The Effect of Laser Assisted Thinning and Drilling Techniques on Assisted Reproductive Outcomes

The Effect of Laser Assisted Thinning and Drilling Techniques on Assisted Reproductive Outcomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Effect of Laser Assisted Thinning and Drilling Techniques on Assisted Reproductive Outcomes İbrahim Pala, Funda Göde This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7577202/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: To compare the effect of two different assisted hatching laser protocols thinning assisted hatching laser (TAH) vs drilling of assisted hatching laser (DAH) and non-assisted hatching control group (NAC) on clinical pregnancy and live birth rates in frozen thawed cleavage stage embryo transfer cycles. Patients and methods: This study included 310 infertile patients who underwent frozen embryo transfer (FET) cycles from 2021 to 2022 at the ART Unit of the Medical Point Hospital of Izmir University of Economics, Izmir. Patients included in the study were those between 20 and 40 years of age, who had undergone frozen thawed embryo transfer after ‘freeze-all’ protocols. The exclusion criteria were azoospermia and degenerated embryos. In TAH, laser thinning was performed by making 4-5 at a depth of 50% of the thickness of the zona pellusida (ZP). In DAH, the laser opening was made from the outer part of the ZP to the inner part. In the last group in NAC, assisted hatching was not performed. Clinical pregnancy and ongoing pregnancy rates were compared between the TAH, DAH, and NAC cycles. Results : There was no difference in terms of the age of the woman, the BMI and the sperm parameters in the three groups. There were no statistical differences between the groups in terms of the number of oocytes, embryos, and the quality of the transferred embryos. Clinical pregnancy in TAH, DAH, NAC cycles (38% vs 39% vs 45% p =0.842, respectively), ongoing pregnancy (34% vs 32% vs 39%; p =0.670, respectively) and live birth rates (34% vs 29% vs 35,4%; p = 0.586, respectively) were similar in three groups. Conclusion: In conclusion, no significant differences were found between the TAH, DAH and NAC groups in terms of ART outcomes. laser-assisted thinning laser-assisted opening zona pellucida cleavage-stage frozen thawed embryo transfer clinical outcomes Figures Figure 1 Introduction The zona pellucida (ZP), surrounded by a specialized extracellular matrix composed of glycoproteins, plays a vital role in facilitating subsequent fertilization events by allowing initial recognition and binding of the sperm to the oocyte (Zhao et al., 2002). Under specific physiological conditions, proteolytic enzymes gradually degrade the ZP, leading to its thinning as the embryo develops, ultimately facilitating its hatching from the ZP and successful implantation (Cohen, 1991 ). Meanwhile, a diverse array of factors regulate implantation, a complex physiological process that includes embryo invasion, attachment, localization, and hatching. The effectiveness of cryopreservation techniques contributes to increased cumulative pregnancy and implantation rates in in vitro fertilization (IVF) procedures (Andersen et al., 2008 ). Cryopreservation-induced "ZP hardening" has been shown to impede human embryo hatching (Kirienko et al., 2019 ). Current research suggests that cryopreservation may initiate the zona hardening (Cohen et al., 1999). In 1988, an additional technique called "assisted hatching (AH)" was developed to address this issue. AH involves artificially disrupting the zona pellucida (ZP) (Cohen et al., 1988 ). AH techniques aim to create an artificial opening or thinning of the ZP, making easy the hatching and implantation of embryos that may strive to escape the ZP otherwise (Cohen et al., 1992 ; Hernandez-Nieto et al., 2015 ). Several techniques have been employed to facilitate embryo hatching, including chemical drilling and thinning of the zona pellucida (ZP) using acidic Tyrode's medium, mechanical piercing known as partial zona dissection (PZD), hydrostatic pressure-induced ZP expansion, creation of piezoelectric pulses on the ZP, and laser-assisted zona drilling (Avella et al., 2019 ). Laser-assisted hatching (LAH) was pioneered in the early 1990s and was recognized for its accuracy, speed, and safety (Tadir et al., 1993 ). LAH can be implemented in two ways: by opening the ZP or thinning its outer wall using laser energy. Studies have compared laser-assisted opening (LAO) and control methods, as well as laser-assisted thinning techniques (Hsieh et al., 2002 ; Valojerdi et al., 2008 ; Kutlu et al., 2010 ; Frydman et al., 2016).Research suggests that human 8 cell embryos undergoing thinning laser assisted hatching exhibit higher pregnancy rates compared to those undergoing drilling laser assisted hatching (Joeng et al., 2018). In this study, we evaluated the effects of two types of LAH groups and a NAC group on clinical outcomes including clinical pregnancy, ongoing pregnancy and live birth rates in frozen-thawed cleavage stage embryo transfer cycles. Materials and Methods Patient characteristics This retrospective study included 310 patients between 20 and 40 years old who underwent FET (frozen embryo transfer) cycles from April 2021 to December 2022 in the ART Unit of Izmir Economy University Medical Point Hospital, Izmir, Turkey and underwent frozen thawed cleavage stage embryo transfer after ‘freeze-all’ protocols and single or double embryos were transferred to the uterus. The choice of the number of embryos transferred was made following the criteria recommended by the Ministry of Health of The Republic of Turkey. Exclusion criteria were patients (i) women 41 years old, (ii) azoospermia, (iii) having partially or fully degenerated cleavage stage embryos. The patients were categorized into 3 groups according to the laser-assisted hatching technique. The thinning laser-assisted hatching group (TAH, 100 patients), the drilling laser-assisted hatching (DAH, 100 patients), and non-assisted control group (NAC, 110 patients). Oocyte stimulation Gonadotropins (follitropin alfa or menotropin 150–450IU) were administered starting on day 3 of the menstrual cycle, with 0.25 mg of the GnRH antagonist cetrorelix acetate (Cetrotide, Merck, Germany) on cycle day 7. Human chorionic gonadotropin was used to trigger ovulation once the dominant follicle reached 18mm and the removal of the oocytes was carried out 35 h after activation. Criteria for Selection of embryos All embryos were cultured in Sage One Step culture medium (Origio, Denmark) in the K-system G 210 incubator (Cooper Surgical, Denmark) in an atmosphere of %5.7 CO 2 and %5.0 O 2 . Cleaving embryos were scored and graded according to the number of blastomeres, their symmetry, and the degree of cytoplasmic fragmentation.The evaluation of each embryo was carried out according to the conventional criteria outlined by the ESHRE Istanbul Consensus Guidelines (2011), which categorizes embryos as follows. • Grade 1: Considered "good" with less than 10% fragmentation, uniform and stage-specific cell sizes, and no multinucleation. • Grade 2: Rated as "fair" with 10-25% fragmentation, predominantly stage-specific cell sizes, and no evidence of multinucleation. • Grade 3: Identified as "poor" with severe fragmentation exceeding 25%, inconsistent cell sizes not aligned with the developmental stage, and multinuclear appearance. Embryo freezing and thawing The embryos were vitrified on day 3 and using a vitrification protocol (Irvine Scientific, USA) with an open cooling system device (Cryotec, Reprolife Inc, Japan) for FET. The SAGE TM Vitrification Warming Kit (Cooper Surgical, USA) was used for the warming procedures. In all groups, good quality embryos in the cleavage stage were thawed on day 3 and cultured in the short culture (2-4 h after thaw culture) in Sage-1 Step (Origio, Denmark). Embryos were considered to survive and are suitable for transfer if their blastomeres were fully intact after thawing. Single or double day 3 embryos were transferred in all groups. Laser-assisted hatching procedure and embryo transfer A computer-controlled non-contact 1480 nm diode laser (Zilos-tkTM, Hamilton Thorne Biosciences, Beverly, MA, USA) was utilized for laser hatching. The laser system was integrated with a 40X objective combination. Mounted on the objective turret of a Leica DMI3000 B inverted microscope (Leica Microsystems CMS GmBH, Germany), the Zilos-tkTM laser was configured with a pulse width of 600 µs and a standard pulse energy of 300 mW/pulse. The laser operated at 100% power. The embryos were subjected to TAH or DAH in 20 µL micro-drops containing handling medium (Origio, Denmark). Minimal laser pulses were used at the lowest feasible power setting to reduce potential damage to the blastomeres. The selection of areas with a substantial perivitelline space between the blastomere and the ZP was made to prevent blastomere damage during the procedure. TAH and DAH were performed on the day of frozen embryo transfer (FET) after embryo thawing. In the TAH thinning technique, the objective was to reduce the thickness of the outer layer of the protective glycoprotein coating by a specific amount. Using a laser shot size of 5 µm, thinning was carried out by administering 4-5 shots without penetrating the inner membrane, reaching a depth of 50% of the thickness of the ZP as shown in Figure-1(A). In the DAH, laser openings were created with 4-5 shots from the outer to the inner side of the ZP as shown in Figure-1(B). In the NAC, which did not receive laser-assisted hatching, are shown in Figure-1(C). Following laser hatching, embryos in the droplets were washed multiple times before being transferred to a four-well Nunc dish (Thermo Scientific, Denmark) containing Sage One-Step culture medium (Origio, Denmark). Embryos were transferred on the third day of development with a Sure-Pro Ultra Embryo Replacement Catheter with an oscillator (Cooper Surgical Company, The Netherlands) and a medium Sage One Step (Origio, Denmark). Endometrial preparation for frozen embryo transfer All patients received luteal support with daily use of a 200 mg Crinone 8% vaginal gel (Dendron Brands Limited, Watford, England) and a 200 mg Lutinus capsule (Ferring GmbH, Wittland, Kiel, Germany). Evaluation of clinical outcomes The pregnancy result was obtained with a blood βhCG test on day 12 after embryo transfer, and the clinical pregnancy was confirmed by detecting the gestational sac on a transvaginal ultrasound. Ongoing pregnancy was defined as the presence of a viable fetus after 12 weeks of pregnancy. Statistical analysis Continuous variables were summarized as mean and standard error and categorical variables as number and percentages. To compare the differences between the groups, we used the Chi-square test for categorical variables and the one-way Anova test for continuous variables. The student t test was performed to compare the difference among the groups. SPSS 29.0 was used in all analyzes and p values <0,05 were considered statistically significant. Results In this study, between April 2021 and December 2022, the results of 310 frozen-thawed ET cycles were evaluated. First or second cleavage stage embryos were transferred to the patients. The demographic characteristics and laboratory parameters are shown in Table 1 . There were no differences in female age of the TAH, DAH, and NAC groups age (33,95 ± 5,260 vs 34,16 ± 4,534 vs 33,83 ± 4,599; p = 0,880), respectively BMI, AMH, duration of infertility, number of previous failed ART cycles ( p > 0.05). Furthermore, the number of oocytes, the number of injected oocytes, and the number of fertilized oocytes were similar between the groups ( p = 0,331, p = 0,672, p = 0,537; respectively). Furthermore, there was no statistical difference between the groups in terms of the number of embryos cryopreserved (2,77 ± 1,58 vs 2,73 ± 1,68 vs 2,59 ± 1,48, p = 0,676, respectively). As mentioned above, there were no statistical differences between the groups in terms of the mean number of transferred embryos (1,74 ± 0,441 vs 1,61 ± 0,490 vs 1,71 ± 0,456; p = 0,116, respectively). As expected, there were no statistical differences between the groups in terms of the number of blastomere cells of transferred embryos (8,53 ± 1,24 vs 8,40 ± 1,23 vs 8,16 ± 1,018; p = 0,069, respectively). Finally, we considered semen parameters that include semen volume, sperm concentration, and total progressive motile sperm count among the three groups and we did not find any statistical difference (p > 0,05) (Table 1 ). Table 1 Cycle characteristics of the participants in three groups. Values are presented as mean ± standard deviation. No differences were significant (P > 0.05) DAH (n = 100) TAH (n = 100) NAC (n = 110) P- value Female age (years) (mean ± SD) 33,95 ± 5,260 34,16 ± 4,534 33,83 ± 4,599 0,880 BMI (mean ± SD) 24,5062 ± 4,41468 24,4648 ± 5,05036 25,0534 ± 4,57221 0,677 AMH (mean ± SD) 0,9369 ± 1,51939 1,2683 ± 1,19267 0,9187 ± 1,07193 0,459 Duration of infertility (years) (mean ± SD) 3,69 ± 2,347 4,23 ± 3,038 4,67 ± 3,349 0,057 Number of previous failed ART cycles (mean ± SD) 0,94 ± 1,362 0,88 ± 1,2 0,94 ± 1,258 0,932 Number of Oocytes collected (mean ± SD) 10,62 ± 8,678 9,76 ± 6,778 11,48 ± 9,327 0,331 Number of Oocytes injected (mean ± SD) 8,00 ± 6,439 7,34 ± 4,904 7,97 ± 6,377 0,672 Number of 2 pro-nuclei (mean ± SD) 5,61 ± 3,923 5,16 ± 3,401 5,77 ± 4,738 0,537 Number of Embryos transferred (mean ± SD) 1,74 ± 0,441 1,61 ± 0,490 1,71 ± 0,456 0,116 Transferred embryo blastomer cell number (mean ± SD) 8,53 ± 1,243 8,40 ± 1,231 8,16 ± 1,018 0,069 Number of Embryos cryopreserved (mean ± SD) 2,77 ± 1,588 2,73 ± 1,68 2,59 ± 1,48 0,676 Semen volume (mean ± SD) 2,77 ± 1,242 2,97 ± 1,356 2,67 ± 1,015 0,200 Sperm concentration (mean ± SD) 28,5070 ± 27,29631 28,7770 ± 22,46404 28,1355 ± 28,33295 0,984 TPMSC (mean ± SD) 36,0996 ± 48,94151 35,1204 ± 37,01602 35,0062 ± 49,25113 0,982 The clinical pregnancy (%38 vs %39 vs %40,9; p = 0,842), ongoing pregnancy (%34 vs %32 vs %35,4; p = 0,670,) miscarriage (%14 vs %11vs %9,09; p = 0,587) (p > 0.05) and the live birth rates (%34 vs %29 vs %35,4; p = 0,586) among the groups were non-significant (Table 2 ). Table 2 Clinical outcomes in frozen thawed good stage cleavage embryo cycles in three groups. DAH (n = 100) TAH (n = 100) NAC (n = 110) P -value Pregnancy Rate(%) 53 (%53) 46 (%46) 56 (%50,9) 0,596 Clinical Pregnancy Rate(%) 38 (%38) 39 (%39) 45 (%40,9) 0,842 Ongoing Pregnancy Rate(%) 34 (%34) 32 (%32) 39 (%35,4) 0,670 Miscarriage Rate (%) 14(%14) 11 (%11) 10 (%9,09) 0,587 Live Birth Rate 34 (%34) 29 (%29) 39 (%35,4) 0,586 Discussion The vitrification process causes the zona pellucida to harden, which can cause difficulties in embryo hatching (Zeng et al., 2018 ). There is a negative correlation between zona thickness and embryo implantation (Edi Osaige et al., 2003). This scenario has led to the question whether LAH could improve clinical outcomes in patients whose embryos have hardened ZP. The use of laser assisted hatching techniques has been shown to enhance the rates of implantation in assisted reproduction (Martins et al., 2011 ). The use of laser-assisted hatching techniques, which involve drilling or thinning of the ZP, has shown a significant elevation of both implantation and clinical pregnancy rates (Ali et al., 2003 ; Zeng et al., 2018 ). In a large-scale retrospective cohort study conducted in 2023, which included 5,779 frozen embryo transfer (FET) cycles, laser-assisted hatching (LAH) was associated with a significantly higher live birth rate compared to cycles without assisted hatching (34.9% vs. 31.4%; p = 0.024). However, no significant differences were observed in implantation or clinical pregnancy rates between the two groups (Yang et al., 2023 ). A meta-analysis by Zhang et al.(2024), which included 2,634 patients from eight studies, demonstrated that laser-assisted hatching (LAH) significantly improved implantation rates. However, LAH was also associated with an increased risk of miscarriage in frozen embryo transfer (FET) cycles. Additionally, a prospective cohort study conducted between 2014 and 2016 found that zona pellucida thinning significantly improved implantation (31.2% vs. 24.6%) and clinical pregnancy rates (49.3% vs. 38.9%) in patients with previous implantation failures. However, no significant difference was observed in live birth rates between the groups (Lee et al., 2019 ). However, in a randomized study, Ng et al. did not show any advantageous impact of LAH on the clinical pregnancy rate when comparing NAC assisted hatching after frozen embryo transfer (Ng et al., 2005 ). Primi et al. also failed to show any advantage of assisted hatching in frozen-thawed ET cycles (Primi et al., 2004 ). A 2024 systematic review by Jin et al. analyzing nine studies found no statistically significant difference between zona drilling (D-LAH) and zona thinning (T-LAH) regarding clinical pregnancy, implantation, or live birth rates. However, D-LAH was associated with a higher singleton pregnancy rate. It is still unclear whether the methods can be used interchangeably on day 3 FET. In this study, the clinical outcomes of frozen embryos frozen on day 3 that underwent TAH, DAH, and NAC were examined. Our results have not shown any significant difference in terms of clinical pregnancy rates in the assisted hatching group based on the thickness of the ZP between the methods. The starting point of this study was the findings of previous research. In general, these findings were similar and there were no superiority to each other in differences studies (Ali et al., 2003 ; Primi et al., 2004 , Ng et al., 2005 ; Zeng et al., 2018 ). The blastocyst must escape from the ZP prior to implantation in the endometrium. The underlying hypothesis of LAH is that altering the ZP can enhance the rates of implantation for embryos that are incapable of hatching naturally (Cohen et al.,1989; Cohen et al., 1992 ). Despite the efforts of numerous researchers, there is still an interest in the results of LAH, and several studies have noted that some patients did not observe any benefits from the use of LAH (Bider et al., 1997 ; Edirisinghe et al., 1999 ; Fryman et al., 2006 ). Unlike other methods, LAH can be applied through an opening or thinning and has the advantage of allowing the simple removal of the ZP by approaching the embryos in a non-contact mode without mechanical or mutagenic side effects (Germond et al., 1995 ; Mantoudis et al., 2001 ). Similar studies conducted on mouse models suggest that thinning at the 8-cell stage does not enhance or promote in vitro hatching (Uppangala et al., 2016 ). Similarly, Lee et al. investigated the impact of thinning versus opening on clinical outcomes based on maternal age in patients experiencing repeated failures of implantation with human embryos, and both groups had similar outcomes (Lee et al., 2019 ). In our study, we evaluated TAH, LAH and NAC in good-quality cleavage-stage embryos and did not show any advantage in clinical outcomes after FET. Our results of frozen thawed cycles showed a trend toward lower clinical pregnancy rates and higher miscarriage rates in the TAH and DAH groups compared to NAC. However, this did not result in a statistical difference. Comparison of frozen thawed embryos DAH and LAH did not show any benefit in performing AH based on ZP hardening, and the results were similar between groups. Several previous studies have shown higher miscarriage rates following LAH groups but had indistinct outcomes on the potential risk of miscarriage due to insufficient statistical power. In comparison to other methods, laser-assisted hatching stands out as the preferred and widely used technology due to several key advantages. First, it saves time and reduces the number of laser shots needed. Second, embryos spend less time outside the incubator. Lastly, the risk of temperature increase near the embryos from laser thermal shock is minimized (Wan et al., 2014 ). Human embryos possess a zona pellucida (ZP) consisting of two layers: an outer layer that is easily dissolved and a denser and more resistant inner layer (Tucker et al., 1993 ). The complete breach of ZP could increase the risk of bacterial infection and other negative environmental exposure. Furthermore, during cleavage, blastomeres can become more susceptible to separation if the ZP is completely breached (Tucker et al., 1993 ). In contrast, thinning the ZP by removing the outer layer while preserving the inner layer appears to minimize the risk to the embryos. Despite these considerations, the advantages of laser-assisted hatching, whether through drilling or thinning the ZP, remain controversial. Previous studies have suggested potential drawbacks of opening the zona pellucida in mouse embryos, including the risk of losing blastomeres during contractions of the female reproductive tract or inhibiting the natural expansion of the blastocyst (Nichols et al., 1989). Conclusion In conclusion, our study has demonstrated that laser-assisted hatching techniques do not damage cleavage stage embryos compared to the non-assisted control group. However, more studies involving a larger number of embryos in a prospective randomized clinical trial design are needed to resolve these issues. Declarations Acknowledgments Ibrahim Pala for the writing of the article has nothing to disclose. Funda Gode, data analysis, proofreading, has nothing to disclose. Author contributions IP designed, planned, and wrote the manuscript; collected data and applied the treatment in the laboratory; FG drafted the manuscript and supervised the manuscript writing. All authors read and approved the final manuscript. Funding The authors declare that no funds, grants, or other support was received during the preparation of this manuscript. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Ethical approval This study was carried out in accordance with the principles of the Declaration of Helsinki. The Ethics Committee of Izmir Economy University granted approval. Clinical Trial Numbers: B.30.2.İEÜSB.0.05.05-20-228 Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish: Participants signed their informed consent to publish their data. Data availability The data that support the findings of this study are available from the corresponding author (I.P.) upon reasonable request. 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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. Hum. Reprod. 2004;19, 2325– 33. DOI: 10.1093/humrep/deh430 Tadir Y, Neev, Ho P, Berns MW. Lasers for gamete micromanipulation: basic concepts. J Assist Reprod Genet.1993;10(2):121-5. DOI: 10.1007/BF01207733 The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology . Hum Reprod 2011 Jun;26(6):1270-83. DOI: 10.1093/humrep/der037. Tucker MJ, Biol MI, Wiker SR, Kort HI. Embryonal zona pellucida thinning and uterine transfer. Assist Reprod Rev 1993; 3: 168–171. DOI.https://doi.org/10.1111/j.1447-0578.2006.00145.x Tucker MJ, Luecke N, Wilker S, Wright G. Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate. 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Y. et al. Laser-assisted hatching improves clinical outcomes of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos: a prospective randomized study. Reprod Biomed Online 2014;28, 582–589. DOI: 10.1016/j.rbmo.2014.01.006 Zeng M, Su S, Li L. Comparison of pregnancy outcomes after vitrification at the cleavage and blastocyst stage: a meta-analysis. J Assist Reprod Genet 2018;35(1):127-134. DOI: 10.1007/s10815-017-1040-1 Zhao M, Dean J. The zona pellucidaa in folliculogenesis, fertilization and early development. Rev. Endocr Metab Disord 2002;3:19-26. DOI: 10.1023/a:1012744617241 Zhang Y, Liu Yan, Wang Jiali, Zhao Xinyu, Sun Xuefeng Laser assisted hatching and clinical outcomes in frozen thawed cleavage embryo transfers of patients with previous repeated failure . Reproductive Biology and Endocrinology , 2024;22:116. DOI: https://doi.org/10.1186/s12958-024-01285-9 Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":370681,"visible":true,"origin":"","legend":"\u003cp\u003eThe thinning laser-assisted hatching (TAH) (A), The drilling laser-assisted hatching (DAH) (B), Non- assisted control group (NAC) (C)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7577202/v1/2df747623f7cf30a73bd02e7.png"},{"id":98240777,"identity":"eb06639f-7d81-4ee0-9d79-6e26fcac7c23","added_by":"auto","created_at":"2025-12-15 15:24:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1047589,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7577202/v1/b0a36a7c-8d3b-4d52-b658-37c4e24ee547.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effect of Laser Assisted Thinning and Drilling Techniques on Assisted Reproductive Outcomes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe zona pellucida (ZP), surrounded by a specialized extracellular matrix composed of glycoproteins, plays a vital role in facilitating subsequent fertilization events by allowing initial recognition and binding of the sperm to the oocyte (Zhao et al., 2002). Under specific physiological conditions, proteolytic enzymes gradually degrade the ZP, leading to its thinning as the embryo develops, ultimately facilitating its hatching from the ZP and successful implantation (Cohen, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Meanwhile, a diverse array of factors regulate implantation, a complex physiological process that includes embryo invasion, attachment, localization, and hatching.\u003c/p\u003e\u003cp\u003eThe effectiveness of cryopreservation techniques contributes to increased cumulative pregnancy and implantation rates in in vitro fertilization (IVF) procedures (Andersen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Cryopreservation-induced \"ZP hardening\" has been shown to impede human embryo hatching (Kirienko et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Current research suggests that cryopreservation may initiate the zona hardening (Cohen et al., 1999). In 1988, an additional technique called \"assisted hatching (AH)\" was developed to address this issue. AH involves artificially disrupting the zona pellucida (ZP) (Cohen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). AH techniques aim to create an artificial opening or thinning of the ZP, making easy the hatching and implantation of embryos that may strive to escape the ZP otherwise (Cohen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Hernandez-Nieto et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral techniques have been employed to facilitate embryo hatching, including chemical drilling and thinning of the zona pellucida (ZP) using acidic Tyrode's medium, mechanical piercing known as partial zona dissection (PZD), hydrostatic pressure-induced ZP expansion, creation of piezoelectric pulses on the ZP, and laser-assisted zona drilling (Avella et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Laser-assisted hatching (LAH) was pioneered in the early 1990s and was recognized for its accuracy, speed, and safety (Tadir et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). LAH can be implemented in two ways: by opening the ZP or thinning its outer wall using laser energy. Studies have compared laser-assisted opening (LAO) and control methods, as well as laser-assisted thinning techniques (Hsieh et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Valojerdi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kutlu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Frydman et al., 2016).Research suggests that human 8 cell embryos undergoing thinning laser assisted hatching exhibit higher pregnancy rates compared to those undergoing drilling laser assisted hatching (Joeng et al., 2018).\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the effects of two types of LAH groups and a NAC group on clinical outcomes including clinical pregnancy, ongoing pregnancy and live birth rates in frozen-thawed cleavage stage embryo transfer cycles.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study included 310 patients between 20 and 40 years old who underwent FET (frozen embryo transfer) cycles from April 2021 to December 2022 in the ART Unit of Izmir Economy University Medical Point Hospital, Izmir, Turkey and underwent frozen thawed cleavage stage embryo transfer after ‘freeze-all’ protocols and single or double embryos were transferred to the uterus. The choice of the number of embryos transferred was made following the criteria recommended by the Ministry of Health of The Republic of Turkey. Exclusion criteria were patients (i) women 41 years old, (ii) azoospermia, (iii) having partially or fully degenerated cleavage stage embryos.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe patients were categorized into 3 groups according to the laser-assisted hatching technique. The thinning laser-assisted hatching group (TAH, 100 patients), the drilling laser-assisted hatching (DAH, 100 patients), and non-assisted control group (NAC, 110 patients).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOocyte stimulation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGonadotropins (follitropin alfa or menotropin 150–450IU) were administered starting on day 3 of the menstrual cycle, with 0.25 mg of the GnRH antagonist cetrorelix acetate (Cetrotide, Merck, Germany) on cycle day 7. Human chorionic gonadotropin was used to trigger ovulation once the dominant follicle reached 18mm and the removal of the oocytes was carried out 35 h after activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCriteria for Selection of embryos\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll embryos were cultured in Sage One Step culture medium (Origio, Denmark) in the K-system G 210 incubator (Cooper Surgical, Denmark) in an atmosphere of %5.7 CO\u003csub\u003e2\u003c/sub\u003e and %5.0 O\u003csub\u003e2\u003c/sub\u003e. Cleaving embryos were scored and graded according to the number of blastomeres, their symmetry, and the degree of cytoplasmic fragmentation.The evaluation of each embryo was carried out according to the conventional criteria outlined by the ESHRE Istanbul Consensus Guidelines (2011), which categorizes embryos as follows.\u003c/p\u003e\n\u003cp\u003e• Grade 1: Considered \"good\" with less than 10% fragmentation, uniform and stage-specific cell sizes, and no multinucleation.\u003c/p\u003e\n\u003cp\u003e• Grade 2: Rated as \"fair\" with 10-25% fragmentation, predominantly stage-specific cell sizes, and no evidence of multinucleation.\u003c/p\u003e\n\u003cp\u003e• Grade 3: Identified as \"poor\" with severe fragmentation exceeding 25%, inconsistent cell sizes not aligned with the developmental stage, and multinuclear appearance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmbryo freezing and thawing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe embryos were vitrified on day 3 and using a vitrification protocol (Irvine Scientific, USA) with an open cooling system device (Cryotec, Reprolife Inc, Japan) for FET. The SAGE\u003csup\u003eTM\u003c/sup\u003e Vitrification Warming Kit (Cooper Surgical, USA) was used for the warming procedures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn all \u003cem\u003egroups,\u003c/em\u003e good quality embryos in the cleavage stage were thawed on day 3 and cultured in the short culture (2-4 h after thaw culture) in Sage-1 Step (Origio, Denmark). Embryos were considered to survive and are suitable for transfer if their blastomeres were fully intact after thawing. Single or double day 3 embryos were transferred in all groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaser-assisted hatching procedure and embryo transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA computer-controlled non-contact 1480 nm diode laser (Zilos-tkTM, Hamilton Thorne Biosciences, Beverly, MA, USA) was utilized for laser hatching. The laser system was integrated with a 40X objective combination. Mounted on the objective turret of a Leica DMI3000 B inverted microscope (Leica Microsystems CMS GmBH, Germany), the Zilos-tkTM laser was configured with a pulse width of 600 µs and a standard pulse energy of 300 mW/pulse. The laser operated at 100% power. The embryos were subjected to TAH or DAH in 20 µL micro-drops containing handling medium (Origio, Denmark). Minimal laser pulses were used at the lowest feasible power setting to reduce potential damage to the blastomeres. The selection of areas with a substantial perivitelline space between the blastomere and the ZP was made to prevent blastomere damage during the procedure. TAH and DAH were performed on the day of frozen embryo transfer (FET) after embryo thawing.\u003c/p\u003e\n\u003cp\u003eIn the TAH thinning technique, the objective was to reduce the thickness of the outer layer of the protective glycoprotein coating by a specific amount. Using a laser shot size of 5 µm, thinning was carried out by administering 4-5 shots without penetrating the inner membrane, reaching a depth of 50% of the thickness of the ZP as shown in Figure-1(A). In the DAH, laser openings were created with 4-5 shots from the outer to the inner side of the ZP as shown in Figure-1(B). In the NAC, which did not receive laser-assisted hatching, are shown in Figure-1(C). Following laser hatching, embryos in the droplets were washed multiple times before being transferred to a four-well Nunc dish (Thermo Scientific, Denmark) containing Sage One-Step culture medium (Origio, Denmark).\u003c/p\u003e\n\u003cp\u003eEmbryos were transferred on the third day of development with a Sure-Pro Ultra Embryo Replacement Catheter with an oscillator (Cooper Surgical Company, The Netherlands) and a medium Sage One Step (Origio, Denmark).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndometrial preparation for frozen embryo transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients received luteal support with daily use of a 200 mg Crinone 8% vaginal gel (Dendron Brands Limited, Watford, England) and a 200 mg Lutinus capsule (Ferring GmbH, Wittland, Kiel, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of clinical outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pregnancy result was obtained with a blood βhCG test on day 12 after embryo transfer, and the clinical pregnancy was confirmed by detecting the gestational sac on a transvaginal ultrasound. Ongoing pregnancy was defined as the presence of a viable fetus after 12 weeks of pregnancy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContinuous variables were summarized as mean and standard error and categorical variables as number and percentages. To compare the differences between the groups, we used the Chi-square test for categorical variables and the one-way Anova test for continuous variables. The student t test was performed to compare the difference among the groups. SPSS 29.0 was used in all analyzes and \u003cem\u003ep\u003c/em\u003e values \u0026lt;0,05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, between April 2021 and December 2022, the results of 310 frozen-thawed ET cycles were evaluated. First or second cleavage stage embryos were transferred to the patients. The demographic characteristics and laboratory parameters are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There were no differences in female age of the TAH, DAH, and NAC groups age (33,95\u0026thinsp;\u0026plusmn;\u0026thinsp;5,260 vs 34,16\u0026thinsp;\u0026plusmn;\u0026thinsp;4,534 vs 33,83\u0026thinsp;\u0026plusmn;\u0026thinsp;4,599; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,880), respectively BMI, AMH, duration of infertility, number of previous failed ART cycles (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Furthermore, the number of oocytes, the number of injected oocytes, and the number of fertilized oocytes were similar between the groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,331, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,672, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,537; respectively). Furthermore, there was no statistical difference between the groups in terms of the number of embryos cryopreserved (2,77\u0026thinsp;\u0026plusmn;\u0026thinsp;1,58 vs 2,73\u0026thinsp;\u0026plusmn;\u0026thinsp;1,68 vs 2,59\u0026thinsp;\u0026plusmn;\u0026thinsp;1,48, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,676, respectively). As mentioned above, there were no statistical differences between the groups in terms of the mean number of transferred embryos (1,74\u0026thinsp;\u0026plusmn;\u0026thinsp;0,441 vs 1,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,490 vs 1,71\u0026thinsp;\u0026plusmn;\u0026thinsp;0,456; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,116, respectively). As expected, there were no statistical differences between the groups in terms of the number of blastomere cells of transferred embryos (8,53\u0026thinsp;\u0026plusmn;\u0026thinsp;1,24 vs 8,40\u0026thinsp;\u0026plusmn;\u0026thinsp;1,23 vs 8,16\u0026thinsp;\u0026plusmn;\u0026thinsp;1,018; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,069, respectively). Finally, we considered semen parameters that include semen volume, sperm concentration, and total progressive motile sperm count among the three groups and we did not find any statistical difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0,05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCycle characteristics of the participants in three groups. Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. No differences were significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAH (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTAH (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNAC (n\u0026thinsp;=\u0026thinsp;110)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP-\u003c/em\u003evalue\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale age (years) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e33,95\u0026thinsp;\u0026plusmn;\u0026thinsp;5,260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e34,16\u0026thinsp;\u0026plusmn;\u0026thinsp;4,534\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e33,83\u0026thinsp;\u0026plusmn;\u0026thinsp;4,599\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,880\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBMI (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e24,5062\u0026thinsp;\u0026plusmn;\u0026thinsp;4,41468\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e24,4648\u0026thinsp;\u0026plusmn;\u0026thinsp;5,05036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e25,0534\u0026thinsp;\u0026plusmn;\u0026thinsp;4,57221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,677\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAMH (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0,9369\u0026thinsp;\u0026plusmn;\u0026thinsp;1,51939\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1,2683\u0026thinsp;\u0026plusmn;\u0026thinsp;1,19267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0,9187\u0026thinsp;\u0026plusmn;\u0026thinsp;1,07193\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,459\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDuration of infertility (years) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3,69\u0026thinsp;\u0026plusmn;\u0026thinsp;2,347\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4,23\u0026thinsp;\u0026plusmn;\u0026thinsp;3,038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4,67\u0026thinsp;\u0026plusmn;\u0026thinsp;3,349\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,057\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of previous failed ART cycles (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0,94\u0026thinsp;\u0026plusmn;\u0026thinsp;1,362\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0,88\u0026thinsp;\u0026plusmn;\u0026thinsp;1,2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0,94\u0026thinsp;\u0026plusmn;\u0026thinsp;1,258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,932\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of Oocytes collected (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e10,62\u0026thinsp;\u0026plusmn;\u0026thinsp;8,678\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9,76\u0026thinsp;\u0026plusmn;\u0026thinsp;6,778\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11,48\u0026thinsp;\u0026plusmn;\u0026thinsp;9,327\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,331\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of Oocytes injected (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8,00\u0026thinsp;\u0026plusmn;\u0026thinsp;6,439\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7,34\u0026thinsp;\u0026plusmn;\u0026thinsp;4,904\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7,97\u0026thinsp;\u0026plusmn;\u0026thinsp;6,377\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,672\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of 2 pro-nuclei (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5,61\u0026thinsp;\u0026plusmn;\u0026thinsp;3,923\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5,16\u0026thinsp;\u0026plusmn;\u0026thinsp;3,401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5,77\u0026thinsp;\u0026plusmn;\u0026thinsp;4,738\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,537\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of Embryos transferred (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1,74\u0026thinsp;\u0026plusmn;\u0026thinsp;0,441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,490\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1,71\u0026thinsp;\u0026plusmn;\u0026thinsp;0,456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,116\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTransferred embryo blastomer cell number (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8,53\u0026thinsp;\u0026plusmn;\u0026thinsp;1,243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e8,40\u0026thinsp;\u0026plusmn;\u0026thinsp;1,231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e8,16\u0026thinsp;\u0026plusmn;\u0026thinsp;1,018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,069\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of Embryos cryopreserved (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2,77\u0026thinsp;\u0026plusmn;\u0026thinsp;1,588\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2,73\u0026thinsp;\u0026plusmn;\u0026thinsp;1,68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2,59\u0026thinsp;\u0026plusmn;\u0026thinsp;1,48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,676\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSemen volume (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2,77\u0026thinsp;\u0026plusmn;\u0026thinsp;1,242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2,97\u0026thinsp;\u0026plusmn;\u0026thinsp;1,356\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2,67\u0026thinsp;\u0026plusmn;\u0026thinsp;1,015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,200\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSperm concentration (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e28,5070\u0026thinsp;\u0026plusmn;\u0026thinsp;27,29631\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e28,7770\u0026thinsp;\u0026plusmn;\u0026thinsp;22,46404\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e28,1355\u0026thinsp;\u0026plusmn;\u0026thinsp;28,33295\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,984\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTPMSC (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e36,0996\u0026thinsp;\u0026plusmn;\u0026thinsp;48,94151\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35,1204\u0026thinsp;\u0026plusmn;\u0026thinsp;37,01602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e35,0062\u0026thinsp;\u0026plusmn;\u0026thinsp;49,25113\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,982\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe clinical pregnancy (%38 vs %39 vs %40,9; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,842), ongoing pregnancy (%34 vs %32 vs %35,4; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,670,) miscarriage (%14 vs %11vs %9,09; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,587) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and the live birth rates (%34 vs %29 vs %35,4; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0,586) among the groups were non-significant (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical outcomes in frozen thawed good stage cleavage embryo cycles in three groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAH (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTAH (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNAC (n\u0026thinsp;=\u0026thinsp;110)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePregnancy Rate(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53 (%53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46 (%46)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56 (%50,9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,596\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical Pregnancy Rate(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38 (%38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39 (%39)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45 (%40,9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,842\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOngoing Pregnancy Rate(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34 (%34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32 (%32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39 (%35,4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,670\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMiscarriage Rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14(%14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (%11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 (%9,09)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,587\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLive Birth Rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34 (%34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29 (%29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39 (%35,4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0,586\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe vitrification process causes the zona pellucida to harden, which can cause difficulties in embryo hatching (Zeng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). There is a negative correlation between zona thickness and embryo implantation (Edi Osaige et al., 2003). This scenario has led to the question whether LAH could improve clinical outcomes in patients whose embryos have hardened ZP. The use of laser assisted hatching techniques has been shown to enhance the rates of implantation in assisted reproduction (Martins et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The use of laser-assisted hatching techniques, which involve drilling or thinning of the ZP, has shown a significant elevation of both implantation and clinical pregnancy rates (Ali et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In a large-scale retrospective cohort study conducted in 2023, which included 5,779 frozen embryo transfer (FET) cycles, laser-assisted hatching (LAH) was associated with a significantly higher live birth rate compared to cycles without assisted hatching (34.9% vs. 31.4%; p\u0026thinsp;=\u0026thinsp;0.024). However, no significant differences were observed in implantation or clinical pregnancy rates between the two groups (Yang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A meta-analysis by Zhang et al.(2024), which included 2,634 patients from eight studies, demonstrated that laser-assisted hatching (LAH) significantly improved implantation rates. However, LAH was also associated with an increased risk of miscarriage in frozen embryo transfer (FET) cycles.\u003c/p\u003e\u003cp\u003eAdditionally, a prospective cohort study conducted between 2014 and 2016 found that zona pellucida thinning significantly improved implantation (31.2% vs. 24.6%) and clinical pregnancy rates (49.3% vs. 38.9%) in patients with previous implantation failures. However, no significant difference was observed in live birth rates between the groups (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, in a randomized study, Ng et al. did not show any advantageous impact of LAH on the clinical pregnancy rate when comparing NAC assisted hatching after frozen embryo transfer (Ng et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Primi et al. also failed to show any advantage of assisted hatching in frozen-thawed ET cycles (Primi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). A 2024 systematic review by Jin et al. analyzing nine studies found no statistically significant difference between zona drilling (D-LAH) and zona thinning (T-LAH) regarding clinical pregnancy, implantation, or live birth rates. However, D-LAH was associated with a higher singleton pregnancy rate.\u003c/p\u003e\u003cp\u003eIt is still unclear whether the methods can be used interchangeably on day 3 FET. In this study, the clinical outcomes of frozen embryos frozen on day 3 that underwent TAH, DAH, and NAC were examined. Our results have not shown any significant difference in terms of clinical pregnancy rates in the assisted hatching group based on the thickness of the ZP between the methods.\u003c/p\u003e\u003cp\u003eThe starting point of this study was the findings of previous research. In general, these findings were similar and there were no superiority to each other in differences studies (Ali et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Primi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Ng et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The blastocyst must escape from the ZP prior to implantation in the endometrium. The underlying hypothesis of LAH is that altering the ZP can enhance the rates of implantation for embryos that are incapable of hatching naturally (Cohen et al.,1989; Cohen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Despite the efforts of numerous researchers, there is still an interest in the results of LAH, and several studies have noted that some patients did not observe any benefits from the use of LAH (Bider et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Edirisinghe et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Fryman et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Unlike other methods, LAH can be applied through an opening or thinning and has the advantage of allowing the simple removal of the ZP by approaching the embryos in a non-contact mode without mechanical or mutagenic side effects (Germond et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Mantoudis et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Similar studies conducted on mouse models suggest that thinning at the 8-cell stage does not enhance or promote in vitro hatching (Uppangala et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, Lee et al. investigated the impact of thinning versus opening on clinical outcomes based on maternal age in patients experiencing repeated failures of implantation with human embryos, and both groups had similar outcomes (Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, we evaluated TAH, LAH and NAC in good-quality cleavage-stage embryos and did not show any advantage in clinical outcomes after FET. Our results of frozen thawed cycles showed a trend toward lower clinical pregnancy rates and higher miscarriage rates in the TAH and DAH groups compared to NAC. However, this did not result in a statistical difference. Comparison of frozen thawed embryos DAH and LAH did not show any benefit in performing AH based on ZP hardening, and the results were similar between groups. Several previous studies have shown higher miscarriage rates following LAH groups but had indistinct outcomes on the potential risk of miscarriage due to insufficient statistical power.\u003c/p\u003e\u003cp\u003eIn comparison to other methods, laser-assisted hatching stands out as the preferred and widely used technology due to several key advantages. First, it saves time and reduces the number of laser shots needed. Second, embryos spend less time outside the incubator. Lastly, the risk of temperature increase near the embryos from laser thermal shock is minimized (Wan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Human embryos possess a zona pellucida (ZP) consisting of two layers: an outer layer that is easily dissolved and a denser and more resistant inner layer (Tucker et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The complete breach of ZP could increase the risk of bacterial infection and other negative environmental exposure. Furthermore, during cleavage, blastomeres can become more susceptible to separation if the ZP is completely breached (Tucker et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, thinning the ZP by removing the outer layer while preserving the inner layer appears to minimize the risk to the embryos. Despite these considerations, the advantages of laser-assisted hatching, whether through drilling or thinning the ZP, remain controversial. Previous studies have suggested potential drawbacks of opening the zona pellucida in mouse embryos, including the risk of losing blastomeres during contractions of the female reproductive tract or inhibiting the natural expansion of the blastocyst (Nichols et al., 1989).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study has demonstrated that laser-assisted hatching techniques do not damage cleavage stage embryos compared to the non-assisted control group. However, more studies involving a larger number of embryos in a prospective randomized clinical trial design are needed to resolve these issues.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eIbrahim Pala for the writing of the article has nothing to disclose. Funda Gode, data analysis, proofreading, has nothing to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eIP designed, planned, and wrote the manuscript; collected data and applied the treatment in the laboratory; FG drafted the manuscript and supervised the manuscript writing. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe authors declare that no funds, grants, or other support was received during the preparation of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis study was carried out in accordance with the principles of the Declaration of Helsinki. The Ethics Committee of Izmir Economy University granted approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Numbers:\u003c/strong\u003e B.30.2.İEÜSB.0.05.05-20-228\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all individual participants included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e Participants signed their informed consent to publish their data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The data that support the findings of this study are available from the corresponding author (I.P.) upon reasonable request.\u003c/p\u003e\n\u003cp\u003eThis research was presented as an oral presentation at the 11th International Turkish Society of Reproductive Medicine Congress, held Nov 16-19, 2023, Antalya, Türkiye.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAli J, Rahbar S, Burjaq H, Sultan AM, Al Flamerzi M, Shahata MA. Routine laser assisted hatching results in significantly increased clinical pregnancies. J Assist Reprod Genet 2003;20(5):177\u0026ndash; 181. \u003cu\u003eDOI: \u003c/u\u003e10.1023/A:1023665909123\u003c/li\u003e\n\u003cli\u003eAndersen AN, Goossens V, Ferraretti AP, Bhattacharya S, Felberbaum R, de Mouzon , et al. Assisted reproductive technology in Europe, 2004: results generated from European registers by ESHRE. Hum Reprod. 2008;23:756-71.\u003cu\u003eDOI: \u003c/u\u003e10.1093/humrep/den014\u003c/li\u003e\n\u003cli\u003eAvella MA, Milne KA, Dawood S, Dawood A, Tucker MJ. Assisted hatching of human embryos for successful implantation. In: Nagy Z, Varghese A, Agrawal A (eds). In Vitro Fertilisation. Springer, Cham, 2019.\u003c/li\u003e\n\u003cli\u003eBider D, Livshits A, Yonish M, Yemini Z, Mashiach S, Dor J. Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod 1997;12(2):317\u0026ndash;320. \u003cu\u003eDOI: \u003c/u\u003e10.1093/humrep/12.2.317\u003c/li\u003e\n\u003cli\u003eCohen J, Alikani M, Trowbridge , and Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos wwith poor prognosis. Hum. Reprod. 1992;7,685-91. \u003cu\u003ehttps://doi.org/10.1093/oxfordjournals.humrep.a137720\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eCohen J, Lindheim S, and Sauer M. Assisted Hatching causes beneficial effects on the outcome fo subsequent frozen embryos transfers of donor oocyte cycle. Ferti. Steril. 2005;72(Suppl. 1 ), S5. DOI: 10.1017/S0967199416000058\u003c/li\u003e\n\u003cli\u003eCohen J, Malter H, Wright G, Kort H, Massey J, Mitchell D. Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated. Hum Reprod 1989;4(4):435\u0026ndash;442. DOI: 10.1093/oxfordjournals.humrep.a136923\u003c/li\u003e\n\u003cli\u003eCohen J, Malter HH, Fehilly C, Wright G, Elsner C, Kort H, et al. Implantation of embryos after partial opening of zona pellucida to facilitate sperm penetration. Lancet. 1988;2(8603):162. DOI: 10.1016/s0140-6736(88)90710-6\u003c/li\u003e\n\u003cli\u003eCohen J. Assisted hatching of human embryos. J Assist Reprod Genet 1991;8(4):179-190. DOI: 10.1007/BF01130802\u003c/li\u003e\n\u003cli\u003eEdi-Osagie E, Hooper L, Seif MW. The impact of assisted hatching on live birth rates and outcomes of assisted conception: a systematic review. Hum Reprod 2003;18(9):1828\u0026ndash;1835. DOI: 10.1093/humrep/deg334\u003c/li\u003e\n\u003cli\u003eEdirisinghe WR, Ahnonkitpanit V, Promviengchai S, Suwajanakorn S, Pruksananonda K, Chinpilas V, Virutamasen P. A study failing to determine significant benefits from assisted hatching: patients selected for advanced age, zonal thickness of embryos, and previous failed attempts. J Assist Reprod Genet 1999;16(6):294\u0026ndash;301. DOI: 10.1023/a:1020497714495\u003c/li\u003e\n\u003cli\u003eFryman N, Madoux S, Hesters L, Duvernoy C, Feyereisen E, Le Du A, Tachdian G, Frydman R, Fanchin. A randomized double-blind controlled study on the efficacy of laser zona pellucida thinning on live birth rates in case of advanced female age. Hum Reprod 2006;21(8):2131-2135. DOI: 10.1093/humrep/del124\u003c/li\u003e\n\u003cli\u003eGermond M, Nocera D, Senn A, Rink K, Delacr\u0026eacute;taz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48\u0026mu;m diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995;64(3):604\u0026ndash;611. DOI: 10.1016/s0015-0282(16)57800-5\u003c/li\u003e\n\u003cli\u003eHernandez-Nieto CA, SotoCossio LE, Basurta-Diaz D. Assisted hatching for improving embryo implantation A bibliographical review. Ginecologia y obstetricia de Mexico 2015;83(4):223-9.\u003c/li\u003e\n\u003cli\u003eHsieh Y, Huang C, Cheng T, Chang C, Tsai H, Lee M. Laser-assisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate inn women with advanced age. Fertil Steril 2002;78(1):179-182. DOI: 10.1016/s0015-0282(02)03172-2\u003c/li\u003e\n\u003cli\u003eJeong JE, Joo BS, Kim CW, Kim HG, Joo JK, Lee KS. Effects of three-area laser-assisted zona thinning in 8-cell human embryos on pregnancy outcomes in vitro fertilisation. Clin Exp Reprod Med 2018;45(1):25-30. DOI: 10.5653/cerm.2018.45.1.25\u003c/li\u003e\n\u003cli\u003eJin Y, Wang X, Liu Z, Li W, Zhang L, Zhao Y.\u003cbr\u003e\u003cem\u003eTwo laser assisted hatching methods of embryos in ART: a systematic review and meta-analysis\u003c/em\u003e. \u003cem\u003eBMC Pregnancy and Childbirth\u003c/em\u003e. 2024;24(1):300\u003cbr\u003e\u003cstrong\u003eDOI:\u003c/strong\u003e https://doi.org/10.1186/s12884-024-06380-8\u003c/li\u003e\n\u003cli\u003eKirienko KV, Apryshko VP, Naumova AA, Yakavenko SA et al. Mechanical zona pellucida removal of vitrified-warmed human blastocysts does not affect the clinical outcome. Reprod BioMed Online 2019;39(5): 745-749. DOI: 10.1016/j.rbmo.2019.06.003\u003c/li\u003e\n\u003cli\u003eKutlu P, Atvar O, Vanlioglu OF. Laser assisted ona thinning technique has no beneficial effect on the ART outcomes of two differenet maternal age groups. Assist Reprod Genet 2010;27(8):457-461. DOI: 10.1007/s10815-010-9431-6\u003c/li\u003e\n\u003cli\u003eLee, JW., Cha, JH., Shin, SH. et al.Effects of laser-assisted thinning versus opening on clinical outcomes according to maternal age in patients with repeated implantation failure. Lasers Med Sci 2019;34, 1889\u0026ndash;1895. DOI: 10.1007/s10103-019-02787-4\u003c/li\u003e\n\u003cli\u003eMantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 2001;16(10):2182\u0026ndash;2186. DOI: 10.1093/humrep/16.10.2182\u003c/li\u003e\n\u003cli\u003eMartins WP, Rocha IA, Ferriani RA, Nastri CO. Assisted hatching of human embryos: a systematic review and meta-analysis of randomized controlled trials. Hum Reprod Update 2011;17(4):438\u0026ndash; 453. DOI: 10.1093/humupd/dmr012\u003c/li\u003e\n\u003cli\u003eNg, E., Naveed, F., Lau, R., Yeung, W.S., Chan, C.C., Tang, O.C. \u0026amp; Ho, P.C. A randomized double blind controlled study of the efficacy of laser assisted hatching on implantation and pregnancy rates of frozen\u0026ndash;thawed embryo transfer at the cleavage stage. Hum. Reprod. 2005;20, 979\u0026ndash;85. DOI: 10.1093/humrep/deh724\u003c/li\u003e\n\u003cli\u003eNichols, J. \u0026amp; Gardner, R. L. Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse. Hum Reprod 1989;4, 180\u0026ndash;187. DOI: 10.1093/oxfordjournals.humrep.a136868\u003c/li\u003e\n\u003cli\u003ePrimi, M., Senn, A., Montag, M., Van der Ven, H., Mandelbaum, J., Veiga, A., Barri, P. \u0026amp; Germond, M. 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. Hum. Reprod. 2004;19, 2325\u0026ndash; 33. DOI: 10.1093/humrep/deh430\u003c/li\u003e\n\u003cli\u003eTadir Y, Neev, Ho P, Berns MW. Lasers for gamete micromanipulation: basic concepts. J Assist Reprod Genet.1993;10(2):121-5. DOI: 10.1007/BF01207733\u003c/li\u003e\n\u003cli\u003eThe Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. \u003cstrong\u003eAlpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology\u003c/strong\u003e. Hum Reprod 2011 Jun;26(6):1270-83. \u003cu\u003eDOI: 10.1093/humrep/der037.\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eTucker MJ, Biol MI, Wiker SR, Kort HI. Embryonal zona pellucida thinning and uterine transfer. Assist Reprod Rev 1993; 3: 168\u0026ndash;171. DOI.https://doi.org/10.1111/j.1447-0578.2006.00145.x\u003c/li\u003e\n\u003cli\u003eTucker MJ, Luecke N, Wilker S, Wright G. Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate. J Assist Reprod Genet 1993; 10: 187\u0026ndash;191. DOI: 10.1007/BF01239219\u003c/li\u003e\n\u003cli\u003eUppangala S, D\u0026rsquo;Souza F, Pudakalakatti S, Atreya HS, Raval K, Kalthur G, Adiga SK. Laser assisted zona hatching does not lead to immediate impairment in human embryo quality and metabolism. Syst Biol Reprod Med 2016;62(6):396\u0026ndash;403. DOI: 10.1080/19396368.2016.1217952\u003c/li\u003e\n\u003cli\u003eValojerdi MR, Eftekhari-Yazdi P, Karimian L, Ashtiani SK. 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. Fertil Steril 2008;90(1):84-91. DOI: 10.1016/j.fertnstert.2007.06.005\u003c/li\u003e\n\u003cli\u003eYang X, Sun L, Huang W, Li W, Hu J.\u003cbr\u003e\u003cem\u003eLaser assisted hatching improves pregnancy outcomes in frozen thawed embryo transfer cycles of cleavage stage embryos: a large retrospective cohort study with propensity score matching\u003c/em\u003e. \u003cem\u003eJournal of Assisted Reproduction and Genetics\u003c/em\u003e.2023;40(2):417\u0026ndash;427.\u003cstrong\u003e DOI:\u003c/strong\u003e https://doi.org/10.1007/s10815-022-02711-w\u003c/li\u003e\n\u003cli\u003eWan, C. Y. et al. Laser-assisted hatching improves clinical outcomes of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos: a prospective randomized study. Reprod Biomed Online 2014;28, 582\u0026ndash;589. DOI: 10.1016/j.rbmo.2014.01.006\u003c/li\u003e\n\u003cli\u003eZeng M, Su S, Li L. Comparison of pregnancy outcomes after vitrification at the cleavage and blastocyst stage: a meta-analysis. J Assist Reprod Genet 2018;35(1):127-134. DOI: 10.1007/s10815-017-1040-1\u003c/li\u003e\n\u003cli\u003eZhao M, Dean J. The zona pellucidaa in folliculogenesis, fertilization and early development. Rev. Endocr Metab Disord 2002;3:19-26. DOI: 10.1023/a:1012744617241\u003c/li\u003e\n\u003cli\u003eZhang Y, Liu Yan, Wang Jiali, Zhao Xinyu, Sun Xuefeng\u003cbr\u003e\u003cem\u003eLaser assisted hatching and clinical outcomes in frozen thawed cleavage embryo transfers of patients with previous repeated failure\u003c/em\u003e. \u003cem\u003eReproductive Biology and Endocrinology\u003c/em\u003e, 2024;22:116. \u003cstrong\u003eDOI:\u003c/strong\u003e https://doi.org/10.1186/s12958-024-01285-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"laser-assisted thinning, laser-assisted opening, zona pellucida, cleavage-stage frozen thawed embryo transfer, clinical outcomes","lastPublishedDoi":"10.21203/rs.3.rs-7577202/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7577202/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e\u003c/em\u003e To compare the effect of two different assisted hatching laser protocols thinning assisted hatching laser (TAH) vs drilling of assisted hatching laser (DAH) and non-assisted hatching control group (NAC) on clinical pregnancy and live birth rates in frozen thawed cleavage stage embryo transfer cycles.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePatients and methods:\u003c/strong\u003e\u003c/em\u003e This study included 310 infertile patients who underwent frozen embryo transfer (FET) cycles from 2021 to 2022 at the ART Unit of the Medical Point Hospital of Izmir University of Economics, Izmir. Patients included in the study were those between 20 and 40 years of age, who had undergone frozen thawed embryo transfer after ‘freeze-all’ protocols. The exclusion criteria were azoospermia and degenerated embryos. In TAH, laser thinning was performed by making 4-5 at a depth of 50% of the thickness of the zona pellusida (ZP). In DAH, the laser opening was made from the outer part of the ZP to the inner part. In the last group in NAC, assisted hatching was not performed. Clinical pregnancy and ongoing pregnancy rates were compared between the TAH, DAH, and NAC cycles.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e:\u003c/em\u003e There was no difference in terms of the age of the woman, the BMI and the sperm parameters in the three groups. There were no statistical differences between the groups in terms of the number of oocytes, embryos, and the quality of the transferred embryos. Clinical pregnancy in TAH, DAH, NAC cycles (38% vs 39% vs 45%\u003cem\u003e p\u003c/em\u003e=0.842, respectively), ongoing pregnancy (34% vs 32% vs 39%; \u003cem\u003ep\u003c/em\u003e=0.670, respectively) and live birth rates (34% vs 29% vs 35,4%; \u003cem\u003ep = 0.586,\u003c/em\u003e respectively) were similar in three groups.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/em\u003e In conclusion, no significant differences were found between the TAH, DAH and NAC groups in terms of ART outcomes.\u003c/p\u003e","manuscriptTitle":"The Effect of Laser Assisted Thinning and Drilling Techniques on Assisted Reproductive Outcomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 07:39:58","doi":"10.21203/rs.3.rs-7577202/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"edb70ad1-6ed0-479f-acb5-879055b5852b","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T15:23:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 07:39:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7577202","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7577202","identity":"rs-7577202","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}