Lasers in Medical Science (2019) 34:939–945 https://doi.org/10.1007/s10103-018-2681-8 ORIGINAL ARTICLE The impacts of laser zona thinning on hatching and implantation of vitrified-warmed mouse embryos Zhengyuan Huang1 & Jinghao Liu2 & Lei Gao1 & Qingrui Zhuan1 & Yuxi Luo 1 & Shien Zhu1 & Kaiyu Lei3 & Xiangwei Fu1 Received: 3 September 2018 /Accepted: 2 November 2018 /Published online: 13 December 2018 # Springer-Verlag London Ltd., part of Springer Nature 2018 Abstract Embryo vitrification has advantages in assisted reproduction yet it also induces zona hardening. Laser zona thinning (LZT) is considered as a solution yet its efficacy and security have not been well studied. In this study, we used vitrified-warmed morulae from 2-month-old and 10-month-old ICR female mice as model to investigate the impacts that LZT treatment brings to the in vitro hatching process and implantation by analyzing hatching rate, implantation rate, and blastocyst quality. The results showed that the fully hatched rate was significantly higher after LZT treatment for both young (25.7% vs. 16.2%, P < 0.05) and aged (36.6% vs. 13.2%, P < 0.01) mice. For zona-thinned morulae in young mice, its onset of hatching occurred earlier (28.6% vs. 8.8%, P < 0.01) at D4 and with a greater percentage of U-shaped hatching at D5 (48.3% vs. 33.0%, P < 0.05). LZT treatment did not induce expression change of apoptosis-related genes in all groups (P > 0.05), but for young mice, the total cell number of day 5 blastocyst in zona-thinned group was significantly less than that of the control group (40.6 ± 5.1 vs. 59.9 ± 14.5, P <0.01). At last, there was an increasing implantation rate in zona-thinned compared to the control group for young (63.8% vs. 52.5%, P > 0.05) and aged (55.6% vs. 47.2%; P > 0.05) mice after embryos were bilaterally transferred in the same recipient. In conclusion, the significant increase of fully hatched rate after LZT treatment is related to the advanced onset of hatching as well as the enhancement of superior hatching structure, and LZT also lead to a better implantation after embryo transfer. Keywords Mouse embryo . Vitrification . Laser zona thinning . Hatching . Implantation Introduction superfluous embryos after in vitro fertilization in vitro culture, and effectively prevent the occurrence of ovarian hyper- As one of the milestone for assisted reproductive technology, stimulation syndrome, after ovarian recovery, frozen-thawed human embryo was firstly successfully cryopreserved in 1983 embryos are able to transfer to the uterus horn after one or [1]. Embryo cryopreservation can long-term preserve those several programmed or natural physiological cycles [2]. However, it has been accepted that zona pellucida (ZP) will undergo problematic hardening during the freeze-thaw proce- Zhengyuan Huang and Jinghao Liu contributed equally to this work. dure [3], which is commonly occurred in ART, exerting ad- verse effect on embryo hatching and implantation [4]. To be Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10103-018-2681-8) contains supplementary more specific, cryopreservation induces microstructural material, which is available to authorized users. change on embryo ZP after freezing-thawing [5] and study has demonstrated that the vitrification solution containing * Xiangwei Fu DMSO and EG which could induce ZP hardening [6]. Apart [email protected] from being induced by vitrification, zona hardening is thought to be a phenomenon that is more prevalent among senior 1 National Engineering Laboratory for Animal Breeding and Key women [7]. A study found out that ZP thickness decreased Laboratory of Animal Genetics, Breeding and Reproduction, Beijing Key Laboratory for Animal Genetic Improvement, College of while the density of the ZP increased in two age groups (older, Animal Science and Technology, China Agricultural University, >38years;younger,≤ 38 years) with extended culture; how- Beijing, China ever, the older group had significantly denser ZP compared 2 Laboratory Animal Center, Peking University, Beijing, China with the younger group [8], indicating that the microstructure 3 BGI Clinical Laboratories (Shenzhen) Co., Ltd, Shenzhen, China of the ZP is also influenced by age. 940 Lasers Med Sci (2019) 34:939–945 For this situation, Jacques Cohen suggested using Vitrified-warmed mouse embryos collection assisted hatching to cope with this problem [9]. This is a methodology using various approaches to drill a hole on CD-1 (ICR) mice purchased from the Vital River Laboratory ZP, which is believed to facilitate the embryo’s escape Animal Technology Co. Ltd. (Beijing, China) were used to from the ZP, and this strategy has been suggested as a collect oocyte and spermatozoa. Mice were under free feed mean of improving pregnancy rates in patients with recur- intake, housing with a 12-h light/dark photoperiod and main- rent implantation failure [10] or in frozen embryo transfer tained in an air-conditioned (22–25 °C) and 40% humidity cycles [3]. Later, as a non-invasive technique, laser grad- room. Female ICR mice (2 months of age, 31.3 ± 1.8 g; ually introduced to assist embryo hatching in early 1990s, 10 months of age, 42.8 ± 5.0 g) were injected with 7.5 IU which is considered to be safer compared to other assisted pregnant mare serum gonadotrophin intraperitoneally hatching techniques [11]. Although laser zona thinning (PMSG; Tianjin Animal Hormone Factory, Tianjin, China) (LZT) is not included as an approach of assisted hatching then 7.5 IU human chorionic gonadotrophin (hCG; Ningbo [12], it is still a commonly used technique in assisted Animal Hormone Factory, Ningbo, China) after 48 h. After reproduction [13] in view of a functional ZP is critical being sacrificed by cervical dislocation, sperm from the caudal in protecting the embryos from detrimental factors likes epididymis of adult male ICR mice (10–14 weeks of age) were toxins, microorganisms, and antibodies in IVF media suspended in human tubal fluid (HTF) medium (Millipore, [14–16], and zona-opening could lead to the loss of em- MA, USA) and incubated at 37 °C under 5% CO2 and 95% bryonic autocrine growth factors [17], which will bring humidity for 1 h in advance. Oocytes were collected from the potential risk to embryo development. However, whether oviducts of female mice 13–15 h after hCG injection. The LZT is necessary to improve the implantation ability of capacitated sperm suspension was gently added to the frozen-thawed, embryo is still under debate [18]. Some cumulus-oocyte complexes at a final motile sperm concentra- researchers suggested that cryopreservation will induce tion of 106 cells/ml. The oocytes and sperm were incubated in zona change, so LZT can increase the implantation of HTF medium for fertilization. After 4 h, the fertilized oocytes frozen-thawed embryos [19–21], while other researchers were washed for three times and transferred to fresh culture have opposing views because they could not obtain posi- medium. Double pronuclei were confirmed with phase- tive outcome [12, 22–25] and two prospective randomized contrast microscopy. All zygotes were cultured in micro drops controlled trials [26, 27] even had controversial opinions (80 μL) of medium in culture dishes (Nunc, Roskilde, about the effectiveness of LZT. The different manipula- Denmark) overlaid with mineral oil for 3 days. Embryo cul- tion condition in different assisted reproductive center ac- ture, capacitation and fertilization dishes were prepared 4 h count for those controversial comments; therefore, reliable prior to embryo culture so as to allow gas and temperature outcomes should be obtained from refined randomized equilibration. controlled trials. Morulae in good quality were collected then vitrified after To our knowledge, this prospective randomized study 3-day culture. The quality of morula on day 3 was assessed was for the first time to investigate what impacts will according to the previous criteria [28]. For vitrification, pre- LZT treatment bring to hatching dynamics of vitrified- treatment solution contained 10% dimethyl sulfoxide warmed mouse embryo. Moreover, vitrified-warmed em- (DMSO) and 10% ethylene glycol (EG) in PBS medium. bryos from laser-thinned and control groups were bilat- Vitrification solution (EDFS35) contained 17.5% DMSO (v: erally transferred to the same recipient to investigate the v), 17.5% EG (v: v), 30% Ficoll (w: v) and 0.5 M sucrose in impact of LZT treatment on implantation. Furthermore, PBS medium. Embryos were vitrified in EDFS35 by the open- age is also taken into account as a factor to study pulled straws (OPS) method [29]. First, embryos were rinsed whether LZT treatment has distinct effectiveness on dif- in pretreatment solution for 30 s, then transferred to vitrifica- ferent ages. This study can provide valuable information tion solution in the narrow end of the OPS, and held for 25 s. to the enhancement of human-assisted reproductive Then the straws were immediately plunged into liquid nitro- technology. gen (LN2). For thawing, the embryos were rinsed in 0.5 M sucrose for 5 min, then rinsed three times in M2 medium. Materials and methods Laser zona thinning Unless otherwise indicated, all chemicals and media were Laser manipulation were performed according to previous re- purchased from Sigma-Aldrich (MO, USA). The protocols ports [30, 31]. We used the XYClone Laser System (Hamilton for the animal studies were approved by and performed in Thorne, MA, USA), including an integrated class I laser accordance with the requirements of the Institutional Animal (1.46 μm, 300 mW) with 20 × objective, and a 640-μm pilot Care and Use Committee of China Agricultural University. light on an inverted microscope with heated stage (TL3-220P, Lasers Med Sci (2019) 34:939–945 941 Olympus, Japan). The pilot light was calibrated to a target on a warmed embryos × 100 (%).