- Open Access
Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers
Reproductive Biology and Endocrinology volume 11, Article number: 21 (2013)
It is still debatable whether a full-thickness assisted hatching (AH) is better than the partial zona thinning. In this research, we used a mouse model to study the effect of partial and complete laser-AH on the rate of completely hatched blastocyst and their cell numbers.
In experiment 1, mouse morulae had 0, 1, 2 or 3 full-thickness openings of 10 microns created in the zona pellucida with an infrared laser beam. In the second experiment, 0, 1 and 2 openings of 20 microns were studied. In the third experiment, a full-thickness opening of 20 microns or quarter-thinning of the zonal circumference to a depth of 90% was compared with non-AH controls.
No difference in blastocyst formation was found in laser-treated groups and in the controls. In experiment 1, the rate of completely hatched blastocysts was significantly lower than the controls. In experiment 2 when the size of the opening was increased, blastocysts completely hatched at a significantly higher rate than that in the controls. In experiment 3, the rate of completely hatched blastocysts was the highest in the full-thickness group. Cell numbers in completely hatched blastocysts from both AH groups were significantly fewer than those in the controls.
Full-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased.
Zona pellucida (ZP) must be shed within a time period when the uterus is receptive in order for a pregnancy to occur. Assisted hatching (AH) has been proposed to increase the hatching and embryo implantation rate, probably by enhancing early communication between the embryo and endometrium . An infrared laser system appears to be the most suitable method for assisted hatching, as it has good repeatability and consistency between operators and is simple to perform [1–3].
It is debatable whether a full-thickness opening is better than the partial zona thinning. There was a concern that a hole in the zona might deprive the embryo of its protective coat against infectious or immunologic insult . Blastomeres could be lost through an opening in the zona during a difficult embryo transfer or during contraction of the uterus after the transfer [3, 4]. Zonal thinning was believed to be optimal in promoting complete hatching without the potential drawbacks of the full-thickness opening . In the Cochrane Systematic Review , subgroup analysis by the extent of assisted hatching showed that there was a significant increase in the clinical pregnancy rate per woman randomized in both the zona thinning group (OR 1.33, 95% CI 1.04, 1.72) and the full-thickness AH (OR 1.23, 95% CI 1.01, 1.50). To date, there have been only one randomized trial  and one retrospective study  that compared laser zonal breaching with thinning in the human, and one study in the mouse , but the results were inconclusive.
Many studies showed that AH significantly increased the implantation and pregnancy rates in women with poor prognosis, such as advanced age [5, 10–12], recurrent implantation failure [5, 12–16], or in the frozen-thawed cycles [17–25]. However, other studies were unable to confirm the beneficial effects of AH in the general populations [26–30], or in specific groups, such as advanced age [31–37], recurrent implantation failure [21, 36], or in frozen-thawed cycles [7, 36, 38–41]. Of more concern was that some studies reported a lower pregnancy rate after complete AH  or partial zona thinning  than that in the controls. The current opinion is that the routine use of assisted hatching for all patients is unwarranted in view of the lack of evidence of universal benefit and the potential risks [3, 43].
In this research, we used a mouse model to study the effect of partial and complete AH on the rate of blastocyst formation, hatching and cell numbers. In a previous study, Montag et al. showed that the mean numbers of embryonic cells in hatching blastocyst in the full-thickness laser AH were significantly lower than those in the non-AH controls. However, they did not report whether the decrease was predominantly in the trophectoderm or inner cell mass. Moreover, the number of cells in completely hatched blastocysts in the two groups on the same day of embryonic development was not reported. We postulated that both the complete and partial AH might have a detrimental effect on the number of cells in the hatched blastocysts.
International Cancer Research (ICR) mice were purchased form the National Animal Institute, Mahidol University, Bangkok, Thailand. They were kept in our animal husbandry unit, at 25±2°C, under 60-70% humidity with controlled 12 hour light/12 hour dark cycles. Before the experiment, the mice were left undisturbed for five days to avoid the effects of stress from transportation. The Animal Ethics Committee of the Faculty of Medicine, Chiang Mai University approved the use of mice in our study (protocol no. 40/2553). All the procedures were performed in compliance with the EC Directive 86/609/EEC for animal experiments.
Five- to seven-week-old ICR females were super-ovulated by an intra-peritoneal injection of 10 IU of equine gonadotropin (Sigma, St. Louis, USA), followed 48 hours later by 10 IU of human chorionic gonadotropin (hCG; Pregnyl, Organon, Oss, The Netherlands). After hCG injection, females were paired with nine- to twenty-week males. They were checked for mating 16 hours later, and sacrificed by cervical vertebrae dislocation 36 hours after hCG injection. Two-cell embryos were flushed from the oviducts, and washed twice in cleavage medium (Cook, Brisbane, Australia) under paraffin oil (Medicult, Jyllinge, Denmark). They were cultured in groups of ten in 10 μL drops of cleavage medium for 48 hours without medium renewal until they became morulae.
The XYClone® laser system (Hamilton Thorne Biosciences, Beverly, MA, USA) was attached to an inverted microscope (Eclipse TE300, Nikon, Tokyo, Japan), equipped with a heated stage (Kitazato, Fujinomiya, Japan). The machine emitted an infrared laser beam with a wavelength of 1.48 μm, at a power of 140 mW. The pulse was set to create a 10-μm hole in the mouse ZP.
Up to five morulae were placed in a 10 μL drop of medium under oil and put on the microscopic stage. The laser beam was fired once at the ZP to create a full-thickness opening of 10 μm, or twice in adjacent area for an opening of 20 μm. For ZP thinning, the same setting of laser was used to fire in a consecutive area along the periphery of zona to leave a thin rim (~ 10%) of the original thickness, covering an area of 1/4 of the ZP circumference. Control morulae were placed on the stage for the same duration as the experimental groups, but laser drilling was not performed.
In experiment 1, four hundred and forty mouse morulae were divided into a control and three laser-AH groups (1 hole, 2 holes or 3 holes evenly apart, each of 10 μm in size) (Table 1).
In experiment 2, three hundred and sixty morulae were divided into a control and two laser-AH groups (1 or 2 holes at opposite pole, each measured 20 μm in size).
In experiment 3, five hundred and forty morulae were divided into a control and two laser-AH groups (full-thickness opening of 20 μm or quarter-thinning of the zonal circumference to a depth of 90%).
Culture condition and assessment of embryo development
After the intervention, groups of ten morulae were cultured together in 10 μL drops of blastocyst medium (Cook, Brisbane, Australia) under paraffin oil, in an atmosphere of 6% CO2, 5% O2 and 89% N2 at 37°C. Embryos were cultured in this medium for up to 72 hours without medium renewal. Embryo development was observed at 24, 48 and 72 hours after AH.
Differential staining of inner cell mass (ICM) and trophectoderm (TE) cells
Differential staining was performed on all completely hatched blastocysts, using the protocol described by Pampfer et al.. In brief, the blastocysts were washed three to four times in calcium- and magnesium-free buffer, before exposure to rabbit anti-mouse antibody (Sigma M 5774; concentration 1:50) for 30 minutes at 37°C. After washing, they were transferred into guinea pig complement serum (Sigma S 1639) with propidium iodide (Sigma P 4170) and bisbenzimide (Sigma B 2261) at 37°C for 10–15 minutes. The blastocysts were washed and transferred onto glass slides to allow air dry. The slides were mounted in glycerol, and the number of ICM and TE cells were counted under a Nikon E600 epifluorescence microscope, equipped with LUCIA FISH program (Laboratory Imaging, Prague, Czech Republic).
The STATA program version 8.2 (College Station, Texas, USA) was used to perform Chi-square tests to compare the proportions of morulae that became blastocysts, and proportions of hatching and completely hatched blastocysts in the experimental and control groups at 24, 48, and 72 hours after AH, respectively. Fisher exact test was used when any of the expected cell frequencies was <5. The mean numbers of inner cell mass (ICM) and trophectoderm (TE) cells of hatched blastocysts from different groups were compared by one-way ANOVA, with Scheffe post-hoc tests as appropriate. A two-tailed P<0.05 was considered statistically significant. Bonferroni correction was used as a safeguard against multiple tests of statistical significance on the same data by dividing the cut-off P-value of <0.05 by the number of tests done on that data set.
A total of 1340 morulae were included in the study. In experiment 1, the proportions of morulae that became blastocysts 24 hours after the interventions in the control (108/110) and 3 laser AH groups (109/110, 109/110 and 110/110 for the 1-, 2- and 3-hole groups, respectively) were not statistically different (P=0.905). The proportions of hatching blastocysts at 48 hours were significantly higher in the AH groups (94/109, 99/109 and 102/110, respectively) than controls (51/108; P=0.000). There was a higher hatching rate when the number of holes was increased. In contrast, the rate of completely hatched blastocysts at 72 hours was significantly higher in the control (44/108) than the AH groups (19/109, 14/109 and 12/110, respectively; P=0.000). A higher proportion of blastocysts were entrapped when the number of holes was increased. There was a significant difference in the number of ICM, TE and total cells, but not in ICM/TE ratio between AH and non-AH blastocysts (Table 2).
In experiment 2, the proportions of morulae that became blastocysts in the controls (118/120) and 2 laser AH groups (119/120 and 118/120 in the 1- and 2-hole groups, respectively) were not different (P=1.000). The proportion of completely hatched blastocysts was significantly higher in both AH groups (66/119 and 63/118, respectively) than that in the controls (44/118; P=0.009). There was a significant difference in the number of ICM, TE, and total cells between control and laser-AH blastocysts, but not in ICM/TE ratio (Table 3).
In experiment 3, the proportions of morulae that became blastocysts were not different between control and the two laser-AH groups (Table 4). After 48 hours, the proportions of hatching blastocysts in the two laser-AH groups were higher than that in the control. The proportion of completely hatched blastocyst was highest in the full-thickness AH group, compared to those in the control and quarter laser-thinning group. The rate of blastocyst trapping was likewise lowest in the full-thickness AH group. There was a significant difference in the number of ICM, TE, and total cells in both AH groups when compared with controls (Table 5).
In the first experiment, we confirmed previous studies that a small opening in the zona, although associated with early hatching, ended up with a higher rate of blastocyst entrapment [19, 46–48]. The entrapment increased with increasing numbers of openings. In experiment 2, laser AH of adequate size was associated with not only early but also a higher rate of complete blastocyst hatching. In this case, two opposite openings did not result in an increase entrapment. Cohen and Feldberg  clearly showed that embryos with multiple openings in their zona preferentially hatched through the largest one. Both their study and ours indicated that the size was more important than the number of zonal opening.
Several previous studies supported the practice of zona thinning rather than total breaching [8, 46]. Zonal thinning was believed to be optimal in promoting complete hatching without the potential drawbacks of the full-thickness opening. Moreover, increasing the area of the zona thinning might encompass the site of natural hatching. In our study, we chose to perform quarter zonal thinning rather than thinning of half of the ZP circumference. The reason was that it would save time as well as decrease the number of laser shots. The embryos would be outside the incubator shorter and the risk of temperature increase in the immediate vicinity of the embryos from laser thermal shock would be minimized. It was disappointing that quarter laser thinning of the ZP did not increase the percentage of completely hatched blastocysts. On the contrary, it even increased the chance of trapping. Our result was consistent with that reported by Tinney et al. . They performed zonal thinning through seven consecutive shots in the zona, and found an increase in the rate of incompletely hatched blastocysts. Interestingly, a randomized trial of laser quarter zona thinning by Valojerdi et al.  showed a significant decrease in clinical pregnancy and implantation rates in vitrified/warmed human embryos, compared with non-hatched controls.
In this study we performed differential counts of ICM and TE cells in all completely hatched blastocysts. The mean numbers of embryonic cells (ICM+TE) in our study were higher than those reported by Montag et al.  and Fathi et al. . This was because they counted cells in hatching  or expanding  blastocysts rather than completely hatched blastocysts. Our study and that by Montag et al.  agreed that blastocyst cell counts significantly decreased after laser AH, when compared with the control. Fathi et al.  also observed a similar decrease in the number of cells in the ICM, TE and total cells, but the difference only reached statistical significant in the case of the ICM. In our study, we found that the decrease occurred proportionately in the ICM and TE cells, with no change in the ICM/TE ratio. The decrease was also observed in the case of partial zona thinning, albeit to a lesser degree than that seen in complete AH. Montag et al.  reasoned that the presence of a laser-drilled opening in the zona allowed hatching to occur through the opening as soon as the blastocyst started to expand. In contrast, in the untreated control, hatching was delayed until sufficient number of embryonic cells was available to overcome the resistance of the zona. However, this assumption alone could not explain why the number of cells in the completely hatched blastocysts in the AH group was significantly fewer than controls on the same day of embryonic development. Perhaps, an alternative explanation could be that blastocyst expansion stimulated the proliferation of cells in the blastocysts. Without blastocyst expansion in the case of full-thickness AH, or with partial expansion in zonal thinning, the number of cells in the resulting blastocysts decreased in a dose–response manner. Given this assumption, one could explain why the routine use of AH in normal embryos might not be beneficial or even be detrimental. On the other hand, AH may be useful in cases with real zona hardening, despite the lower number of cells in the resulting blastocysts.
The limitation of our study was that we did not transfer the laser-treated embryos into the uterus, and hence implantation and pregnancy rates could not be determined. Moreover, the hatching process in vitro and in vivo might be different. There could also be species difference, and one should be very cautious in projecting data from the mouse directly to the human.
Although the use of an in vitro mouse model was our weakness, it was also our strength. We were able to study a large numbers of embryos, which was not possible with humans due to ethical reasons. Our in vitro study allowed us to follow the fate of all embryos to the end. The percentage of trapped and completely hatched blastocysts could be accounted for, and the number of ICM and TE cells could be enumerated in all completely hatched blastocysts.
In conclusion, the size of the zonal opening was more important than its number. A full-thickness opening allowed more complete blastocyst hatching than quarter zona thinning. The numbers of cells in the ICM and TE of hatched blastocysts after full-thickness or partial AH were lower than those in the non-manipulated controls, but there was no significant change in the ICM to TE ratio. The blastocyst cell number was slightly lower in the complete than the partial AH group, but the difference was not statistically significant.
Human chorionic gonadotropin
Inner cell mass
International Cancer Research
Feng HL, Hershlag A, Scholl GM, Cohen MA: A retroprospective study comparing three different assisted hatching techniques. Fertil Steril. 2009, 91: 1323-1325. 10.1016/j.fertnstert.2008.02.133.
Veiga A, Boiso I, Belil I: Assisted hatching. Textbook of assisted reproductive technologies. Edited by: Gardner DK, Weissman A, Howles CM, Shoham Z. 2009, London: Informa Healthcare, 181-190. 3
Hammadeh ME, Fischer-Hammadeh C, Ali KR: Assisted hatching in assisted reproduction: a state of the art. J Assist Reprod Genet. 2011, 28: 119-128. 10.1007/s10815-010-9495-3.
Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, Wiemer K: Impairment of hatching process following IVF in the human and improvement of implantation by assisted hatching using micromanipulation. Hum Reprod. 1990, 5: 7-13.
Petersen CG, Mauri AL, Baruffi RL, Oliveira JBA, Massaro FC, Elder K, Franco JG: Implantation failures: success of assisted hatching with quarter-laser zona thinning. Reprod Biomed Online. 2005, 10: 224-229. 10.1016/S1472-6483(10)60944-3.
Carney SK, Das S, Blake D, Farquhar C, Seif MM, Nelson L: Assisted hatching on assisted conception (in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). Cochrane Database Syst Rev. 2012, 12: 10.1002/14651858.CD001894.pub5. CD001894
Ng EHY, Lau EYL, Yeung WSB, Cheung TM, Tang OS, Ho PC: Randomized double-blind comparison of laser zona pellucida thinning and breaching in frozen-thawed embryo transfer at the cleavage stage. Fertil Steril. 2008, 89: 1147-1153. 10.1016/j.fertnstert.2007.05.016.
Mantoudis 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: 2182-2186. 10.1093/humrep/16.10.2182.
Khalifa EA, Tucker MJ, Hunt P: Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse. Hum Reprod. 1992, 7: 532-536.
Meldrum DR, Wisot A, Yee B, Garzo G, Yeo L, Hamilton F: Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J Assist Reprod Genet. 1998, 15: 418-421. 10.1007/BF02744934.
Schoolcraft WB, Schlenker T, Jones GS, Jones HW: In vitro fertilization in women age 40 and older: the impact of assisted hatching. J Assist Reprod Genet. 1995, 12: 581-584. 10.1007/BF02212578.
Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Aicardi G, Montanaro N: Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod. 1998, 13: 1331-1335. 10.1093/humrep/13.5.1331.
Nakayama T, Fujiwara H, Yamada S, Tastumi K, Honda T, Fujii S: Clinical application of a new assisted hatching method using a piezo-micromanipulator for morphologically low-quality embryos in poor-prognosis infertile patients. Fertil Steril. 1999, 71: 1014-1018. 10.1016/S0015-0282(99)00131-4.
Antinori S, Selman HA, Caffa B, Panci C, Dani GL, Versaci C: Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy. Hum Reprod. 1996, 11: 2488-2492. 10.1093/oxfordjournals.humrep.a019145.
Chao KH, Chen SU, Chen HF, Wu MY, Yang YS, Ho HN: Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)-embryo transfer (ET), but not that of IVF-tubal ET in patients with repeated IVF failures. Fertil Steril. 1997, 67: 904-908. 10.1016/S0015-0282(97)81404-5.
Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia J, Fisch B: Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril. 1995, 63: 838-841.
Ge HS, Zhou W, Zhang W, Lin JJ: Impact of assisted hatching on fresh and frozen-thawed embryo transfer cycles: a prospective, randomized study. Reprod Biomed Online. 2008, 16: 589-596. 10.1016/S1472-6483(10)60466-X.
Balaban B, Urman B, Yakin K, Isiklar A: Laser-assisted hatching increases pregnancy and implantation rates in cryopreserved embryos that were allowed to cleave in vitro after thawing: a prospective randomized study. Hum Reprod. 2006, 21: 2136-2140. 10.1093/humrep/del097.
Hiraoka K, Fuchiwaki M, Hiraoka K, Horiuchi T, Murakami T, Kinutani M, Kinutani K: Effect of the size of zona pellucida opening by laser assisted hatching on clinical outcome of frozen cleaved embryos that were cultured to blastocyst after thawing in women with multiple implantation failures of embryo transfer: a retrospective study. J Assist Reprod Genet. 2008, 25: 129-135. 10.1007/s10815-008-9214-5.
Tucker MJ, Cohen J, Massey JB, Mayer MP, Wiker SR, Wright G: Partial dissection of the zona pellucida of frozen-thawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol. 1991, 165: 341-344.
Valojerdi 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: 84-91. 10.1016/j.fertnstert.2007.06.005.
Check JH, Hoover L, Nazari A, O'Shaughnessy A, Summers D: The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril. 1996, 65: 254-257.
Gabrielsen A, Agerholm I, Toft B, Hald F, Petersen K, Aagaard J, Feldinger B, Lindenberg S, Fedder J: Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study. Hum Reprod. 2004, 19: 2258-2262. 10.1093/humrep/deh434.
Tao J, Tamis R: Application of assisted hatching for 2-day-old, frozen-thawed embryo transfer in a poor-prognosis population. J Assist Reprod Genet. 1997, 14: 128-130. 10.1007/BF02765783.
Vanderzwalmen P, Bertin G, Debauche C, Standaert V, Bollen N, van Roosendaal E, Vandervorst M, Schoysman R, Zech N: Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod. 2003, 18: 1504-1511. 10.1093/humrep/deg298.
Cohen J, Alikani M, Trowbridge J, Rosenwaks Z: Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod. 1992, 7: 685-691.
Hellebaut S, De Sutter P, Dozortsev D, Onghena A, Qian C, Dhont M: Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? A prospective randomized study. J Assist Reprod Genet. 1996, 13: 19-22. 10.1007/BF02068864.
Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD: Assisted hatching does not enhance IVF success in good-prognosis patients. J Assist Reprod Genet. 1998, 15: 62-64. 10.1007/BF02766826.
Mansour RT, Rhodes CA, Aboulghar MA, Serour GI, Kamal A: Transfer of zona-free embryos improves outcome in poor prognosis patients: a prospective randomized controlled study. Hum Reprod. 2000, 15: 1061-1064. 10.1093/humrep/15.5.1061.
Tucker MJ, Luecke NM, Wiker SR, 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: 187-191. 10.1007/BF01239219.
Frydman N, Madoux S, Hesters L, Duvernoy C, Feyereisen E, Le Du A, Tachdjian G, Frydman R, Fanchin R: A randomized double-blind controlled study on the efficacy of laser zona pellucida thinning on live birth rates in cases of advanced female age. Hum Reprod. 2006, 21: 2131-2135. 10.1093/humrep/del124.
Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S, Muasher SJ: A prospective, randomized, double-blind study for the evaluation of assisted hatching in patients with advanced maternal age. Hum Reprod. 1998, 13: 409-413. 10.1093/humrep/13.2.409.
Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW: Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril. 1994, 62: 551-554.
Ali J, Rahbar S, Burjaq H, Sultan AM, Al Flamerzi M, Shahata MA: Routine laser assisted hatching results in significantly increased clincal pregnancies. J Assist Reprod Genet. 2003, 20: 177-181. 10.1023/A:1023665909123.
Bider 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: 317-320. 10.1093/humrep/12.2.317.
Edirisinghe 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: 294-301. 10.1023/A:1020497714495.
Makrakis E, Angeli I, Agapitou K, Pappas K, Dafereras A, Pantos K: Laser versus mechanical assisted hatching: a prospective study of clinical outcomes. Fertil Steril. 2006, 86: 1596-1600. 10.1016/j.fertnstert.2006.05.031.
Primi MP, Senn A, Montag M, Van der Ven H, Mandelbaum J, Veiga A, Barri P, 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: 2325-2333. 10.1093/humrep/deh430.
Sifer C, Sellami A, Poncelet C, Kulski P, Martin-Pont B, Bottero J, Porcher R, Cedrin-Durnerin I, Hugues JN, Wolf JP: A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers. Hum Reprod. 2006, 21: 2384-2389. 10.1093/humrep/del149.
Petersen CG, Mauri AL, Baruffi RL, Oliveira JB, Felipe V, Massaro FC, Franco JG: Laser-assisted hatching of cryopreserved-thawed embryos by thinning one quarter of the zona. Reprod Biomed Online. 2006, 13: 668-675. 10.1016/S1472-6483(10)60657-8.
Ng EH, Naveed F, Lau EY, Yeung WS, Chan CC, Tang OS, Ho P: A randomized double-blind controlled study of the efficacy of laser-assisted hatching on implantation and pregnancy rates of frozen-thawed embryo transfer at the cleavage stage. Hum Reprod. 2005, 20: 979-985.
Valojerdi MR, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B: Effect of laser zona thinning on vitrified-warmed embryo transfer at the cleavage stage: a prospective, randomized study. Reprod Biomed Online. 2010, 20: 234-242. 10.1016/j.rbmo.2009.11.002.
The Practice Committee of the Society for Assisted Reproductive Technology and the Practice Committee of the American Society for Reproductive Medicine: The role of assisted hatching in in vitro fertilization: A review of the literature. A Committee opinion. Fertil Steril. 2008, 90: S196-S198.
Montag M, Koll B, Holmes P, Van der Ven H: Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo. Biol Reprod. 2000, 62: 1738-1744. 10.1095/biolreprod62.6.1738.
Pampfer S, De Hertogh R, Vanderheyden I, Michiels B, Vercheval M: Decreased inner cell mass proportion in blastocysts from diabetic rats. Diabetes. 1990, 39: 471-476. 10.2337/diabetes.39.4.471.
Baruffi RL, Mauri AL, Petersen CG, Ferreira RC, Coelho J, Franco JG: Zona thinning with noncontact diode laser in patients aged < or = 37 years with no previous failure of implantation: a prospective randomized study. J Assist Reprod Genet. 2000, 17: 557-560. 10.1023/A:1026481729632.
Schmoll F, Schneider H, Montag M, Wimmers K, Rink K, Schellander K: Effects of different laser-drilled openings in the zona pellucida on hatching of in vitro-produced cattle blastocysts. Fertil Steril. 2003, 80 (Suppl 2): 714-719.
Cohen J, Feldberg D: Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol Reprod Dev. 1991, 30: 70-87. 10.1002/mrd.1080300110.
Tinney GM, Windt ML, Kruger TF, Lombard CJ: Use of a zona laser treatment system in assisted hatching: optimal laser utilization parameters. Fertil Steril. 2005, 84: 1737-1741. 10.1016/j.fertnstert.2005.05.048.
Fathi R, Valojerdi MR, Eftekhari-Yazdi P: Effext of laser-assisted hatching and necrotic blastomere removal on the develoment of vitrified-warmed four-cell mouse embryos. J Assist Repprod Genet. 2008, 25: 333-339. 10.1007/s10815-008-9238-x.
This research was supported by the Faculty of Medicine Endowment Fund for Medical Research, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, Grant no. 31/2554. The funding agency played no role in the study design, data collection, analysis, interpretation and writing of the report or in the decision to submit the article for publication. The Faculty of Medicine, Chiang Mai University provided a professional reviewer, who is a native English speaker, to help in language editing.
The authors declare that they have no competing interests.
CC participated in the design, carried out experiments 1–3, and helped in data analysis and revising the manuscript critically for important intellectual content. US and SS helped CC in carrying out experiments 1–3, read, revised and approved the final manuscript. WP participated in the design and coordination of the study, took part in the analysis, interpretation revised and approved the manuscript. TV made substantial contributions to conception, design, analysis and interpretation of the data, and wrote the manuscript. All authors read and approved the final manuscript.
About this article
Cite this article
Chailert, C., Sanmee, U., Piromlertamorn, W. et al. Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers. Reprod Biol Endocrinol 11, 21 (2013) doi:10.1186/1477-7827-11-21
- Assisted hatching
- Differential staining
- Zona drilling
- Zona pellucida