Laser-assisted Hatching Research Articles

Human oocyte zona pellucida abnormalities: evaluation of clinical impact for different zona pellucida abnormalities and role of using assisted hatching.

Zona pellucida (ZP) plays an important role in oocyte development and fertilisation, as well as in early embryo development. However, there is currently no exploration of systematic categorising and clinical treatment measures for patients with abnormal ZP, especially the fast and easy method that can be used clinically. In this study, 185 patients with abnormal ZP (ZPA) and 222 patients with normal ZP (ZPN) were enrolled and analysed. Patients with ZPA exhibited altered hormone levels and lower laboratory outcomes in in vitro fertilisation (IVF), such as reduced MII rate, cleavage rate, high-quality embryo rate, and blastocyst formation rate compared to ZPN group. ZPA patients were sub-categorised into ZPA-A/B/C/D group according to the status of oocyte polar body and the width of perivitelline space (PVS). There are also differences in the degree of sperm binding to ZP derived from different ZPA patients and the elasticity of the oocyte membrane. ZPA subgroup analysis revealed further disparities in various IVF parameters and pregnancy outcomes. In addition, by performing different treatments on oocytes derived from ZPA patients, we found that ZP assisted hatching before the first oocyte cleavage on D1 of embryonic development in the ZPA-A/B groups and before blastocyst expansion on D4 of embryonic development in the ZPA-C/D groups were beneficial to improve embryonic development. The degree of the impact on clinical outcomes is correlated with the types of ZPA, and laser-assisted hatching of the ZP helps to improve embryonic development in patients with ZPA.

P-109 Non-invasive Genetic Testing (niPGTA) and Laser Assisted Hatching (LAH) may improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age

Abstract Study question Does niPGTA and LAH improve pregnancy and ongoing pregnancy rates in patients of advanced maternal age? Summary answer niPGTA increases pregnancy and clinical pregnancy rates in patients aged ≥38 years compared to those without tested embryos, while LAH may further improve pregnancy outcomes. What is known already Embryos release cell-free DNA (cf-DNA) in the IVF culture media, which can be analyzed for chromosomal constitution. The benefit that niPGTA holds over PGT-A is that it is a non-invasive test and could potentially produce comparable results. Therefore, the time to pregnancy and the risk of miscarriage could be reduced in comparison to an IVF treatment with non-tested embryos. Previous studies have shown high concordance with trophectoderm biopsies in PGT-A patients and improved outcomes with LAH in frozen-thawed cycles. However, there is a lack of studies investigating clinical pregnancy rates with niPGTA. Study design, size, duration Retrospective cohort study including 74 patients aged ≥38 years, who underwent Single Frozen Embryo Transfer (FET) from May 2021 to February 2023. Patients who had Endometrial Receptivity Assay (ERA) were excluded. Participants/materials, setting, methods 32 couples had niPGTA; Spent Blastocyst Media (SBM) for each blastocyst were collected on Day 6 and analyzed using the EMBRACE test. Embryos were vitrified. 42 couples had their blastocysts vitrified on Day 5/6 without testing. LAH was introduced to all patients in October 2021. Before that, no LAH was performed. Embryos were transferred to patients 2 hours post-warming after LAH. Pregnancy test was performed 10 days after FET and clinical pregnancy was established. Main results and the role of chance There was a statistical significant increase of pregnancy rate (p = 0.017, Pearson’s Chi-Squared test) and a trend towards higher clinical pregnancy rate for niPGTA patients versus those who had a transfer of not tested embryos. Regarding LAH, there was a trend towards higher pregnancy and clinical pregnancy rates in both niPGTA and not tested group. Results for pregnancies and clinical pregnancies are presented in Table 1. Limitations, reasons for caution More patients are required to confirm clinical significance of pregnancy and clinical pregnancy rates of niPGTA over not tested embryos and if LAH can improve the outcomes. A review of patients who had previous implantation failures and miscarriages in addition to ERA could yield clearer conclusions. Wider implications of the findings The results of this study are encouraging for niPGTA combined with LAH for patients aged ≥38 years. Further studies on niPGTA and modifications of the protocol for increased concordance rates and shorter time in embryo culture should be explored. Trial registration number Not applicable

P-193 Does the day of laser-assisted hatching affects the clinical outcomes of single vitrified blastocyst transfer cycles? A propensity score–matched study

Abstract Study question What is the impact of different days of laser-assisted hatching on the clinical outcomes of single vitrified blastocyst transfer cycles? Summary answer Clinical pregnancy of single vitrified blastocyst were similar, irrespective of day of laser-assisted hatching. What is known already Assisted hatching aims to create a weakness in the ZP to aid embryo hatching and implantation. Day 5 assisted hatching is typically performed shortly before the embryo transfer while day 3 assisted hatching is performed before blastocyst culture expansion and vitrification. Despite over 25 years of use, there is still no standardized protocol for AH, and published data on its efficacy are controversial and inconclusive. While efforts have been made to study the link between AH techniques and pregnancy outcomes, there is limited consensus for optimizing AH protocols like the day of performance. Study design, size, duration This was a retrospective study based on data collected between January 2022 and October 2023. Propensity score matching (PSM) was performed in a 1:1 matching ratio. 123 patients of day 3 laser assisted hatching group (D3 LAH group) were successfully matched to 123 cycles of day 5 laser assisted hatching group (D5 LAH group). The D5 LAH group was from March 2023 to October 2023, and the D3 LAH from January 2022 to February 2023. Participants/materials, setting, methods Single vitrified day 5 embryo transfer, embryos survived after warming, and patient agreed to receive LAH were include in this study. We excluded donation cycles, missing data, severe female factors. Primary outcome is clinical pregnancy. Comparisons between groups were performed with chi-square test, Fisher’s exact test, Mann–Whitney U test, and Student’s t-test where appropriate, with a P value <0.05 considered statistically significant. A multivariable analysis in the GEE model was adjusted for the confounding factors. Main results and the role of chance After propensity score matching, there was no significant difference in patient characteristics between the groups, creating similar cohorts. For example, the frequency of primary infertility was significantly different between groups initially (61% vs 46.7% in D5 LAH vs D3 LAH respectively, P = 0.004). Following propensity score matching the rate of primary infertility was similar in both groups (with SMD=0.08). Standardized mean difference (SMD) < 0.1 suggests adequate variable balance after propensity matching. Clinical pregnancy rate were similar between two groups: 66/123 (53.7%) D3 LAH cycles and 75/123 (61%) D5 LAH cycles. When generalized estimating equation were performed: only TE grade, but not the day of laser assisted hatching, was noted to be an independent predictor of clinical pregnancy (OR = 0.068; 95% CI: 0.005-0.977; p = 0.048). Poor TE grade had a weak negative relationship with clinical pregnancy of single blastocyst cycles. Limitations, reasons for caution The main limitation of the study is its retrospective design and small sample size. Wider implications of the findings Clinical outcomes are comparable between day 3 laser assisted hatching and day 5 laser assisted hatching, although clinical pregnancy can be slightly improved with higher TE grade. Trial registration number not applicable

P-152 The impact of one-step fast warming protocol on vitrified-warmed blastocysts on reproductive outcome

Abstract Study question Does the implementation of one-step fast warming protocol affect the post warming quality of embryos and/or the reproductive outcome in frozen embryo replacement cycles? Summary answer The implementation of a one-step fast warming protocol provides similar survival, pregnancy, and clinical pregnancy rates with standard warming protocols. What is known already Cryopreservation process presents a significant challenge for embryos, as the formation of ice crystals can be lethal to their cells. Therefore, effective cryopreservation protocols are essential to mitigate this risk. Vitrification method protects the cells during cryopreservation by transforming water from a liquid state to a glass-like phase, thereby preventing the ice crystals formation. Moreover, the warming procedure can itself contribute to ice crystal formation, particularly through recrystallization. Studies have shown that embryo survival is related to warming rate and a one-step fasting warming protocol can be equally or more effective than the conventional protocol. Study design, size, duration This prospective randomized study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between July and December 2023 and included 118 frozen embryo transfers. Both study and control group included only expanded blastocysts of good quality (AA or AB according to Gardner’s criteria). Control group was matched to the study group according to treatment, time period, woman age, quality of embryos and number of transferred embryos. The embryos were vitrified using open system. Participants/materials, setting, methods Fast warming group included embryos that were warmed using the one-step warming protocol: the embryos were exposed to 1M sucrose solution for 1’ at 37 °C and then immediately placed into culture media where laser-assisted hatching was performed. Control group included embryos that were warmed using conventional protocol: 1’ in 1M, 2’ in 0.5M, 2’ in 0.25M and 3’ in washing solution at room temperature and then placed into culture media for laser-assisted hatching. Main results and the role of chance The analyzed cases were 118 in total, 59 in the fast-warming group and 59 in the control group. Survival rate was 100% in both groups. Pregnancy rate (positive b-HCG) was not significantly different between the two groups: 72.41% in the fast-warming group and 67.79% in the control group (p = 0.687). Clinical pregnancy rate was also not significant between the two groups: 78.26% in the fast-warming group and 81.81% in the control group (p = 0.753). Limitations, reasons for caution The study included a limited number of cases, and information on the live birth rate was still pending. To establish the efficacy of fast warming conclusively, it is recommended that additional prospective randomized studies be conducted to validate its potential benefits. Wider implications of the findings One-step fast warming protocol resulted in similar survival rates post warm compared to the conventional warming protocol, while pregnancy and clinical pregnancy rates were not significantly different. Furthermore, it is worth noting that by minimizing the duration of the warming protocol, the workflow efficiency in the IVF laboratory is enhanced. Trial registration number N/A

THE EFFECT OF LASER-ASSISTED HATCHING ON PREGNANCY OUTCOMES OF VITRIFICATION FROZEN EMBRYO TRANSFER

Highlights LAH might effectively increase pregnancy outcomes for frozen-thawed embryo transfer. Zona pellucida drilling might help increase pregnancy outcomes in FET. Abstract Background: Zona Pellucida (ZP) thickness of less than 16 mm is better for embryo implantation inside the endometrium. Laser Assisted Hatching (LAH) is commonly used, especially in non-contact mode, using a 1.48-um infrared diode laser beam because of its short exposure time, accurate positioning, simple operation, indirect contact, safety, and effectiveness. Objective: This paper describes the potential of laser-assisted hatching in biochemical pregnancy outcomes in Frozen Embryo Thawed transfers. Material and Method: The total number of patients enrolled in this study was 141. Patients were prospectively treated during embryo transfers at Pusat Fertilitas Bocah Indonesia, Primaya Hospital at Tangerang, Indonesia, from December 2020 until December 2021. Result: There were no significant differences between the LAH and no-LAH groups regarding average age, infertility duration, infertility type, and etiology of infertility (p>0.05). In the same line, the blastocyst (0.76 ± 0.87; 1.25 ± 1.08) compared with cleavage (0.72 ± 0.84; 0.67 ± 0.98) (p<0.534 and p< .032). There was no significant difference, even though clinically, the proportions of live births, multiple pregnancies, biochemical pregnancies, and ongoing pregnancies in the LAH group were not exceptions to the outcomes of failures and miscarriages. Conclusion: LAH did not appear to increase the pregnancy rate in this study. However, the methodology seems to reduce bias in this study by considering methodology for selecting FET embryos based on the cryopreserved condition, embryo quality, and precise LAH.

Case Report: The combined effect of endometrial receptivity array and laser assisted hatching on embryo implantation in repeated implantation failure case

This case report describes a couple with nine years of primary infertility and three failed in vitro fertilization (IVF) attempts that showed no reproductive abnormality. Negative results were observed following the conventional IVF protocol. The couple then insisted on laser-assisted hatching (LAH) and endometrial receptivity analysis (ERA). The ERA results showed a specific window for embryo implantation, and accordingly, the treatment was carried out, which resulted in seven good-quality oocytes, leading to the formation of four blastocyst-stage embryos. These embryos were then frozen, single embryo transfer (ET) was performed following the ERA results, and the embryos were successfully implanted. The βhCG level was 245 mIU/mL, indicating that the female had successfully conceived. This case highlights the challenges of a thick zona pellucida and recurrent implantation failure (RIF), defining how a personalized method combining both LAH and ERA could lead to successful implantation.

Two laser-assisted hatching methods of embryos in ART: a systematic review and meta-analysis

BackgroundLaser-assisted hatching (LAH) stands as the predominant technique for removing the zona pellucida (ZP) in embryos, primarily consisting of two methods: drilling laser-assisted hatching (D-LAH) and thinning laser-assisted hatching (T-LAH). Presently, both methods have limitations, and their comparative efficacy for embryo implantation and clinical pregnancy remains uncertain.AimEvaluate the impact of D-LAH and T-LAH on clinical pregnancy rates within assisted reproductive technology (ART).MethodsWe systematically searched electronic databases including PubMed, Web of Science, and Cochrane Library until July 20, 2022. This study encompassed observational studies and randomized controlled trials (RCTs). A 95% confidence interval (CI) was utilized for assessing the risk ratio (RR) of pregnancy outcomes. The level of heterogeneity was measured using I2 statistics, considering a value exceeding 50% as indicative of substantial heterogeneity.ResultsThe meta-analysis scrutinized 9 studies involving 2405 clinical pregnancies from D-LAH and 2239 from T-LAH. Findings suggested no considerable variation in the clinical pregnancy rates between the two techniques (RR = 0.93, 95% CI: 0.79–1.10, I2 = 71%, P = 0.41). Subgroup analyses also revealed no substantial differences. However, D-LAH exhibited a notably higher occurrence of singleton pregnancies compared to T-LAH (RR = 2.28, 95% CI: 1.08–4.82, I2 = 89%, P = 0.03). There were no noteworthy distinctions observed in other secondary outcomes encompassing implantation rate, multiple pregnancies, ongoing pregnancy, miscarriage, premature birth, and live birth.ConclusionBoth the primary findings and subgroup analyses showed no marked variance in clinical pregnancy rates between D-LAH and T-LAH. Therefore, patients with varying conditions should select their preferred LAH technique after assessing their individual situation. However, due to the restricted number of studies involved, accurately gauging the influence of these laser techniques on clinical outcomes is challenging, necessitating further RCTs and high-quality studies to enhance the success rate of ART.Trial registrationPROSPERO: CRD42022347066.

The effect of laser-assisted hatching on vitrified/warmed blastocysts: the ALADDIN randomized controlled trial

ObjectiveTo evaluate whether laser-mediated assisted hatching (AH) performed on vitrified/warmed blastocysts before embryo transfer can improve live birth rate. DesignThe “pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN)” is a 2-center comparative study with a parallel randomized controlled design. SubjectsParticipants were recruited between September 2018 and November 2021. They were aged 18 to 39 years, underwent non-donor IVF cycles, and were scheduled for elective single embryo transfer with vitrified/warmed blastocysts. Those with uterine abnormalities, BMI >35 kg/m2, severe male factor infertility, or performing preimplantation genetic testing were excluded. InterventionAssisted hatching was performed using a 1480 nm diode laser, removing approximately one-third of the zona pellucida with continuous 0.2 ms pulses applied from the 1 to 5 o’clock positions. Main Outcome MeasuresThe primary outcome was the live birth rate. Secondary endpoints included clinical pregnancy, miscarriage, multiple pregnancies, preterm births, obstetrical and neonatal complications, and congenital anomalies. ResultsOverall, 698 participants met the inclusion criteria and were randomized: 352 patients were assigned to the AH arm and 346 to the control arm. One-hundred and five (29.8%) and 101 (29.2%) participants achieved a live birth following treatment, respectively (p=0.87). The relative risk of live birth in patients with vitrified/warmed blastocysts treated with AH was 1.02 (95%CI: 0.86-1.19). Exploratory subgroup analyses for women’s age, recruiting centers, indications for IVF, method of insemination, blastocyst quality, and days of blastocyst development failed to highlight any clinical situation that could benefit from AH in thawed blastocysts. ConclusionIn patients undergoing frozen embryo transfer with vitrified/warmed blastocysts, laser AH does not improve the live birth rate. Further studies are required to rule out milder but potentially interesting benefits in specific subgroups of patients.

Clinical and neonatal outcomes of complete zona pellucida removal by laser-assisted hatching after single vitrified-warmed blastocyst transfer.

Laser-assisted hatching (LAH) is a widely used and unavoidable technique in assisted reproductive technology after frozen embryo transfer (FET). We aimed to investigate the safety and efficacy of completely ZP (zona pellucida) removed by an assisted laser group compared to an intact group after FET. This retrospective study involved 320 SVBT single vitrified-warmed blastocyst transfers of 213 patients who underwent clomiphene citrate (CC) based minimal stimulation protocol. This study compared 160 SVBT with ZP that were completely removed using laser-assisted (laser beams of 10-15, a wavelength of 1.48μm, and duration of 1.8-2.2ms) to 160 cases of intact SVBT. Fisher's exact test, chi-squared test, and logistic regression are used for statistical analysis. The women's age, cause of infertility, BMI, transferred embryo day, insemination methods, and blastocyst grade were not significantly different between two groups. The implantation rate (66.2% vs. 51.2%, P < 0.01), the clinical pregnancy rate (CPR, 52.5% vs. 39.3%, P = 0.01), and the live birth rate (LBR, 43.7% vs. 29.3%, P < 0.01) were significantly higher in the completely ZP removed than the intact group. In logistic regression analysis, the laser-assisted hatching group showed about 2 times higher implantation rate and CPR and LBR than the control group. But miscarriage, gestational weeks, gender, birth weight, and twin births were insignificant between two groups. Our study suggests ZP free embryo transfer by assisted laser is more effective and safer than intact embryo transfer.

Efficacy of Laser-Assisted Hatching on Pregnancy and Live-Birth Rates of Human Frozen-Thawed Embryos

Efficacy of Laser-Assisted Hatching on Pregnancy and Live-Birth Rates of Human Frozen-Thawed Embryos

Optimal Exposure Parameters for Microsurgery of Embryo Zona Pellucida Using Femtosecond Laser Pulses

We applied femtosecond laser pulses for microsurgery of the zona pellucida (ZP) of mouse embryos in terms of assisted reproductive technologies. The embryos were subjected to a series of laser pulses (wavelength of 514 nm, pulse duration of 280 fs, repetition rate of 2.5 kHz). Optical microscopy was used to study the dependence of the ZP cut width on the pulse energy E and velocity v of the laser beam. It is shown that the same value of the cut width can be obtained for different combinations of these parameters. The boundaries of admissible values were found to be E = (19–52) nJ, v = (0.001–0.03) mm/s; recommendations on their proper choice are given. An analytical expression of the cut width of the ZP for a given combination of laser-pulse energy, beam velocity, and pulse repetition rate is proposed. This simple and easy-to-use equation allows for quick prediction of ZP cut width in embryo microsurgery procedures of laser-assisted hatching and embryo laser tagging using femtosecond laser pulses.

Dual laser-assisted hatching: an effective technique to salvage low-grade cleavage-stage embryos and harvest day 7 blastocysts

To investigate whether repeating laser-assisted hatching (LAH) procedure on day 6 low-grade cleavage-stage embryos (LGCEs) helps blastulation. A total of 579 cycles with LGCEs from 2019 to 2022 was retrospectively reviewed. In 323 cycles, single LAH producing small holes (10 μm) was performed on LGCEs on day 4 (D4-LAH). In 256 cycles with persistent LGCEs despite D4-LAH, a repeat LAH procedure was performed on day 6 (Dual-LAH) with a bigger hole (30 μm). We compared day 7 blastocyst formation rate, usable blastocyst rate, and good grade blastocyst rate from these day 6 LGCEs between the two groups. Compared to the D4-LAH group, the Dual-LAH group had both higher day 7 blastocyst formation rate (9.4% vs. 3.0%, p < 0.001) and higher day 7 usable blastocyst rates (7.4% vs. 2.1%, p < 0.001). For persistent LGCEs despite single LAH, performing a repeat LAH on day 6 increased day 7 blastocyst formation rate.

Efficacy of Laser-Assisted Hatching on Pregnancy and Live-Birth Rates of Human Frozen-Thawed Embryos

Efficacy of Laser-Assisted Hatching on Pregnancy and Live-Birth Rates of Human Frozen-Thawed Embryos

P-230 Does laser-assisted hatching improve pregnancy rates : a retrospective analysis

Abstract Study question Is there any effect of laser-assisted zona drilling in improving pregnancy outcomes in women undergoing vitrified-warmed embryo transfers? Summary answer Our data suggests that there is evidence of possible improvement in pregnancy rates with laser-assisted hatching of embryos among women &amp;gt;40 years of age. What is known already Mammalian embryo is surrounded by a glycoprotein shell called zona pellucida. The embryo must hatch out of this shell for it to implant in the uterus. However, certain factors such as sub-optimal in vitro culture conditions, advanced maternal age, zona-hardening due to vitrification and warming, among others, negatively impact the ability of the embryo to escape from the matrix surrounding it. Laser-assisted hatching is the safest and easiest technique available to overcome the problem of hatching by artificially disrupting the zona, though there isn’t sufficient data available on whether this has any actual benefit or is just a fancy add-on. Study design, size, duration Retrospective cohort study comparing embryo transfer results of 88 women whose embryos underwent LAH prior to transfer with that of a control group of women. Data was collected from embryo transfers that took place between March 2021 to December 2022. Participants/materials, setting, methods Beta-hCG results of women (N = 88) whose embryos underwent LAH prior to transfer was compared with the results of transfer without LAH in women (N = 88) of similar age and clinical history. Patient age, clinical indications and embryo grades were comparable across both groups. LAH was performed immediately post-warming of blastocysts using a 1.48 µm diode to drill the zona at a point opposite the ICM. Data was analyzed using independent t test and chi square test. Main results and the role of chance Women from both test and control groups were further stratified into the following sub-groups based on age – A (25-29 years), B (30-34 years), C (35-39) and D ( &amp;gt;40 years). Beta-hCG results were assessed and was found to be lower in sub-groups A (57.1% vs. 78.5%) and B (46.4% vs. 67.7%) of the test group as compared to control. It was marginally higher in test group C as compared to that of control (50% vs. 47.3%). However, we found an increase in pregnancy outcomes of test sub-group D in comparison to control (83.3% vs. 20%). Limitations, reasons for caution This is a retrospective, preliminary study to assess initial results of effect of LAH on vitrified-warmed embryo transfer outcomes. A prospective study with a larger sample size is needed to confirm our results in both frozen and fresh embryos. Wider implications of the findings Our results indicate that laser-assisted hatching does not provide any advantage except possibly in older women above the age of 40 years. Thus we can select the exact group of patients who would benefit with this add-on and offer it to them routinely to improve their pregnancy rates. Trial registration number Not applicable

O-239 A model based on artificial intelligence for the non-invasive prediction of embryo aneuploidy

Abstract Study question Can the automated artificial intelligence (AI) model iDAScore predict embryo aneuploidy? Summary answer The AUC for aneuploidy prediction of iDAScore was AUC 0.640, with optimal cut-off score 7.75, sensitivity 61.0% and specificity 60.9%. What is known already Embryo morphokinetics and image analysis using AI have been proposed as alternative approaches for non-invasive embryo aneuploidy prediction. The automated AI model iDAScore, as well as the morphokinetic embryo selection model KIDScoreD5 have been shown to have correlation with embryo ploidy status. The aim was to study the performance of aneuploidy prediction using the iDAScore, KIDScoreD5 and morphology grading (Gardner criteria). Study design, size, duration Retrospective study including 382 blastocysts of known ploidy from 92 patients undergoing PGT-A in a single centre during January-December 2022. For statistical analysis, continuous and categorical data were compared using ANOVA and Fisher’s exact test/Chi-square, respectively. ROC analysis was used to estimate the discrimination performance of variables to predict aneuploidy. Stepwise logistic regression was used to identify confounding variables and construct a model for aneuploidy prediction. Participants/materials, setting, methods Embryos were cultured in Embryoscope, underwent laser assisted hatching on Day3, were biopsied on Day5-Day6 and were vitrified. Whole-genome amplification and next-generation sequencing were used for PGT-A. iDAScore values were calculated using v1.2.0. KIDScoreD5 v.3 was calculated after annotation of the required morphokinetic parameters in Embryoviewer. For Gardner criteria, inner cell mass (ICM) and trophectoderm (TE) were classified into grades A to C. The expansion in all biopsied blastocysts was &amp;gt; =grade 4. Main results and the role of chance Out of 382 embryos analysed, 127 (33.2%) were euploid and 255 (66.8%) were aneuploid. Euploid embryos were associated with significantly higher iDAScore (7.69, 95% CI 7.44-7.95 vs 6.86, 95% CI 6.66-7.09, p &amp;lt; 0.001), higher KIDScoreD5 (6.41, 95% CI 6.07-6.75 vs 5.74, 95% CI 5.51-5.97, p = 0.001) and lower maternal age (37.10 years, 95% 36.39-37.82 vs 39.83 years, 95% CI 39.36-40.30, p &amp;lt; 0.001) compared to aneuploid embryos. Significantly more embryos with grade A ICM (A:39.4%, B:26.0%, C:20.0%, p = 0.013) and grade A TE (A: 45.9%, B:29.6%, C:23.6%, p = 0.002) were euploid compared with B and C grades, respectively. Blastocysts biopsied on Day 5 and Day 6 had similar proportions of euploidy (34.9% vs 27.6%, p = 0.125). The AUCs for aneuploidy prediction were: iDAScore: 0.640 (95% CI 0.582-0.699), KIDScoreD5: 0.604 (95% CI 0.542-0.665), ICM grade: 0.581 (95% CI 0.521-0.640), TE grade: 0.596 (0.536-0.657), maternal age: 0.694 (0.640-0.748). The optimal cut-off (Youdex index) for aneuploidy prediction of iDAScore was 7.75 (sensitivity 61.0%, specificity 60.9%). Following stepwise multivariate logistic regression, the variables that entered the model were iDAScore, maternal age and TE grade. The model iDAScore+age+TE grade increased the ability of aneuploidy prediction (AUC 0.747, 95% CI 0.696-0.796). KIDScoreD5, ICM grade and day of biopsy failed to enter the model. Limitations, reasons for caution This is a retrospective study and therefore the presence of bias cannot be excluded. iDAScore and KIDScore are models originally developed for the prediction of implantation and not for the diagnosis of embryo aneuploidy. Wider implications of the findings iDAScore appears to associate with embryo aneuploidy. Combination of iDAScore+age+TE increased the AUC for aneuploidy prediction. iDAScore can used as a decision-support tool for prioritising embryos for transfer, cryopreservation or biopsy. The analysis of more embryos is needed to confirm the present findings. Trial registration number not applicable

Laser-assisted hatching improves pregnancy outcomes in frozen-thawed embryo transfer cycles of cleavage-stage embryos: a large retrospective cohort study with propensity score matching

IntroductionLaser-assisted hatching (LAH) is a commonly used adjunct technique; however, its effectiveness has not been fully established.ObjectiveWe evaluated the effects of LAH on pregnancy outcomes in frozen-thawed embryo transfer (FET) cycles of cleavage-stage embryos.Materials and methodsThis retrospective study involved 5779 FET cycles performed at the Reproductive and Genetic Center in the Affiliated Hospital of Shandong University of Traditional Chinese Medicine between January 2016 and December 2020. After propensity score matching, 3535 FET cycles were included, out of which 1238 were subjected to LAH while the remaining 2297 cycles were non-LAH (NLAH). The primary outcomes were clinical pregnancy rate (CPR) and live birth rate (LBR) while secondary outcomes included implantation rate (IR), biochemical pregnancy rate (BPR), ectopic pregnancy rate (EPR), pregnancy loss rate (PLR), multiple pregnancy rate (MPL), and monozygotic twinning rate (MTR). Logistic regression analysis was conducted to adjust for possible confounders. Subgroup analysis was also performed based on the endometrial preparation regimen.ResultsThe LAH group exhibited a higher LBR, compared to the NLAH group (34.9% vs. 31.4%, OR = 1.185, 95% CI = 1.023, 1.374, P = 0.024). Additionally, the LAH group showed a decreasing trend in PLR and EPR; however, differences were insignificant (P = 0.078, P = 0.063 respectively). Differences in IR (24.6% vs. 24.3%), BPR (41.8% vs. 40.4%), CPR (40.7% vs. 38.3%), MPR (14.1% vs. 17.3%), and MTR (1.4% vs. 1.1%) were insignificant. Subgroup analysis revealed that LAH may be more conducive for pregnancy outcomes in hormone replacement cycles.ConclusionsIn summary, LAH has an increased chance of achieving live births. However, further prospective studies should be performed to confirm our findings.

Исследование влияния микродиссекции блестящей оболочки эмбрионов млекопитающих на ее толщину

The zona pellucida (ZP) is a dynamically changing object that plays an important role during the preimplantation stage of embryogenesis. The ZP thickness may affect the implantation success and pregnancy rate, it is considered as a prognostic factor in a number of studies. The study was aimed to assess the dynamic changes in the mouse embryonic ZP thickness after laser assisted hatching (LAH) that involved breaching the ZP integrity at the blastocyst stage. Femtosecond laser pulses were used to perform the zona microsurgery. The zona thickness was measured both at the stage of blastocyst microsurgery (~Е3.5, i.e. 3.5 days of embryogenesis) and at the hatching stage (~Е5). Significant differences in the ZP thickness were revealed in the control group of embryos: from 6.21 µm (Е3.5) to 5.4 µm (Е5). The changes in thickness from 6.6 µm (Е3.5) to 6.2 µm (Е5) observed in the group subjected to LAH were non-significant. Tracing the ZP thickness of a particular embryo from the blastocyst stage to the hatching stage made it possible to estimate the thinning coefficients in the experimental and control groups. The findings that indicate lower tensile strength of the zona in case of LAH can provide the basis for further research on the ZP properties in case of using the embryo cryopreservation protocols.

The effect of laser assisted hatching on vitrified-warmed blastocysts: a multicentric randomized controlled trial

Background and Aim: The Human Fertilization and Embryology Authority declared that assisted hatching (AH) is an unproven add-on intervention. Inline, the guidelines of the National Institute for Clinical Excellence do not recommend the use of AH to improve pregnancy rates. Of note, available data are mainly focused on the effect of AH in fresh embryo transfers while data regarding the effect of laser AH (LAH) on blastocysts, especially after cryopreservation, are inconclusive. This study aimed to investigate the benefits of the laser-mediated partial removal of the zona pellucida in vitrified/warmed blastocysts.

EXPANSION GRADE OF POST THAW EMBRYOS AND IMPLANTATION POTENTIAL

Preimplantation genetic testing for aneuploidy (PGT-A) includes laser-assisted hatching which affects the rate of embryo expansion. The study evaluated the relationship between expansion grades of post thaw, euploid embryos and implantation potential.

P-286 Can laser assisted hatching help implantation of warmed blastocysts in presence of fragmented cells ?

Abstract Study question Does laser assisted hatching (LAH) following warming of blastocysts containing fragmentated cells improves blastocyst expansion and implantation? Summary answer The application of LAH does not improve blastocyst expansion and implantation regardless the presence or absence of fragmented cells. What is known already The absence of natural hatching is one of the hypotheses of implantation failure after cryopreservation, a process that could lead to hardening of the zona pellucida. Another theory is the fact that cellular fragments inside the zona pellucida, surrounding the trophectoderm, could impairs the exit of the blastocyst through the zona decreasing its chances of implantation. Available data regarding the effect of LAH on blastocysts after vitrification are inconclusive and limited to small samples. Evaluating the effectiveness of LHA performed at the time of blastocyst warming when cellular fragments are present could elucidate its impact on hatching and implantation Study design, size, duration A bicentric prospective randomized study including 344 successive FET cycles from January 2020 until March 2021. Patients were enrolled only once in the study. Patients underwent a natural cycle or hormonal replacement treatment for FET. Blastocysts graded ≥ BL3BB (Gardner scoring) underwent artificial collapse and were vitrified on D5 or D6. Only blastocysts surviving post-warming were considered in the analysis. Primary end point was clinical pregnancy rate. Participants/materials, setting, methods Patients ≤42 years with ≤3 previous oocyte retrievals scheduled for the first elective single embryo transfer (eSET) with vitrified/warmed blastocysts. Survived blastocysts were randomized immediately after warming to LAH group (n = 172) or to control group (no-LAH, n = 172). Cellular fragmentation was annotated as a percentage of the total volume of the embryo (0%, ≤25%, ≤50%, &amp;gt;50%) and LHA was performed on the opposite side. Embryo expansion was annotated at time of transfer, at 3 hours post-warming. Main results and the role of chance Patients age were similar between LHA (33.1±9.3) and controls (34.8±7.5). Patients in LAH and controls had similar pregnancy rates (hCG &amp;gt;100) (46% versus 52%, respectively), CPR (37% versus 36%, respectively) (NS) and miscarriage rates. No difference was observed in CPR in relation to patients age. A significant increase in implantation was observed when blastocyst expansion took place 3 hours after warming, independently whether allocated in LAH (47% versus 17%, p &amp;lt; 0.01) or no-LAH group (51% versus 33%, p &amp;lt; 0.01). LAH did not influence cell expansion (83% in LAH versus 85% in no-LAH), however more blastocysts underwent hatching in LAH group (27% versus 12% in no-LAH, p &amp;lt; 0.06). Significantly more embryos that had hatched in LAH group led to pregnancy compared to no-LHA (83% versus 67%, respectively) (p = 0.05). Extra-cellular (EC) fragmentation not did not impact implantation in neither of the groups. LAH group had 46% of embryos implanted when absence of fragmentation, 40% when EC was present at ≤ 25%, 58% when EC was present at &amp;gt; 25% (NS). In no-LAH group, 52% of embryos implanted when absence of fragmentation, 49% when EC was present at ≤ 25%, 65% when EC was present at &amp;gt; 25% (NS). Limitations, reasons for caution A sample size of 700 blastocysts was first chosen calculating a 10% difference in clinical pregnancy rate (CPR) between LAH-group and no-LAH group. The study was interrupted following this interim analysis. Live birth outcomes should be considered in a further analysis to conclude on the null impact of LHA post-warming. Wider implications of the findings This study adds to the evidence of the existence of a limited potential of the application of LAH on vitrified-warmed blastocysts and its impact in terms of clinical pregnancy rates. Trial registration number not applicable