Analysis of embryo intactness and developmental potential following slow freezing and vitrification

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1 Systems Biology in Reproductive Medicine ISSN: (Print) (Online) Journal homepage: Analysis of embryo intactness and developmental potential following slow freezing and vitrification Lan Yu, Chanwei Jia, Yonglian Lan, Rui Song, Liying Zhou, Ying Li, Yu Liang & Shuyu Wang To cite this article: Lan Yu, Chanwei Jia, Yonglian Lan, Rui Song, Liying Zhou, Ying Li, Yu Liang & Shuyu Wang (2017) Analysis of embryo intactness and developmental potential following slow freezing and vitrification, Systems Biology in Reproductive Medicine, 63:5, , DOI: / To link to this article: Published online: 10 Aug Submit your article to this journal Article views: 12 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 06 September 2017, At: 05:00

2 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 2017, VOL. 63, NO. 5, RESEARCH COMMUNICATION Analysis of embryo intactness and developmental potential following slow freezing and vitrification Lan Yu, Chanwei Jia, Yonglian Lan, Rui Song, Liying Zhou, Ying Li, Yu Liang, and Shuyu Wang Department of Reproductive Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China ABSTRACT The aim of this study was to identify the parameters that are related to intactness and developmental potential of a day 3 embryo after warming to improve the selection criteria used to cryopreserve and transfer embryos. We also sought to compare slow freezing and vitrification methods of cryopreservation and to evaluate the viability of non-intact embryos. Embryos warmed following slow freezing (n=220) or vitrification (n=522) were divided into 3 groups according to the proportion of surviving blastomeres (I<50%; II=50-99%; and III=100%). The developmental potential of embryos, including the mitosis resumption rate, blastocyst formation rate, and formation rate of grade A blastocysts (i.e., fully expanded blastocysts with an inner cell mass and grade A or B trophectoderm) were retrospectively assessed in embryos. Cleavage-stage embryos with <50% blastomere survival were analyzed using next-generation sequencing (NGS). Logistic regression analysis showed that vitrification and grade 1 were independent predictive factors of embryo intactness and developmental potential (all p<0.05). On day 3, embryos with 4-6 cells or blastomere damage had lower developmental potential than those with 7-9 cells or intact blastomeres (all p<0.05). NGS results showed that the chromosomal status was completely normal in 8 embryos that developed into expanded blastocysts, whereas 4 out of 5 embryos in which development was arrested were abnormal. The results of this study suggest that vitrification is a better choice than slow freezing for embryo cryopreservation. Embryos showing poor quality (fragmentation >30% and/or a non-stage-specific cell size) and lower cell numbers (4-6 cells) on day 3 should be cultured to the blastocyst stage and then vitrified if they develop into goodquality blastocysts. The developmental potential of non-intact embryos is lower than that of intact embryos; however, after they are cultured to the fully expanded blastocyst stage, embryos with <50% blastomere survival appear to be better candidates for transfer. Abbreviations ART: assisted reproductive technology; grade A blastocyst: fully expanded blastocyst with an inner cell mass and grade A or B trophectoderm; NGS: next-generation sequencing; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; FET: frozen-thawed embryo transfer Introduction Cryopreservation has become an integral part of assisted reproductive technology (ART) [He et al. 2016]. Most reports regarding the developmental potential of embryos after warming have involved the slow-freezing method. The developmental potential of cryopreserved cleavagestage embryos treated with slow freezing has been investigated, and higher blastocyst formation rates and implantation rates have been obtained using fully intact embryos than when using damaged day 2 embryos [El-Toukhy et al. 2003; Archer et al. 2003]. One study suggested that pregnancy and implantation rates were lower when the blastomere lysis rate in day 3 embryos was greater than 25% but not greater than 50%, [Tang et al. 2006]. However, another study reported that the developmental potential of partially damaged frozen and thawed ARTICLE HISTORY Received 2 February 2017 Revised 8 July 2017 Accepted 10 July 2017 KEYWORDS Blastocyst culture; cryopreservation; embryo; next-generation sequencing; non-intact embryo embryos might be equivalent to that of fully survived embryos [Rienzi et al. 2005]. Previous studies have shown that during slow freezing, the presence of one or two lysed blastomeres in a thawed day 3 embryo did not appear to have a negative influence on the further development of sibling intact cells [Rienzi et al. 2005]. In recent years, vitrification has progressively replaced slow freezing in in vitro fertilization (IVF) laboratories [Fernandez Gallardo et al. 2016; Guanetal.2016; VanLanduytetal. 2013]. One study indicated that the overnight cleavage rate in day 3 embryos is lower in damaged than in fully intact embryos after both vitrification and slow freezing [Van Landuyt et al. 2013]. However, few reports have compared development to the blastocyst stage between CONTACT Shuyu Wang yushu572000@126.com Department of Reproductive Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Road 251, Chaoyang, Beijing , China Taylor & Francis

3 286 L. YU ET AL. intact and non-intact embryos that were submitted to vitrification and slow freezing. Generally, during frozen embryo transfer (FET), an embryo possessing >50% viable blastomeres following warming is considered a survivor [Gardner et al. 2012] and is therefore transferred. While most studies have focused on embryos with % viable cells after warming, in our study, the embryos were distributed into groups with blastomere survival rates of <50% and 50%-99% after warming, and the embryos were then cultured to determine their developmental potential. Cells in cleavage-stage embryos are totipotent [Van de Velde et al. 2000]. So even if a part of the embryo is lost, the cleavage-stage embryo remains capable of producing an intact individual. However, few previous reports have evaluated the viability of such embryos. We analyzed these embryos using next-generation sequencing (NGS), which is a large-scale, high-throughput, and massive paired-end mapping method that can be used to detect structural variations in genomes [Liang et al. 2017]. The Gardner grading system [Gardner et al. 2012] is capable of effectively predicting clinical outcomes regarding implantation, clinical pregnancy, and live birth [Richardson et al. 2015]; blastocysts with an expansion grade of 4, 5, or 6 (i.e., fully expanded), an inner cell mass, and grade A or B trophectoderm (i.e., a grade A blastocyst) are associated with better clinical outcomes. To determine the influence of blastomere loss on the developmental potential of cryopreserved cleavage-stageembryos,wecompared the effects of blastomere loss in embryos that were subjected to slow freezing and vitrification on day 3 and subsequently cultured. We evaluated the development potential (i.e., mitosis resumption, blastocyst formation, and grade A blastocyst formation) of embryos with different degrees of damage (i.e., I: <50%, II: 50-99%, and III: intact survival), different grades (i.e., 1-3 based on cell size and fragmentation), and different cell numbers before freezing. Furthermore, to evaluate viability, blastocysts that developed from embryos with a blastomere survival rate <50% were sequenced and analyzed using NGS. Results A total of 742 cryopreserved-warmed embryos were used in this study; they were cultured to days 5-6 to determine whether they could develop into expanded blastocysts. These included embryos with a blastomere survival rate <50% (5.4%, group I, n=40), 50-99% blastomere survival (23.0%, group II, n=171), and intact survival (71.6%, group III, n=531). Results from multivariate logistic analysis of the factors associated with the cell survival rate (I: <50%; II: 50-99%; and III: 100%) are shown in Table 1. Age (30.1 ± 3.9, 30.6 ± 3.7, and 30.4 ± 3.9 years old, respectively) and the IVF (vs. ICSI) rate (80.0%, 84.8%, and 81.2%, respectively) were not independently associated with intactness (p>0.05). Grade 1 was an independent protective factor that was more associated with the maintenance of intactness of embryos than grade 3 (I vs. III, odds ratio 0.32, 95% confidence interval ; and II vs. III, odds ratio 0.38, 95% confidence interval ). Cell number was negatively associated with the cell survival rate (p>0.05), and the vitrification cryopreservation method (p<0.05) was positively associated with intactness (Table 1). Among the embryos included in this analysis (n=742), 29.6% (n=220) were subjected to the slow freezing method, and 70.4% (n=522) were subjected to the vitrification method. There was no significant difference in the average age between the slow freezing (32.6±4.2) and the vitrification (31.1±3.9) groups Table 1. Multivariate logistic regression analysis of different cell survival rates. I(n=40) II (n=171) III (n=531) OR (95% CI) I vs. III OR (95% CI) II vs. III Age (y) 30.1 ± ± ± ( ) 1.03( ) IVF 32(80.0) 145(84.8) 430(81.2) 0.98( ) 1.33( ) ICSI 8(20.0) 26(16.2) 101(18.8) ref ref Grade 1 9(22.5) 43(25.1) 217(40.9) *0.32( ) *0.38( ) Grade 2 21(52.5) 88(51.5) 238(44.8) 0.67( ) 0.70( ) Grade 3 10(25.0) 40(23.4) 76(14.3) ref ref Cell number 1 8(20.0) 53(31.0) 170(32.0) 0.73( ) 1.13( ) Cell number 2 28(70.0) 100(58.5) 288(54.2) 1.83( ) 1.45( ) Cell number 3 4(10.0) 18(10.5) 73(13.7) ref ref Slow freezing 23(57.5) 86(50.3) 111(20.9) *5.84( ) *4.11( ) vitrification 17(42.5) 85(49.7) 420(79.1) ref Ref Proportion of surviving blastomeres: group I (<50%), group II (50%-99%), and group III (100%); Grade 1: fragmentation <10% and a stage-specific cell size; grade 2: fragmentation 10-30% with/without a stage-specific cell size for the majority of cells; grade 3: fragmentation 30-50% with/without a non-stagespecific cell size; Cell number: 1: cell number between 4-6; cell number 2: cell number between 7-9; and cell number 3: cell number greater than 9. Data are presented as n (%); *P<0.05.

4 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 287 (p>0.05). The survival rate (89.5%, 197/220 vs. 96.7%, 505/522; odds ratio 0.29, 95% confidence interval ) and the rate of intactness (50.5%, 111/220 vs. 80.7%; 421/522, OR 0.24, 95% confidence interval ) were significantly lower in the slow-freezing group than in the vitrification group (p<0.05). Similarly, the mitosis resumption rate (odds ratio 0.61, 95% confidence interval ), the blastocyst formation rate (odds ratio 0.60, 95% confidence interval ), and the grade A blastocyst formation rate (odds ratio 0.59, 95% confidence interval ) were significantly lower in the slow-freezing group than in the vitrification group (p<0.05, Table 2). Logistic regression analysis showed that the cryopreservation procedure used on embryos (i.e., slow freezing or vitrification) and grade of embryo had independent effects (all p<0.05, Table 3) but not an interactive effect on the rates of mitosis resumption, blastocyst formation, and grade A blastocyst formation (all p>0.05, Table 2). In contrast, the cryopreservation procedure and intactness of the embryos had an interactive effect on the developmental potential, as did the cryopreservation procedure and cell number of embryos (all p<0.05, Table 3). In the group of intact embryos (group III), logistic regression analysis showed that the cryopreservation procedure (slow freezing or vitrification) had no independent effect on the rates of mitosis resumption (odds ratio 1.49, 95% confidence interval ) and grade A blastocyst formation (odds ratio 1.36, 95% confidence interval ) (all p>0.05). In contrast, the cryopreservation procedure had an independent effect on the rate of blastocyst formation (odds ratio 1.58, 95% confidence interval , p<0.05). In the 7-9-cell group, logistic regression analysis showed that the cryopreservation procedure (slow freezing or vitrification) had no independent effect on the rates of mitosis resumption (odds ratio 0.22, 95% confidence interval ), blastocyst formation (odds ratio 0.25, 95% confidence interval ), and grade A blastocyst formation (odds ratio 1.32, 95% confidence interval ) (all p>0.05). Grade 2 embryos had significantly lower rates of mitosis resumption (251/347, 72.3%, odds ratio 0.66, 95% confidence interval ), blastocyst formation (202/347, 58.2%, odds ratio 0.61, 95% confidence interval ), and grade A blastocyst formation (133/347, 38.3%, odds ratio 0.61, 95% confidence interval ) than grade 1 embryos (215/269, 79.9%; 187/269, 69.5%; and 136/269, 50.6% in rate of mitosis resumption, blastocyst formation, and grade A blastocyst formation, respectively). The intact embryo rates were not significantly different among embryos with 4-6 cells, 7-9 cells, and >9 Table 2. Developmental potential of different grades of embryos. Method Slow freezing (n=220) Vitrification (n=522) OR (95% CI) Grade (n,%) 1(71, 32.3) 2(121, 55.0) 3(28, 12.7) 1(198, 37.9) 2(226, 43.3) 3(98, 18.8) Survival % 65(91.5) 107(88.4) 25(89.3) 195(98.5) 219(96.9) 91(92.9) 0.53( ) Intact % 37(52.1) 58(47.9) 16(57.1) 180(90.9) 181(80.1) 60(61.2) 0.39( )* Mitosis resumption % 51(71.8) 80(66.1) 15(53.6) 335(79.0) 171(75.7) 60(61.2) 0.83( ) Blastocyst formation % 42(59.2) 62(51.2) 8(28.6) 145(73.2) 140(61.9) 38(38.8) 0.85( ) Grade A blastocysts % 29(40.8) 38(31.4) 2(7.1) 107(54.0) 95(42.0) 19(19.4) 1.02( ) OR: interactions between cryopreservation method and different graded embryos. Data are presented as n (%), *p<0.05. Table 3. Logistic regression analysis of development. Mitosis resumption % Blastocyst formation % Grade A blastocyst % OR (95% CI) OR (95% CI) OR (95% CI) I vs. III 0.18( )* 0.22( )* 0.07( )* II vs. III 0.65( )* 0.60( )* 0.51( )* II vs. I 3.61( )* 2.73( )* 7.85( )* Cell 2 vs ( )* 2.21( )* 3.00( )* Cell 3 vs ( ) 1.59( ) 2.09( )* Cell 2 vs ( )* 1.39( ) 1.44( ) Grade 2 vs ( )* 0.61( )* 0.61( )* Grade 3 vs ( )* 0.25( )* 0.20( )* Grade 2 vs ( )* 2.42( )* 3.11( )* Slow freezing vs. vitrification 0.61( )* 0.60( )* 0.59( )* Interaction ( )* 1.23( )* 1.27( )* Interaction ( )* 1.25( )* 1.29( )* Interaction 1: interactions between the cryopreservation method and the intactness of the embryo; Interaction 2: interactions between the cryopreservation method and the cell number of the embryo. Data are presented as n (%); *p<0.05.

5 288 L. YU ET AL. cells (p>0.05). Logistic regression analysis showed that the mitosis resumption (154/231, 66.7% vs. 324/416, 77.9%), blastocyst formation (107/231, 46.3% vs. 273/ 416, 65.6%), and grade A blastocyst formation rates (54/231, 23.4% vs. 199/416, 47.8%) were significantly lower in embryos with 4-6 cells (54/231, 23.4%) than in those with 7-9 cells (37/95, 38.9%; all p<0.05). The grade A blastocyst formation rate was significantly lower in embryos with 4-6 cells than in those with >9 cells (odds ratio 2.04, 95% confidence interval , p<0.05). Among the different blastomere survival groups, logistic regression analysis showed that embryos in group II had a significantly higher mitosis resumption rate (117/171, 68.4%), rate of blastocyst formation (87/171, 50.9%), and grade A blastocyst formation rate (50/171, 29.2%) than those in group I (15/40, 37.5%; 11/40, 27.5%; and 2/40, 5.0% rate of mitosis resumption, blastocyst formation, and grade A blastocyst formation, respectively; all p<0.05), whereas these rates were significantly lower in group II than in group III (409/531, 77.0%; 337/ 531, 63.5%; and 238/531, 44.8% rate of mitosis resumption, blastocyst formation and grade A blastocyst formation, respectively; all p<0.05, Table 3). Thenumberofembryoswithablastomeresurvival rate <50% was so small that for this group, our study also included embryos that were cryopreserved on day 2 and day 3. Thirteen cleavage-stage embryos with a blastomere survival rate <50% were analyzed using NGS, and all were donated. Seven embryos were subjected to slow freezing, and 6 were subjected to vitrification. Seven embryos were evaluated after freezing/thawing. Of these, 2 developed arrest (2/7, 28.6%) and exhibited aneuploidy (2/2, 100%), and 5 (5/7, 71.4%) were normal (5/5,100%) expanded blastocysts. Six were evaluated after being vitrified/ warmed. Of these, 3 developed arrest (3/6, 50.0%), 2 of those exhibited aneuploidy (2/3, 66.7%), and 3 (3/ 6, 50.0%) were normal (3/3, 100%) expanded blastocysts (Tables 4 and 5). The results showed that the chromosomal status was normal in all 8 (8/8) embryos that developed into fully expanded blastocysts, whereas most (4/5) of the Table 4. NGS of cleavage stage embryos with <50% blastomere survival between those with developmental arrest and expanded blastocysts. Developed arrest (n=5) Expanded blastocyst (n=8) Variable Slow freezing Vitrification Slow freezing Vitrification No. embryos Normal % 0(0) 1(33.3) 5(100.0) 3(100.0) Data are presented as n (%). Table 5. Chromosomal status of embryos with <50% blastomere survival. Embryonic stage on Day Method a Before b After b biopsy day Interpretation 3 S cell Complex abnormal 3 S cell 47,XX,+15 3 S 7 3 5BC 46,XY 2 S 4 1 5BA 46,XY 2 S 5 1 6BA 46,XX 2 S 4 1 4CB 46,XY 2 S 4 1 6BA 46,XX 3 V 8 4 5BB 46,XX 2 V 4 1 4CC 46,XX 2 V 4 1 4BC 46,XY 3 V cell 45,XX,-22,del(1p p34.3),dup(5p p15.1) 3 V cell 46,XX 2 V cell 48,XY,+4,+X a Cryopreservation method: S-slow freezing and V-vitrification; b cell number: before (after) cryopreservation. embryos that underwent developmental arrest had an abnormal chromosomal status (Tables 4 and 5). Discussion In this study, we aimed to identify the parameters that are related to day 3 embryo intactness and developmental potential after warming to improve the selection criteria used to cryopreserve and transfer embryos. We also sought to compare the slow freezing and vitrification methods of cryopreservation and evaluate the viability of non-intact embryos. Our results showed that the embryo grade had an independent effect on embryo intactness. Grade 1 (i.e., <10% fragmentation and a stage-specific cell size) was an independent protective factor that was more closely associated with the maintenance of intactness in embryos than grade 3 (30-50% fragmentation and/or a non-stage-specific cell size); however, grade 3 embryos had lower blastocyst and grade A blastocyst formation rates than grade 1 and 2 embryos. Consistent with these findings, a recent study suggested that embryos with 10-25% fragmentation and/or symmetry <75% should be cryopreserved because they display acceptable implantation potential [Fernandez Gallardo et al. 2016]. It has therefore been recommended that embryos with high developmental potential should be identified using morphological criteria, including <30% fragmentation and a stage-specific cell size on day 3. Using these criteria avoids the need for costly treatments that have low success rates. We also compared embryonic intactness and developmental potential after both vitrification and slow freezing. Logistic regression analysis showed that

6 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 289 vitrification had a positive independent effect on intactness, the mitosis resumption rate, the blastocyst formation rate, and the grade A blastocyst rate of embryos (all p<0.05) compared to slow freezing. Our results show that the cryopreservation method and embryo grade did not have interactive effects on the mitosis resumption, blastocyst formation, and grade A blastocyst formation rates, but the cryopreservation method and intactness of the embryo had interactive effects on those parameters, as did the cryopreservation method and cell number (all p<0.05). We also found that in the 7-9-cell group (cell number 2 group) and the intact embryo group (group III) there were no statistically significant differences in the rates of mitosis resumption and grade A blastocyst formation between the slow freezing and vitrification groups (p>0.05). These findings indicated that slow developing cleavage-stage embryos had lower developmental potential. Vitrification preserved cell intactness better than slow freezing. Previous reports have shown that in mouse embryos, slow freezing is more likely to induce chromatin damage than is vitrification [Martino et al. 2013; Somoskoi et al. 2015]. A consensus is slowly emerging regarding the notion that better embryological and clinical outcomes are obtained following the use of vitrification procedures than following the use of slow freezing procedures [De Munck et al. 2013]. Our results show that the intactness rate did not differ significantly among embryos with 4-6 cells, 7-9 cells, and >9 cells, but the rates of mitosis resumption, blastocyst formation, and grade A blastocyst formation were significantly lower in embryos with 4-6 cells than in those with 7-9 cells (p<0.05). The grade A blastocyst formation rate was significantly lower in embryos with 4-6 cells than in those with >9 cells. In partial agreement with these results, other reports have shown that on day 3, the continued development rate [Van Landuyt et al. 2013] and the blastocyst formation rate were higher in embryos with 7-9 cells than in embryos with more than 9 or fewer than 7 cells [Alikani et al. 2000]. Another study showed that having too few cells decreased embryonic development [Stylianou et al. 2012]. In addition, studies have shown that having a high number of cells (>9 cells) on the third day results in the formation of more desirable, high-quality blastocysts (i.e., 4AA or better) [Luna et al. 2008] and that the developmental potential, implantation rate, and live birth rate of day 3 embryos increased with cell number [Kong et al. 2016]. Finally, another report showed that embryos with <5 cells had little developmental potential [Yang et al. 2015]. We analyzed the developmental potential of intact and non-intact embryos after warming. Among groups III, II, and I, we found that the mitosis resumption, blastocyst formation, and grade A blastocyst formation rates were all significantly decreased by this process (all p<0.05). Similar to the findings reported in our study, a previous study showed that blastomere loss following slow freezing impaired preimplantation development and resulted in reduced cell numbers at the periimplantation stages [Archer et al. 2003]. Early reports indicated that partially intact embryos had lower implantation rates because of the toxic influence of the damaged blastomeres on sibling cells and embryos [Rienzi et al. 2002]. Recent studies have shown that there was no difference in the live birth rate between embryos with partial loss of blastomeres (i.e., 1 blastomere in day 2 embryos or 1 or 2 blastomeres in day 3 embryos) following FET cycles. Additionally, the live births were achieved using only good-quality cryopreserved intact embryos (<20% fragmentation and 4-5 blastomeres at day 2 or 7-9 blastomeres at day 3) [DuPont et al. 2015]. According to the current standards for FET, embryos with >50% blastomere loss are not regarded as surviving embryos and should therefore be discarded. In our study, the developmental potential was lower in embryos with <50% surviving blastomeres than in intact embryos. A previous study showed that vitrified and slowly frozen embryos have similar implantation potentials if further cleavage occurs overnight, regardless of the number of cells lost [Van Landuyt et al. 2013]. The NGS results in the present study showed that the chromosomal status was normal in 8 embryos with <50% blastomere survival that were cultured into fully expanded blastocysts, whereas most developmentally arrested embryos (4/5) had chromosomal abnormalities. These results suggest that cleavagestage embryos with <50% blastomere survival could be considered for transfer after they are cultured to fully expanded blastocysts. To our knowledge, our study is the first to use NGS to investigate the chromosomal status of embryos with lower than 50% blastomere survival. Embryos that failed to reach the blastocyst stage were more likely to have poor developmental potential; if these embryos were transferred at the cleavage stage, they would be destined to fail to implant [Zhu et al. 2013]. We found that after freezing and subsequent thawing, 28.6% of embryos experienced developmental arrest, and all of those embryos exhibited aneuploidy; the other 71.4% became fully expanded blastocysts, and all of those embryos were normal. Following vitrification and warming, 50.0% of the embryos experienced developmental arrest, and 66.7% of those embryos exhibited aneuploidy; the other 50.0% became fully expanded blastocysts, and those embryos

7 290 L. YU ET AL. were all normal. Embryos in which fewer than 50% of the blastomeres survived following slow freezing had a higher developmental potential than those subjected to vitrification. This suggests that there were reasons other than aneuploidy for embryonic arrest following vitrification. However, additional studies with a larger sample size are required to verify these findings, and additional studies [Carbone and Chavez 2015 ] investigating the genetic, epigenetic, and chromosomal characteristics of non-intact embryos should be performed. It is crucial to select embryos with the highest implantation potential for transfer [Yin et al. 2016]. Cleavagestage embryos with poor survival that develop into good quality blastocysts should also be transferred. Blastulation depends not only on the survival and mitosis resumption of the embryo (the cryopreservation technique) but also on the capacity of the embryo to develop. Culturing the embryo provides enough time for the embryo to show its potential and has therefore become a valuable tool during embryo selection [Martins et al. 2016]. Increasing the standards used in choosing frozen cleavage-stage embryos will improve outcomes and avoid the need for costly treatments with low success rates [Veleva et al. 2013]. One study showed that slow development (<5 cells on day 3) and fragmentation (>15%) interfered with the formation of apparently normal blastocysts [Alikani et al. 2000]. Therefore, day 3 embryos with 4-6 cells [Balaban et al. 2011] or poor quality embryos should be cultured to the blastocyst stage and vitrified if they develop into good-quality blastocysts [Shaw-Jackson et al. 2013]. It has been observed in the case of vitrification that although embryos are often intact, they do not always resume mitosis within 24 hours. Certain embryos do, nevertheless, implant and lead to healthy births. Extended culture will help in the identification of such embryos. Directly extending fresh embryos to the blastocyst stage prior cryopreservation could be considered. This allows the selection of embryos with implantation potential; survival is less of an issue since the blastocyst has many cells, and embryo selection can be performed in a few hours based on re-expansion. In our study, in vitro observation scores were used to evaluate the relationships among blastomere loss, embryo grade, and development potential. Further studies are required to determine the rates of implantation and live births following embryo transfer. We have demonstrated that vitrification is the better choice for embryo cryopreservation because it results in superior outcomes compared with those obtained with slow freezing. Embryo grades could be used to more effectively select embryos with low fragmentation (<30%) and a stage-specific cell size containing more than 6 cells to be cryopreserved on day 3. Poor-quality embryos and those with low cell numbers on day 3 should be cultured to the blastocyst stage and vitrified if they develop into good-quality blastocysts. After warming, the developmental potential of non-intact embryos is lower than that of intact embryos. However, in our study, NGS showed that most of the embryos in which fewer than 50% of the blastomeres survived appeared normal if they were allowed to develop into fully expanded blastocysts, at which point transfer could be considered. However, additional studies are needed to evaluate the possibility of using embryos with less than 50% blastomere survival in a clinical setting. Materials and methods This study was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital of Capital Medical University. The patient samples used in this study were obtained with informed consent. Study design This was a retrospective, observational study that was conducted in the Department of Reproductive Medicine of the Beijing Obstetrics and Gynecology Hospital (Beijing, China). Analyses of day 3 cryopreserved embryos that were warmed and developed to days 5-6 were performed between August 2013 and January The day 3 embryos were cryopreserved by either slow freezing (n=216) or vitrification (n=522). After they were warmed, the embryos were separated into the following 3 groups based on the proportion of surviving blastomeres: group I (<50%), group II (50-99%), and group III (100%). The embryos were further cultured to determine their developmental potential; blastocyst formation rates and whether they became grade A blastocysts were evaluated. Thirteen embryos in which blastomere survival was <50% were analyzed using NGS. In this study, we evaluated standard morphological characteristics (e.g., the number of blastomeres, symmetry, and fragmentation) before the embryos were cryopreserved at day 3. After they were warmed, all embryos in this study were cultured to day 5 or 6. The embryos that were transferred developed into blastocysts; analyses were performed on those that developed into expanded blastocysts on day 5 or were transferred on day 6. We investigated the intactness, survival, mitosis resumption rate, blastocyst formation rate, and the grade A blastocyst formation rate of day 3 vitrified/warmed and slow frozen/thawed embryos.

8 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 291 Ovarian stimulation and in vitro culture Patients underwent controlled ovarian hyperstimulation using a long GnRH-a protocol or a GnRH-ant protocol. In the long GnRH-a protocol, a down-regulation protocol that combined a gonadotrophin-releasing hormone agonist with recombinant human follicle-stimulating hormone (FSH) was followed by individual dose adjustment, as required, according to the patient s ovarian response. In the GnRH-ant protocol, cetrorelix was injected at a dose of 0.25 mg daily when the leading follicle was 12 to 13 mm in diameter. Treatment was continued until the day human chorionic gonadotrophin (hcg) was injected. Ovulation was triggered by injecting 250 µg of recombinant hcg when the two leading follicles had a mean diameter of 17 mm or wider. Ultrasound-guided transvaginal oocyte retrieval was performed h later. Oocytes were inseminated using either conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) according to routine protocols [Gardner et al. 2012]. Fertilization was considered normal when two pronuclei were present between 16 and 18 h after insemination. In this study, only normally fertilized oocytes were further considered for embryo cryopreservation. All zygotes were cultured in cleavage medium (Sydney IVF, COOK, Brisbane, Australia) covered in oil in a 37 C incubator (C200, Labortect, Gottingen, Germany) with a 6% CO 2,5%O 2, and 89% N 2 atmosphere until day 3 after insemination. Classification of cryopreserved embryos Embryonic development was assessed prior to cryopreservation. Embryo quality was assessed using the manual scoring system of the Department of Reproductive Medicine at the Beijing Obstetrics and Gynecology Hospital of Capital Medical University. This system was based on a visual evaluation of the number and size of blastomeres [Balaban et al. 2011] and the degree of fragmentation. Grade 1 referred to embryos with <10% fragmentation and a stage-specific cell size. It is generally accepted that for embryos at the 4- and 8-cell stages, the blastomeres should be evenly sized. For all other cell stages, one would expect size differences among the cells [Balaban et al. 2011]. Grade 2 referred to embryos with 10-30% fragmentation with or without a stage-specific cell size for the majority of cells. Grade 3 referred to embryos with 30-50% fragmentation with or without a non-stage-specific cell size. Only embryos that were graded 1-3 were included in the study. Cryopreservation and warming protocols Slow freezing Day 3 embryos were sequentially dehydrated in Embryo Freezing Medium (Cook Scientific, Brisbane, Australia) and loaded into a 0.25 ml plastic straw (IMV, France). They were then frozen in a programmable freezer (Planer Products Ltd., UK). The temperature was decreased to -7 C at a rate of 2 C/min from room temperature. Seeding was performed manually at -7 C by touching a straw close to the cotton plug using forceps that were cooled in liquid nitrogen. Then, the embryos were further cooled to -32 C at a rate of -0.3 C/min, cooled to -140 C at a rate of -50 C/min, and then plunged into liquid nitrogen tanks for storage. To thaw the embryos, straws containing slow-cooled day 3 embryos were removed from liquid nitrogen and held in the air at room temperature for 30 s. They were then immersed in a 30 C water-bath for s until the ice melted. Cryoprotectants were removed using sequential dilutions in Embryo Thawing Medium (Cook Scientific). Vitrification procedure The vitrification and warming procedure was conducted according to the protocol recommended in the Kitazato Vitrification Kit. All materials required for vitrification were obtained from Kitazato (Tokyo, Japan). The embryos were equilibrated for 5-15 min at room temperature in equilibration solution until the volume of cells was completely recovered. They were then placed in vitrification solution for s. One or two embryos were picked up into the tip of a transfer pipette using a minimal volume of vitrification solution (0.1 μl or less) and then placed on a Cryotop strip. Within 30 s of suspending the cells in the vitrification medium, the strip with the embryos was plunged into liquid nitrogen. After they were warmed, the embryos were immediately immersed in a 37 C warming solution for 1 min. They were then recovered and rehydrated using a 3-step dilution protocol at room temperature. After the cells were warmed and diluted, they were transferred to blastocyst media for culture. Evaluation of the intactness of embryos after warming After the embryos were warmed, the intactness of the embryos was determined by quantifying the proportion of morphologically intact cells in the embryo. Embryos were considered intact when all of the blastomeres had

9 292 L. YU ET AL. well-defined membranes and cytoplasm with no sign of lysis or degeneration. Morphological survival after warming was expressed as the percentage of warmed embryos that remained fully intact or in which at least 50% of the cells were intact. In our study, embryos were incubated for an additional 3 days to assess blastulation potential. The embryos were subsequently cultured in blastocyst medium (Sydney IVF, COOK) covered with oil in a 37 C incubator (Labortect, C200) with a 6% CO 2,5% O 2, and 89% N 2 atmosphere to the blastocyst stage. Blastocysts were given a numeric score from 1 6 based on the degree of expansion and hatching status. Richardson et al. [2015] noted that when using the Gardner grading system [Gardner et al. 2012], blastocysts that had an expansion grade of 4, 5, or 6 (i.e., were fully expanded), an inner cell mass, and a grade A or B trophectoderm had the highest implantation rate. This grading scheme can be used to effectively predict clinical outcomes including implantation, clinical pregnancy, and live birth. In this study, we called such blastocysts grade A blastocysts. Next-generation sequencing (NGS) Donated cleavage-stage embryos with <50% blastomere survival were cultured to day 5-7 and used to perform NGS. The zona pellucida was separated from whole cells that were obtained from embryos or blastocysts. Template DNA was generated using standard degenerate oligonucleotide primer PCR (DOP-PCR), and the resulting products were sequenced using Illumina sequencing at BGI-Shenzhen. The series procedure [Zhang et al. 2013] and quality controls were all performed by BGI-Shenzhen. Theoretically, all CNVs larger than 1 megabase were detected. Statistical analysis The Statistical Package for Social Sciences (SPSS: version 21.0, IBM, Chicago, USA) was used for all statistical analyses. Descriptive data are presented as the mean ± SD (standard deviation), and variance in the measured data was analyzed using ANOVA. Categorical variables are expressed as percentages. The rate was calculated using the chi-square test, and interactions between fixed factors were calculated using logistic regression analysis. P values <0.05 were considered statistically significant. Declaration of interests This work was supported in part by grants from the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (code: ZYLX201510) and the Beijing Municipal Science and Technology Commission (code: Z ). The authors report no conflicts of interest. Notes on contributors Conceived and designed the experiments: SW; Performed the experiments: LY, RS, LZ; Analyzed the data: LY, CJ; Contributed reagents/materials/analytical tools: YLan, YLi, YLiang; Wrote the manuscript: LY, CJ. All authors approved all revisions and the final paper. ORCID Lan Yu References Alikani, M., Calderon, G., Tomkin, G., Garrisi, J., Kokot, M. and Cohen, J. (2000) Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro. Hum Reprod 15: Archer, J., Gook, D.A. and Edgar, D.H. (2003) Blastocyst formation and cell numbers in human frozen-thawed embryos following extended culture. Hum Reprod 18: Balaban, B., Brison, D., Calderón, G., Catt, J., Conaghan, J., Cowan, L., et al. (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology) (2011) The Istanbul consensus workshop on embryo assessment: Proceedings of an expert meeting. Hum Reprod 26: Carbone, L. and Chavez, S.L. (2015) Mammalian pre-implantation chromosomal instability: species comparison, evolutionary considerations, and pathological correlations. Syst Biol Reprod Med 61: De Munck, N., Verheyen, G., Van Landuyt, L., Stoop, D. and Van de Velde, H. (2013) Survival and post-warming in vitro competence of human oocytes after high security closed system vitrification. J Assist Reprod Genet 30: Dupont, C., Hafhouf, E., Sermondade, N., Sellam, O., Herbemont, C., Boujenah, J., et al. (2015) Delivery rates after elective single cryopreserved embryo transfer related to embryo survival. Eur J Obstet Gynecol Reprod Biol 188:6 11. El-Toukhy, T., Khalaf, Y., Al-Darazi, K., Andritsos, V., Andritsos, V., Taylor, A., et al. (2003) Effect of blastomere loss on the outcome of frozen embryo replacement cycles. Fertil Steril 79: Fernandez Gallardo, E., Spiessens, C., D Hooghe, T. and Debrock, S. (2016) Effect of embryo morphology and morphometrics on implantation of vitrified day 3 embryos after warming: a retrospective cohort study. Reprod Biol Endocrinol 14:40. Gardner, D.K., Weissman, A., Howles, C.M. and Shoham, Z. (2012) Textbook of Assisted Reproductive Techniques, Laboratory and Clinical Perspectives, Third Edition, Informa Healthcare, UK.

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