Abstract
Purpose
This study aimed to compare slow freezing (SF) and vitrification (VT) techniques for day 3 embryo cryopreservation in infertile couples.
Methods
This retrospective cohort study enrolled 5613 infertile patients, with 7862 frozen-thawed day 3 embryos and 3845 vitrified-warmed day 3 embryos, from 2010 to 2014, at a single center. The rates of embryo survival, pregnancy, implantation, miscarriage, live birth, and live birth weight were compared between the two groups.
Results
A total of 5613 cycles with 5520 transfers were analyzed. Using SF, the rates of overall embryo survival and fully intact blastomeres were lower than those in VT (91.5 vs. 97.4 % and 68.7 vs. 92.3 %, respectively). The rate of good quality embryos after thawing/warming was lower in SF than in VT. In single frozen embryo transfer cycles (FETs), the pregnancy and implantation rates were similar between the two groups (35.0 vs. 40.8 % and 34.6 vs. 35.9 %, respectively). In double FETs, the pregnancy rate per cycle was also similar between the groups (58.8 vs. 58.4 %). The implantation rate per embryo transfer was significantly higher with SF than with VT (38.8 vs. 34.6 %). With adjustment for maternal age and the number of good quality embryos, differences in implantation rate remained significant (adjusted P value, SF vs. VT P < 0.05). No independent effect was found for the method of cryopreservation on the pregnancy rate. No significant differences in the rates of miscarriage, live birth, and live birth weight were observed between the two techniques.
Conclusions
Despite the significantly low embryo survival rate, fully intact blastomere rate, and good quality embryo rate in SF, the pregnancy and implantation rates were not adversely affected in single and double FETs. SF yielded an equivalent miscarriage rate, live birth rate, and live birth weight compared with VT. The SF protocol to cryopreserve day 3 embryos still should be considered.
Keywords: Slow freezing, Vitrification, Cryopreservation, Implantation rate, Pregnancy outcome
Introduction
Day 3 embryo cryopreservation is a routine procedure in assisted reproductive technology. Cryopreservation allows the storage of supernumerary embryos for future use and thus enhances the cumulative pregnancy rate in in vitro fertilization (IVF). In addition, cryopreservation avoids the incidence of late ovarian hyperstimulation syndrome in high-risk patients, while it maintains the probability of pregnancy in future embryo transfer cycles [1, 2]. Interestingly, an increasing amount of studies have shown that the success rates after frozen-thawed embryo transfer (FET) are now similar to or better than the success rates of fresh embryo transfer [3]. Therefore, there is an increasing appeal for FET policy. However, while we have attempted to perform the FET strategy, an alternative of the cryopreservation protocol has become a major issue.
The main problem during embryo cryopreservation is the formation of intracellular ice, which can lead to cell damage and development arrest [4, 5]. To overcome these problems, cryopreservation methods and cryoprotective solutions are constantly improving and developing. Since the first publication of frozen embryo transfer pregnancy [6], slow freezing has become the predominant cryopreservation protocol in most IVF centers. During slow freezing, at a controlled rate, embryos are exposed to relatively low concentration of cryoprotectants and dehydration without excessive shrinkage [5]. Although slow freezing is likely still the most widely used cryopreservation method for human embryos, its lower survival rate has forced IVF laboratories to switch from slow freezing to vitrification in the recent years [7]. Vitrification obtains a higher embryo survival rate and fully intact blastomere rate compared with slow freezing [8]. However, vitrification uses high concentration of cryoprotectants [9] and there are still concerns regarding its safety. The effect of vitrification on embryo development potential and birth outcomes after vitrification still needs to be determined.
Vitrification was introduced in our assisted reproductive technology (ART) center in 2012 for day 3 embryos. The decision to switch the cryopreservation protocol was based on the reported high embryo survival rate, low risk of losing the chance of embryo transfer [10, 11], and higher blastulation rates [12] by vitrification. However, some previous studies have found no difference in the pregnancy rate between vitrification and slow freezing [13, 14]. Therefore, there is an obvious need to analyze the results of two cryopreservation protocols before totally abandoning slow freezing.
In the present study, we compared the rates of embryo survival, pregnancy, implantation, miscarriage, and live birth as well as live birth weight of day 3 embryo transfer after cryopreservation using either slow freezing or vitrification.
Materials and methods
Study design
This study was approved by the institutional review board of our hospital. In this retrospective cohort study, 7862 frozen-thawed (slow freezing) day 3 embryos of all IVF/intracytoplasmic sperm injection (ICSI) thawing cycles that were carried out in the assisted reproductive unit of our hospital between January 2010 and December 2013 were included for analysis. The same analysis was performed for 3845 vitrified-warmed day 3 embryos of warming cycles between January 2012 and June 2014. Analysis was limited to only one or two day 3 frozen embryo-transferred cycles. We excluded those cycles without accurate medical record numbers and missing data.
Morphological survival was analyzed according to the cell stage before cryopreservation. The pregnancy and implantation rates were analyzed after one or two frozen embryos were transferred in patients for 3657 slow freezing cycles and 1956 vitrification cycles.
Ovarian stimulation and IVF/ICSI treatment
Patients were stimulated as previously described [15]. Briefly, two ovarian stimulation protocols for IVF treatment (long and short protocols) and two types of recombinant follicle-stimulating hormone (rFSH: Gonal-F; Serono Laboratories, Aubonne, Switzerland or Puregon; NV Organon, Oss, The Netherlands) were used. Follicular development was monitored by ultrasound scanning, and final oocyte maturation was induced by injection of human chorionic gonadotropin (hCG) as soon as three follicles of 18 mm in diameter were observed. Oocyte retrieval was performed 36 h after hCG administration.
Embryo selection for cryopreservation and evaluation after thawing/warming
Day 3 embryos with at least four blastomeres and ≤35 % fragmentation were selected for cryopreservation. Embryo morphology was scored as described by Veeck as follows [16]: grade 1, embryos had blastomeres of equal size and no cytoplasmic fragmentation; grade 2, embryos had blastomeres of equal or unequal size and cytoplasmic fragmentation to ≤20 % of the embryo surface; grade 3, embryos had blastomeres of equal or unequal size and 21–49 % overall cytoplasmic fragmentation; and grade 4, embryos had blastomeres of equal or unequal size and cytoplasmic fragmentation to ≥50 % of the embryo surface. An embryo with 6–12 blastomeres and grades 1 and 2 was defined as good quality. Embryos were evaluated for morphological thaw survival immediately after thawing/warming. Embryos with at least 50 % of their cells intact were considered as survived and able to be transferred.
Cryopreservation protocols
Slow freezing of day 3 embryos was performed as described by Edgar et al. [17], which involved a slow controlled freezing and thawing protocol with propanediol/sucrose (Freezing Kit; Irvine Scientific, Santa Ana, CA, USA) as the cryoprotectant, as described previously [18]. For vitrification of embryos, an open system was selected based on previous studies for its safety [19, 20]. The open vitrification procedure was performed using the Cryotop strip in combination with ethylene glycol-dimethylsulfoxide-sucrose (Kitazato Supply Co., Fujinomiya, Japan) as the cryoprotectant.
Preparation of the FET cycle
In our center, three main types of clinical protocols were used for endometrial preparation: the natural cycle, hormone replacement cycle, or human menopausal gonadotropin (HMG)-stimulated cycles [18]. For patients with regular menstrual cycles, the natural cycle was proposed and embryo transfer was performed 3 days after ovulation, or 4.5 days after the luteinizing hormone peak. In patients with an irregular ovulation or anovulatory cycle, a hormone replacement treatment or HMG-stimulation protocol was used.
Outcome measures
Maternal age (years) was registered at the time of oocyte retrieval. The thaw survival rate was calculated as embryos that survived per the total number of warmed embryos. Biochemical pregnancy was assessed by detecting serum β-hCG levels at 12 days after transfer, and 50 IU/L or greater β-hCG levels were considered as positive. The pregnancy rate was calculated by the number of β-hCG-positive pregnancies per patient. Five weeks after transfer, the number of gestational sacs and presence of fetal heart motion were evaluated by ultrasound. The thawing transfer implantation rate was calculated using the number of gestational sacs per number of embryo transferred.
Statistical analysis
Data analysis was performed using the Statistical Package for the Social Sciences Version 16.0 (IBM Corp., New York, NY, USA). A P value <0.05 indicated significance. Comparison of continuous data was performed using the independent t test. Categorical variables were compared by Pearson’s chi-square test. For analyzing the rate of pregnancy and implantation, logistic regression analysis was used to adjust for potential confounding factors (maternal age and the number of good quality embryos).
Results
Analysis of patients’ clinical parameters
During the 4-year study period, our ART center performed a total of 5613 thawing cycles, of which 3657 (65.2 %) used slow freezing/thawing and 1956 (34.8 %) used vitrification/warming (Table 1). Maternal age was an average of 0.4 years older in the vitrified group than in the slow frozen group (P < 0.01). The number of good quality embryos after thawing/warming was lower with slow freezing than with vitrification. The overall embryo survival rate after vitrification was 97.4 % (3744/3845) and the proportion of embryos with fully intact blastomeres was 92.3 %, which were significantly higher compared with slow frozen/thawed embryo transfer (91.5 and 68.7 %, respectively, P < 0.001).
Table 1.
Comparison of patients’ clinical parameters using slow frozen and vitrified day 3 embryo cryopreservation
| Slow freeze | Vitrified | P value | |
|---|---|---|---|
| No. of cycles | 3657 | 1956 | |
| Maternal age (years) | 31.4 ± 4.3 | 31.8 ± 4.5 | <0.01a |
| Cause of infertility | |||
| Male factor | 622 (17.0 %) | 342 (17.5 %) | |
| Tubar factor | 2523 (69.0 %) | 1230 (62.9 %) | |
| Endometriosis | 139 (3.8 %) | 111 (5.7 %) | |
| Ovarian factor | 205 (5.6 %) | 131 (6.7 %) | |
| Unexplained | 168 (4.6 %) | 142 (7.3 %) | |
| Natural cycles | 153 (4.2 %) | 27 (1.4 %) | |
| HRT cycles | 3241 (88.6 %) | 1803 (92.2 %) | |
| HMG-stimulated cycles | 263 (7.2 %) | 126 (6.4 %) | |
| Insemination methods | |||
| IVF | 2518 (68.9 %) | 1289 (65.9 %) | |
| ICSI | 934 (25.5 %) | 624 (31.9 %) | |
| Half-ICSI | 205 (5.6 %) | 43 (2.2 %) | |
| No. of thawed/warmed embryos | 7862 | 3845 | |
| No. of thawed/warmed embryos per cycle | 2.15 ± 0.716 | 1.97 ± 0.632 | <0.001a |
| No. of good quality embryos before cryopreservation | 5860 (74.5 %) | 2940 (76.5 %) | <0.05b |
| No. of good quality embryos after thawing/warming | 4889 (62.2 %) | 2801 (72.8 %) | <0.001b |
| Embryo survival rates | 7190 (91.5 %) | 3744 (97.4 %) | <0.001b |
| Intact blastomere rates | 5401 (68.7 %) | 3549 (92.3 %) | <0.001b |
| Endometrial thickness (mm) | 9.3 ± 1.7 | 9.5 ± 1.9 | >0.05a |
aIndependent t test
bPearson’s chi-square test
Analysis of pregnancy and implantation rates in single FETs
In total, 526 cycles of frozen-thawed single FET and 485 cycles of single FET vitrified day 3 embryos were analyzed (Table 2). The analyses included only single transfer of surviving embryos (≥50 % of intact cells). Maternal age was comparable between the two groups (P > 0.05). The number of good quality embryos was significantly higher in the vitrified group than in the slow frozen group [(400; 82.5 %) vs. (366; 69.6 %), P < 0.001]. Furthermore, the pregnancy and implantation rates were similar between the groups regarding overall and good quality embryo transfer (P > 0.05).
Table 2.
Comparison between pregnancy and implantation rates of single FETs using slow frozen or vitrified day 3 embryos
| Variable | Slow freeze | Vitrified | P value |
|---|---|---|---|
| No. of cycles | 526 | 485 | |
| Maternal age (years) | 32.3 ± 4.8 | 32.3 ± 4.7 | >0.05a |
| No. of transferred embryos | 526 | 485 | |
| No. of good quality embryos transferred | 366 (69.6 %) | 400 (82.5 %) | <0.001b |
| Pregnancy rates | |||
| Overall embryos | 184 (35.0 %) | 198 (40.8 %) | >0.05b |
| Good quality embryos | 167 (45.6 %) | 186 (46.6 %) | >0.05b |
| Implantation rates | |||
| Overall embryos | 182 (34.6 %) | 174 (35.9 %) | 0.671 b |
| Good quality embryos | 165 (45.1 %) | 162 (40.5 %) | 0.200 b |
aIndependent t test
bPearson’s chi-square test
Analysis of pregnancy and implantation rates in double FETs
The pregnancy and implantation rates after double FETs are shown in Table 3. A total of 2645 pregnancies were obtained, with 1795 (67.9 %) from slow freezing and 850 (32.1 %) from vitrification. The pregnancy rate of overall embryos was similar between the groups (58.8 vs. 58.4 %, P > 0.05), but the implantation rate per embryo transfer was significantly higher in the slow frozen group than in the vitrified group (38.8 vs. 34.6 %, P < 0.001). With regard to the transfer of good quality embryos, the pregnancy and implantation rates after double good quality embryo transfer were significantly higher in the slow frozen group compared with the vitrified group (67.7 vs. 63.6 %, P < 0.05; 46.5 vs. 38.5 %, P < 0.001, respectively). When adjustment was made for maternal age and the number of good quality embryos, the difference in implantation rate remained significant (adjusted P value, slow frozen vs. vitrified P < 0.05). No independent effect was found for the method of cryopreservation on the pregnancy rate (P > 0.05).
Table 3.
Comparison of pregnancy and implantation rates per transfer between slow frozen and vitrified day 3 embryos using double FETs
| Variables | Slow freeze | Vitrified | P value |
|---|---|---|---|
| No. of cycles | 3054 | 1455 | |
| Maternal age (years) | 31.1 ± 4.1 | 31.5 ± 4.3 | <0.05a |
| No. of transferred embryos | 6108 | 2910 | |
| No. of good quality embryos transferred | 4365 (71.5 %) | 2275 (78.2 %) | <0.001b |
| Pregnancy rates per cycle | |||
| Overall embryos | 1795 (58.8 %) | 850 (58.4 %) | >0.05b |
| Good quality embryos | 1185/1751 (67.7 %) | 609/957 (63.6 %) | <0.05b |
| Implantation rates | |||
| Overall embryos per embryo transfer | 2371 (38.8 %) | 1008 (34.6 %) | <0.001b |
| Good quality embryos | 1629/3052 (46.5 %) | 737/1914 (38.5 %) | <0.001b |
aIndependent t test
bPearson’s chi-squared test
Ongoing pregnancy and neonatal outcomes
The number of ongoing pregnancies at the time of analysis was 1343 in the slow frozen group and 189 in the vitrified group (Table 4). The live birth rate was similar between the two groups (P > 0.05). No significant difference was found in spontaneous miscarriages before 12 weeks or after 12 weeks (P > 0.05). Moreover, the live birth weight of neonates from singleton and twin pregnancies was slightly lower with slow freezing than with vitrification, but this difference was not significant (P > 0.05).
Table 4.
Comparison of ongoing pregnancies and neonatal outcomes after transfer of slow frozen or vitrified day 3 embryos
| Variable | Slow freeze | Vitrified | P value |
|---|---|---|---|
| No. of pregnancies | 1343 | 189 | |
| No. of ongoing pregnancies after 12 weeks | 1132 | 152 | |
| Spontaneous miscarriages (% per pregnancy, before 12 weeks) | 12.10 % | 15.30 % | >0.05a |
| Spontaneous miscarriages (% per pregnancy, after 12 weeks) | 2.10 % | 0.70 % | >0.05a |
| Live birth rate per embryo transfer | 1119/3783 (29.6 %) | 115/399 (28.8 %) | >0.05a |
| Live birth weight of singleton pregnancies (g) | 3286.5 ± 570.4 | 3327.5 ± 491.9 | >0.05b |
| Live birth weight of twin pregnancies (g) | 2499.6 ± 477.0 | 2619.6 ± 499.9 | >0.05b |
aPearson’s chi-square test
bIndependent t test
Discussion
It is imperative to consider that the cryopreservation method employed in ART centers has some impact on embryo quality. There are a handful of studies suggesting that vitrification obtains higher embryo survival rate and blastulation rate [12, 21]. In 2012, our ART center gradually shifted from slow freezing to vitrification. Therefore, data on both cryopreservation protocols were available for comparison. The decision to shift protocols was based on the quantity of literature reporting higher embryo survival rate and fully intact blastomere rate in vitrified embryos [5, 10]. The current study compared the pregnancy rate and neonatal outcomes between slow frozen and vitrified day 3 embryo techniques and evaluated their effect on immediate and future development of embryos. In accordance with most previous reports, the current study indicated that the embryo survival rate and fully intact blastomere rate were significantly lower in slow freezing than in vitrification. The pregnancy rate was similar between the two groups in single and double FETs. However, notably, the implantation rate per embryo transfer in double FETs was significantly higher with the frozen-thawed method than with vitrification in the present study. Furthermore, the rates of pregnancy and implantation with transfer of good quality embryos were significantly higher with slow freezing than with vitrification. These data indicate that, although the slow freezing technique resulted in more damaged embryos and lost more embryos, it did not affect the embryo implantation potential and pregnancy outcomes. Clinical outcomes may actually be better with slow freezing because more good quality embryos were obtained with vitrification.
The design of the current study and the different periods for slow freezing and vitrification may have resulted in some biases, which might have affected our results. However, because this study was retrospective, we were able to enroll a large number of cases, providing valuable information on the rate of embryo survival, pregnancy, and implantation. Moreover, neither the embryologist evaluating the embryos and the embryo selection criteria for cryopreservation nor the culture medium and environment have changed over time at our center. Thorough evaluation and taking into consideration many parameters, such as safety and workload, when choosing the best embryo cryopreservation method, are important. Vitrification requires a minimal setup time and is a quick procedure, but it requires more hands-on time per cryopreserved embryo [22]. Slow frozen embryos, which predominantly survive intact after thawing, appear to retain their pre-freeze developmental potential, and optimal dehydration prior to slow freezing can result in high survival rates (90 %) [23–25]. One lysed blastomere of a slow frozen embryo has no significant effect on implantation potential because similar implantation rates are observed when three out of four and four out of four blastomeres survive thawing [26].
A retrospective study by Van Landuyt et al. [8] showed that vitrified embryos had a higher survival rate, better overnight development, and allowed a higher transfer rate but had similar implantation potential, compared with slow frozen embryos. The authors concluded that the number of single-embryo transfers with vitrified embryos was limited, and the results did not prove that vitrified embryos were more viable than slow frozen embryos. In the current study, we found that slow freezing resulted in a 5 % lower survival rate and 23 % lower intact blastomere rate than vitrification but yielded similar pregnancy and implantation rates in single FETs. More interestingly, the implantation rate per embryo transfer was significantly higher with slow freezing than with vitrification in double FETs, despite a decreased rate of survival, blastomeres, and good quality embryos. These results suggested that slow freezing may have a minimal impact on development and implantation potential of embryos.
Vitrification uses high concentrations of cryoprotectants [9] and may have higher potential cell toxicity, which may be one of the major factors affecting development of embryos. Several studies have shown similar pregnancy and implantation rates between slow freezing and vitrification [13, 27]. Vitrified embryo transfer is associated with lower hCG concentrations on day 12 post-embryo transfer compared with slow freezing embryo transfer [18]. In the current study, a higher implantation rate in slow freezing than in vitrification was found. To the best of our knowledge, our study is the first report to suggest a better embryo implantation potential in slow freezing cycles. We had a large number of embryos used for slow freezing and vitrification, as well as a relatively higher thaw survival rate (91.5 %) for slow freezing in our ART center compared with results reported by other ART centers. Importantly, the current study used a traditional method for slow freezing that has been shown to be significantly inferior to a modified method produced by Edgar et al. [24]. This modified method showed a 47 % increase (from 54.6 to 80.4 %) in the proportion of all embryos that survive fully intact [24]. The intact blastomere rate was 68.7 % in the current study. Considering the close association between loss of blastomeres and loss of implantation potential [17], a significant improvement will be obtained if we adopt this modified method.
When appraising the efficacy of two cryopreservation protocols, embryo survival is an important issue but far enough to determine which one is actually better [23]. The rates of pregnancy, implantation, miscarriage, and live birth were used as the most important end-points. In the current study, no significant differences were found in the rates of miscarriage and live birth in slow freezing compared with vitrification. These findings suggested that no method was superior regarding embryo development after implantation. Moreover, there was no difference in live birth weight between the two methods, although a slightly lower birth weight was observed in slow freezing. The birth outcomes from slow frozen embryos where propanediol was used as the permeating cryoprotectant are reassuring [23]. However, the birth outcomes of vitrification need to be further investigated for the use of the more potentially toxic, permeating cryoprotectants (dimethyl sulfoxide and ethylene glycol) [23].
In conclusion, day 3 embryos can be cryopreserved with comparable clinical pregnancy and live birth rates using traditional slow freezing and vitrification. The comparative better implantation potential associated with slow frozen embryos in this retrospective study provided further evidence for the continued use of slow freezing of day 3 embryos. Optimal slow freezing methods would also need to be established because they may alleviate concerns on safety issues relating to exposure to high concentrations of cryoprotectants used in vitrification.
Acknowledgments
The authors sincerely thank the other investigators and physicians who made contributions to this study. This work was supported by grants from the National Natural Science Foundation of China (No. 81270657), the Natural Science Program of Zhejiang (Y14H040012), and the Science and Technology Department Program of Zhejiang (2013C33145).
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Capsule
The comparable clinical pregnancy and live birth rate between slow frozen and vitrified embryos in this retrospective study provides further evidence for the continued use of slow freezing of day 3 embryos.
References
- 1.Michelmann HW, Nayudu P. Cryopreservation of human embryos. Cell Tissue Bank. 2006;7:135–41. doi: 10.1007/s10561-005-0877-1. [DOI] [PubMed] [Google Scholar]
- 2.Wu K, Zhao H, Liu H, Li M, Ma S, Li C, et al. Day 3 ET, single blastocyst transfer (SBT) or frozen-thawed embryo transfer (FET): which is preferable for high responder patients in IVF/ICSI cycles? J Assist Reprod Genet. 2014;31:275–8. doi: 10.1007/s10815-013-0156-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Roy TK, Bradley CK, Bowman MC, McArthur SJ. Single-embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers. Fertil Steril. 2014;101:1294–301. doi: 10.1016/j.fertnstert.2014.01.046. [DOI] [PubMed] [Google Scholar]
- 4.Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update. 2003;9:583–605. doi: 10.1093/humupd/dmg041. [DOI] [PubMed] [Google Scholar]
- 5.Loutradi KE, Kolibianakis EM, Venetis CA, Papanikolaou EG, Pados G, Bontis I, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril. 2008;90:186–93. doi: 10.1016/j.fertnstert.2007.06.010. [DOI] [PubMed] [Google Scholar]
- 6.Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305:707–9. doi: 10.1038/305707a0. [DOI] [PubMed] [Google Scholar]
- 7.Wong KM, Mastenbroek S, Repping S. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertil Steril. 2014;102:19–26. doi: 10.1016/j.fertnstert.2014.05.027. [DOI] [PubMed] [Google Scholar]
- 8.Van Landuyt L, Van de Velde H, De Vos A, Haentjens P, Blockeel C, Tournaye H, et al. Influence of cell loss after vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos. Hum Reprod. 2013;28:2943–9. doi: 10.1093/humrep/det356. [DOI] [PubMed] [Google Scholar]
- 9.Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker MJ. Potential importance of vitrification in reproductive medicine. Biol Reprod. 2002;67:1671–80. doi: 10.1095/biolreprod.102.006833. [DOI] [PubMed] [Google Scholar]
- 10.Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol. 2009;21:270–4. doi: 10.1097/GCO.0b013e3283297dd6. [DOI] [PubMed] [Google Scholar]
- 11.Rezazadeh Valojerdi M, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet. 2009;26(6):347–54. doi: 10.1007/s10815-009-9318-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, et al. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod. 2008;23:1976–82. doi: 10.1093/humrep/den222. [DOI] [PubMed] [Google Scholar]
- 13.Wilding MG, Capobianco C, Montanaro N, Kabili G, Di Matteo L, Fusco E, et al. Human cleavage-stage embryo vitrification is comparable to slow rate cryopreservation in cycles of assisted reproduction. J Assist Reprod Genet. 2010;27:549–54. doi: 10.1007/s10815-010-9452-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.AbdelHafez FF, Desai N, Abou-Setta AM, Falcone T, Goldfarb J. Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis. Reprod Biomed Online. 2010;20:209–22. doi: 10.1016/j.rbmo.2009.11.013. [DOI] [PubMed] [Google Scholar]
- 15.Zhu H, Liu L, Yang L, Xue Y, Tong X, Jiang L, et al. The effect of progesterone level prior to oocyte retrieval on the numbers of oocytes retrieved and embryo quality in IVF treatment cycles: an analysis of 2,978 cycles. J Assist Reprod Genet. 2014;31:1183–7. doi: 10.1007/s10815-014-0291-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Veeck LL. An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. Preembryo grading and degree of cytoplasmic fragmentation. New York: Parthenon Publishing; 1999. pp. 46–51. [Google Scholar]
- 17.Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod. 2000;15:175–9. doi: 10.1093/humrep/15.1.175. [DOI] [PubMed] [Google Scholar]
- 18.Xue Y, Tong X, Jiang L, Zhu H, Yang L, Zhang S. Effect of vitrification versus slow freezing of human day 3 embryos on β-hCG levels. J Assist Reprod Genet. 2014;31:1037–43. doi: 10.1007/s10815-014-0259-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Kyuwa S, Nishikawa T, Kaneko T, Nakashima T, Kawano K, Nakamura N, et al. Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks. Exp Anim. 2003;52:67–70. doi: 10.1538/expanim.52.67. [DOI] [PubMed] [Google Scholar]
- 20.Morris GJ. The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels. Cryobiology. 2005;50:231–8. doi: 10.1016/j.cryobiol.2005.01.005. [DOI] [PubMed] [Google Scholar]
- 21.Fasano G, Fontenelle N, Vannin AS, Biramane J, Devreker F, Englert Y, et al. A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos. J Assist Reprod Genet. 2014;31(2):241–7. doi: 10.1007/s10815-013-0145-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Capalbo A, Rienzi L, Buccheri M, Maggiulli R, Sapienza F, Romano S, et al. The worldwide frozen embryo reservoir: methodologies to achieve optimal results. Ann N Y Acad Sci. 2011;1221:32–9. doi: 10.1111/j.1749-6632.2010.05931.x. [DOI] [PubMed] [Google Scholar]
- 23.Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Human Reprod Update. 2012;18:536–54. doi: 10.1093/humupd/dms016. [DOI] [PubMed] [Google Scholar]
- 24.Edgar DH, Karani J, Gook DA. Increasing dehydration of human cleavage-stage embryos prior to slow cooling significantly increases cryosurvival. Reprod Biomed Online. 2009;19:521–5. doi: 10.1016/j.rbmo.2009.06.002. [DOI] [PubMed] [Google Scholar]
- 25.Jericho H, Wilton L, Gook DA, Edgar DH. A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos. Hum Reprod. 2003;18(3):568–71. doi: 10.1093/humrep/deg106. [DOI] [PubMed] [Google Scholar]
- 26.Edgar DH, Archer J, McBain J, Bourne H. Embryonic factors affecting outcome from single cryopreserved embryo transfer. Reprod Biomed Online. 2007;14:718–23. doi: 10.1016/S1472-6483(10)60674-8. [DOI] [PubMed] [Google Scholar]
- 27.Li Y, Chen ZJ, Yang HJ, Zhong WX, Ma SY, Li M. Comparison of vitrification and slow-freezing of human day 3 cleavage stage embryos: post-vitrification development and pregnancy outcomes. Zhonghua Fu Chan Ke Za Zhi. 2007;42:753–5. [PubMed] [Google Scholar]