female age (> or =37 y), 796 patients with recurrent implantation failure (for > or =2 cycles), and 180 patients with frozen-thawed embryos. INTERVENTION(S): Patients were divided equally into test and control groups. On the day of embryo transfer, the zona pellucida of the selected embryos in the test group were opened about 40 mum by using an infrared optical laser system, whereas in the control group they were all intact. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates and implantation rates. RESULT(S): In the patients with advanced female age or recurrent implantation failure, the clinical pregnancy and implantation rates were similar for the test and control groups. However, in the patients with frozen-thawed embryos, the rates were statistically significantly higher in the test group as compared with those of the control group (31.2% and 12.8% vs. 11.1% and 4.2%, respectively). CONCLUSION(S): The laser-assisted hatching improved the pregnancy and implantation rates in patients with frozen-thawed embryos but had no effect in patients with advanced female age or recurrent implantation failure.

Comparison of laser-assisted hatching and acidified Tyrode's hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial.

OBJECTIVE: To assess two zona drilling methods in terms of blastocyst development rates using sister embryos. DESIGN: Prospective, randomized study. Sister embryos of 14 patients were randomly assigned on day 3 to acidified Tyrode's zona drilling or to laser zona drilling. After biopsy, subsequent embryo culture until the blastocyst stage (day 5) was performed. SETTING: Private fertility center. PATIENT(S): Patients undergoing IVF-preimplantation genetic diagnosis. INTERVENTION(S): Embryo biopsy using either laser-assisted hatching or acidified Tyrode's hatching on sibling embryos and subsequent blastocyst development evaluation. MAIN OUTCOME MEASURE(S): Evaluation of blastocyst development in terms of degree of expansion and cell number in the inner cell mass and trophectoderm. RESULT(S): Blastocyst development rates (and blastocyst quality) were similarly high in both the acidified Tyrode's hatching group and the laser-assisted hatching group. CONCLUSION(S): Laser hatching does not impair embryonic development to the blastocyst stage, demonstrating that laser-assisted hatching is a suitable alternative to the use of acidified Tyrode's solution for zona drilling.

Implantation failures: success of assisted hatching with quarter-laser zona thinning.

Implantation failure after IVF is one of the factors associated with a reduced chance of pregnancy for some patients. Assisted hatching methodologies are designed to facilitate the embryo's escape from the zona pellucida, and this strategy has been suggested as a means of improving pregnancy rates in patients with previous implantation failure. The aim of this prospective and randomized study was to evaluate the efficacy of quarter-laser zona thinning assisted hatching (qLZT-AH) in improving the implantation of embryos in patients with previous implantation failure. A total of 150 patients with a history of previous implantation failure were treated with intracytoplasmic sperm injection, and allocated into two groups: group 1, only one previous implantation failure, and group 2, repeated implantation failures. The patients in each group were randomized at the time of embryo transfer into a control group (no qLZT-AH) or experimental group where qLZT-AH was performed. For patients with repeated implantation failures, the implantation rate in those who received laser-thinned embryos was significantly higher (P = 0.02) than in those whose embryos were not laser-thinned (10.9 and 2.6% respectively). However, this difference was not observed in patients who presented with only one previous implantation failure. The data demonstrate that qLZT-AH is an effective strategy for improving the implantation of embryos in patients with repeated implantation failures.

A European multicentre prospective randomized study to assess the use of assisted hatching with a diode laser and the benefit of an immunosuppressive/antibiotic treatment in different patient populations.

BACKGROUND: Assisted hatching (AH) techniques, designed for facilitating the embryo escape out of the zona pellucida (ZP) have been used in IVF centres since 1992. The initial indications for AH were patient's age, ZP thickness, high basal FSH and repeated IVF failures. Several retrospective and prospective studies assessing AH in these indications have given disparate results. Our aims were to evaluate the benefits of AH and immunosuppressive/antibiotic treatment (IA) in patients with either a poor prognosis of success, previous implantation failures or transfers of cryopreserved embryos. METHODS: Four IVF centres allocated 426 patients, randomized for AH and IA, into four groups of AH indications between 1997 and 1999. AH was performed with a diode laser. ZP thickness, opening size and embryo score were recorded. Outcome measures were implantation and delivery rates. RESULTS: Patients coming for a first or third transfer of cryopreserved embryos and poor prognosis patients admitted for a first trial did not benefit from AH. Even patients with repeated implantation failures of fresh embryos did not gain significantly from AH. CONCLUSIONS: Among AH indications, absence of implantation after several transfers of good quality embryos remains the strongest patient selection criterion. Prescription of an immunosuppressive/antibiotic treatment is essential.