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Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles
ORIGINAL ARTICLES: ASSISTED REPRODUCTION
Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles de los Santos, Ph.D., Damia Castello , Ph.D., Pilar Ga miz, Ph.D., Ana Cobo, Ph.D., María Jose Remohí, M.D. Pilar Campos, M.L.T., and Jose IVI-Valencia, Institut Universitari IVI, Valencia, Spain
Objective: To assess the outcomes achieved after Cryotop vitrification of both early cleavage and blastocyst-stage embryos and to determine whether the embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer had any effect on the final outcome. Design: Observational study. Setting: Private university-affiliated IVF center. Patient(s): Women undergoing 3,150 warming cycles whose embryos were vitrified due to various reasons. Intervention(s): Vitrification by the Cryotop open device. Main Outcome Measure(s): Delivery rate (DR) per warming cycle. Result(s): Survival rate was 95% (5,722 out of 6,019 embryos). The percentage of intact embryos at warming showing 100% blastomere survival was 93% (95% CI 90.1%–95.3%) for day 2 and 95% (95% CI 94.3%–95.7%) for day 3; 3,057 embryo transfers were performed (3% cancellation rate). The DR/warming cycle was 32.5% (95% CI 30.9%–34.2%). Slight differences in survival rate were found [94.9% (95% CI 93.0%–96.8%) for day 2, 94.2% (95% CI 93.4%–94.9%) for day 3, 95.7% (95% CI 94.5%–96.9%) for day 5, and 97.6% (95% CI 96.9%–98.6%) for day 6]. Overall implantation, clinical pregnancy, ongoing pregnancy, and live birth rates per warming cycle were 35.5% (95% CI 33.5%–38.5%), 41.7% (95% CI 39.9%–43.4%), 32.6% (95% CI 31.0%–34.2%), and 38.1% (95% CI 36.4%–39.8%) respectively. The linear regression model considering embryo developmental stage, ovum donation or patient's own oocytes, and hormonal replacement therapy or natural cycle for endometrial preparation (odds ratio 1.179; 95% CI 0.912–1.277) showed no impact on the DR. Conclusion(s): Highly successful cryopreservation of all embryo developmental stages is possible with the use of the Cryotop system. There are no variables clearly exerting a negative effect Use your smartphone on the survival and delivery rates. (Fertil SterilÒ 2012;98:1138–46. Ó2012 by American Society to scan this QR code for Reproductive Medicine.) and connect to the Key Words: Cryopreservation, embryo vitrification, survival rate, delivery rate Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/coboa-vitrified-embryos-cryopreservation-program/
E
mbryo cryopreservation has become an essential therapeutic strategy in assisted reproduction
technology (ART), because it allows several pregnancies to be achieved from a single controlled ovarian stimu-
Received February 23, 2012; revised July 10, 2012; accepted July 11, 2012; published online August 3, 2012. A.C. has nothing to disclose. M.J.d.l.S. has nothing to disclose. D.C. has nothing to disclose. P.G. has nothing to disclose. P.C. has nothing to disclose. J.R. has nothing to disclose. Reprint requests: Ana Cobo, Ph.D., Embryology laboratory, Plaza de la Policía Local 3, Valencia, Valencia 46015, Spain (E-mail: [email protected]). Fertility and Sterility® Vol. 98, No. 5, November 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1107 1138
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lation (COS) cycle, thus contributing to an increase in cumulative outcome. Furthermore, it allows single-embryo transfer (SET) to take place, thereby minimizing the incidence of multiple pregnancies. The first successful pregnancy resulting from the transfer of a slow-cooled human embryo was reported in 1983 (1). Two years later, Lassalle et al. introduced some modifications to the protocol with the use of VOL. 98 NO. 5 / NOVEMBER 2012
Fertility and Sterility® 1,2-propanediol and sucrose as cryoprotectants (CPAs) (2), which led to the establishment of a standard method that has been applied extensively in IVF centers worldwide. Since then, this protocol has undergone very few modifications, with thousands of infants having been born as a result of the original procedure. Currently, vitrification is being increasingly applied to both early cleavage- and blastocyst-stage embryos. Vitrification was first applied in embryology with the use of mouse embryos almost 30 years ago (3), with successfully vitrification in 1991 by Kono et al. (4). The first human pregnancy and delivery of a baby as a result of blastocyst vitrification was published in 2001 (5). In contrast to slow freezing, vitrification protocols have undergone many changes since their introduction into clinical practice. First adaptations to the procedure were made in an attempt to reduce the CPA concentration necessary to achieve vitrification (6). The accomplishment of the glassy state of vitrification is related to the cooling rate and the viscosity of the sample and is inversely proportional to the volume of the solution containing the vitrified samples (7). In this way, by significantly reducing the volume of the sample the cooling rate is increased and the concentration of cryoprotectant can be reduced (8). This is the principle of the minimum drop size system introduced by Arav (9). A wide variety of vitrification tools and procedures are described in the literature and have been applied with varying degrees of success in several species of mammals, including humans (10–17). Most of these tools require direct contact between samples and liquid nitrogen to ensure high cooling rates, although available evidence suggests that their efficacy depends less on the cooling rate and more on an extremely high warming rate (18). In our institution, we have a vast experience in applying the Cryotop method for oocyte vitrification (19– 23). The present observational study describes the outcome achieved after the routine Cryotop vitrification of both early cleavage- and blastocyst-stage embryos in our IVF program. Additionally we have addressed the impact of embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer (ET) on the final outcome.
MATERIALS AND METHODS Patient Population, Embryo Grading, and Policies for Embryo Transfer Oocyte and embryo vitrification was introduced into our laboratory in 2007. Computerized data from all patients whose vitrified embryos underwent warming cycles from January 2007 to December 2010 were analyzed. Due to the retrospective nature of the study, Institutional Review Board approval was not sought. A total of 3,150 warming cycles were subject to analysis, including those of both infertile patients whose embryos were obtained from their own oocytes (1,685 warming cycles) and recipients of donor oocytes (1,465 warming cycles). Embryo cryopreservation had been performed on day 2, day 3, day 5, or day 6 (147, 1,725, 675, and 603 warming cycles, respectively). VOL. 98 NO. 5 / NOVEMBER 2012
Infertility was due to male factor (n ¼ 660; 39.2%), unexplained infertility (n ¼ 241; 14.3%); polycystic ovary (n ¼ 236; 14%), tubal factor (n ¼ 181; 10.7%), female age (n ¼ 146; 8.7%), endometriosis (n ¼ 145; 8.6%), low response to gonadotropins (n ¼ 68; 4.0%), recurrent miscarriage (n ¼ 6; 0.3%), and immunologic factor (n ¼ 2; 0.1%) in patients whose own oocytes were used. Indications for ovum donation were advanced maternal age (n ¼ 853; 58.2%), low response to godadotropins (n ¼ 309; 21.1%), recurrent IVF failure (n ¼ 147; 10.3%), endometriosis (n ¼ 78; 5.3%), genetic factor (n ¼ 22; 1.5%), premature ovarian failure (n ¼ 21; 1.4%), recurrent miscarriage (n ¼ 20; 1.3%), and poor-quality oocytes (n ¼ 15; 1.2%). The ovarian stimulation protocols used in our center for infertile patients whose own oocytes were used and for ovum donation are described elsewhere (22, 24, 25). Policies for embryo cryopreservation depended on the morphologic quality of surplus embryos after cryopreservation of fresh ETs, or in some cases all good-quality embryos were cryopreserved owing to risk of ovarian hyperstimulation syndrome (OHSS), impaired endometrium pattern, or high progesterone (P) levels. Embryos were classified morphologically according to the criteria of the Asociaci on para el Estudio de la Biología de la Reproducci on (ASEBIR), the Spanish embryology society (26), with slight modifications. A summary of the ASEBIR classification system can be found in the Istanbul Consensus Workshop document on embryo assessment published by Alpha Scientists in Reproductive Medicine (27, 28). In short, a type A embryo (with optimum implantation potential) showed <10% focal fragments, no multinucleation, absence of vacuoles, and no irregularities in the zona pellucida (ZP) and had 4 cells on day 2 and 7–8 cells on day 3 (coming from a 4-cell day-2 embryo). Day 2 type B embryos (with good implantation potential) were defined as those having 4 cells with 11%–25% fragmentation or 2 or 5 cells with <26% fragmentation and no multinucleation, absence of vacuoles, and good appearance of the ZP. Day 3 type B embryos were defined as those having 7–8 cells (from a 4-cell embryo on day-2) with 11%–25% fragmentation or 9 cells (from a 2-cell embryo with <25% fragmentation and a maximum of 1 multinucleated cell) and showing the same parameters for vacuoles and ZP evaluation as described above. Embryos defined as type A or B on day 2 and day 3 were selected for vitrification. Some suboptimal embryos were maintained in extended culture and were vitrified only if they developed into good-quality blastocysts. ASEBIR grading for blastocysts is based on the assessment of inner cell mass (ICM) and trophectoderm appearance as proposed by Gardner et al. (29). Type A ICM was well defined, had a compact appearance, and consisted of many cells (1,900–3,800 mm2 in diameter). Type B ICM was the same size as type A, but showing lower compaction. A Type A trophectoderm was well defined, uniform, and formed of many cells, whereas a type B trophectoderm was formed by fewer cells and irregular in appearance. Type A blastocysts were defined as having both type A ICM and type A trophectoderm. Type B blastocysts were defined as those having a type A ICM and 1139
ORIGINAL ARTICLE: ASSISTED REPRODUCTION type B trophectoderm or both type B ICM and type B trophoectoderm. Type A and B blastocysts were selected for vitrification. Artificial collapsing of blastocysts was occasionally performed in hatching embryos. The number of embryos to be replaced varied with each case, but typically two embryos were transferred on day 2 or day 3 and elective SET was considered for blastocyst stage.
Endometrial Preparation for ET For natural cycles, follicular growing was monitored. When a leading follicle was 18 mm in diameter, ovulation was triggered with 6,500 IU hCG (Ovitrelle; Merck Serono). Micronized P was initiated 3 days later (400 mg/d vaginally). Day 2 and day 3 ETs were conducted 4 and 5 days, respectively, after hCG administration. Day 5 and day 6 blastocyst ETs were scheduled 7 and 8 days, respectively, after hCG. The protocol for hormonal replacement therapy (HRT) was that described previously (30), with slight modifications. Women with functioning ovaries were first down-regulated in the luteal phase with a single dose of GnRH agonist depot (Decapeptyl, 3.75 mg; Ipsen Pharm, or Gonapeptyl, 3.75 mg; Ferring) and received 6 mgs oral estradiol valerate (EV; Progynovaw, Schering Spain) after menses. If E2 levels were >150 pg/mL and a triple layer endometrial pattern was confirmed 10–15 days after initiation of EV, administration of micronized P (800 mg/d vaginally; Progeffik; Effik Laboratories) was initiated. Day 2, day 3, day 5, and day 6 ETs were scheduled 1, 2, 4, or 5 days, respectively, after P administration. The GnRH antagonist protocol (Cetrotide; Merck Serono) was very similar, except for the administration of 0.25 mg/d for the first 7 days of the cycle. EV and P were administered as described above.
Embryo Vitrification Protocol The Cryotop method for embryo vitrification was that described by Kuwayama et al. (16), with slight modifications. Early cleavage- and blastocyst-stage embryos were equilibrated in a single 10–12-minute step at room temperature in 7.5% (v/v) ethylene glycol (EG) þ 7.5% dimethylsulfoxide (DMSO) in TCM199 medium þ 20% synthetic serum substitute (SSS). The equilibration time was defined by the reexpansion of the embryos. Typically, it took 10 minutes for day 2 and day 3 embryos to fully reexpand and 12 minutes for blastocysts. The vitrification step was performed in a solution containing 15% EG þ 15% DMSO þ 0.5 mol/L sucrose. Embryos were ‘‘washed’’ continuously in this solution for 45 seconds, at which point embryo collapsing was checked. Subsequently, embryos were taken up into the pipette and placed at the end of it while making sure it contained the lowest possible volume of vitrification solution ahead of the embryo. Embryos were placed on the Cryotop sheet, and the excess solution was removed by aspiration. After checking the minimum volume, the Cryotop was plunged into liquid nitrogen (LN). This step should not be longer than 10 seconds. The Cryotop was loaded with no more than two day 3 embryos and only one blastocyst. 1140
Although all of a patient's embryos could have been equilibrated at the same time (in separate wells), the vitrification step was always performed strictly for the number of embryos designated to be loaded onto the Cryotop. For warming, the Cryotop was removed from the LN and instantly placed in 1.0 mol/L sucrose in TCM199 þ 20% SSS at 37 C. Special care was taken to avoid any manipulation of the embryos and thus protect them from mechanical stress. After 1 minute, oocytes were placed in 0.5 mol/L sucrose in TCM199 þ 20% SSS at room temperature for 3 minutes and were not subjected to any further manipulation. Finally, oocytes were washed for 5 minutes and then for 1 minute with TCM199 þ 20% SSS at room temperature. The embryos were cultured at 37 C for R2 hours before ET. All vitrification materials were obtained from Kitazato. Immediately after warming, embryo survival was determined according to the appearance of the blastomeres and ZP. For early cleavage, blastomere survival of R50% (with clear cellular boundaries and no fragmentation) identified a live embryo. The proportion of intact embryos (100% of survived cells) and embryo morphology were recorded after warming. Blastocyst survival was evaluated according to morphologic appearance after warming and the ability of the blastocele to reexpand before transfer. None of the embryos were subjected to assisted hatching after warming. If embryos had degenerated by the time of ET they were catalogued as ‘‘dead embryos,’’ which represented a change to their original classification as ‘‘surviving embryos.’’
Definition of Outcomes and Statistics Our main measurement of outcome was the delivery rate (DR) per warming cycle. Secondary outcomes were survival, implantation, clinical pregnancy (CPR), ongoing pregnancy (OPR), and live birth (LBR) rates. Clinical pregnancy was confirmed when an intrauterine gestational sac was revealed by transvaginal ultrasound scan R5 weeks after ET (31). Ongoing pregnancy was defined as a clinical pregnancy with a fetal heartbeat (FHB) at R12 weeks (32) and implantation rate (with FHB) referred to implantation per transferred embryo. Survival, delivery, and live birth rates were determined according to the embryo's developmental stage and morphologic quality at vitrification. One-way analysis of variance with Bonferroni, Tukey, and Jaffe post hoc tests was performed for continuous variables. The chi-square test, Student t test, and Fisher exact test were used as appropriate. Logistic regression analysis was carried out to assess the impact of the following confounding variables on the DR: embryo developmental stage, own oocytes/ovum donation cycle, and type of endometrial preparation (HRT or natural cycle). A P value of < .05 was considered to be significant. Statistical analyses were performed using SPSS software (version 15.0).
RESULTS A total of 6,019 embryos were warmed in 3,150 warming cycles; mean patient age was 37.9 years (95% CI 37.5–38.1); 5,722 embryos survived warming (95% survival rate); 3,057 cryotransfers were performed, which resulted in an ET VOL. 98 NO. 5 / NOVEMBER 2012
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(38.2–39.0) (96.9–98.6) (96.4–98.8) (1.5–1.6) (39.2–45.6) (37.7–45.5) (38.6–46.6) (25.8–33.1) (27.0–34.4) (23.4–34.6) (25.9–33.1) (28.8–36.2) (38.5–39.3) (94.5–96.9) (94.7–97.6) (1.5–1.6) (39.3–45.3) (40.0–47.4) (41.7–49.3) (31.3–38.5) (32.5–39.9) (14.4–23.4) (31.2–38.4) (36.9–44.3)
603 952 38.6 5.5b 929 (97.6)b 589 (97.6)b 919 (1.5 0.6)b 390 (42.4)c 251 (41.6) 251 (42.6) 178 (29.5) 178 (30.7) 73 (29) 178 (29.5) 196 (32.5)a,c
Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
Note: CPR ¼ clinical pregnancy rate; OPR ¼ ongoing pregnancy rate; DR ¼ delivery rate; LBR ¼ live birth rate. a,b,c Different superscripts in the same row indicate statistical difference (P< .05).
(36.7–37.8) (93.4–94.9) (96.9–98.3) (1.8–1.9) (32.9–36.3) (38.7–43.3) (39.6–44.4) (30.1–35.3) (31.6–36.1) (14.5–20.1) (30.9–35.3) (36.9–41.5) (35.6–36.4) (93.0–96.8) (87.4–96.2) (1.9–2.0) (22.0–32.4) (32.2–48.0) (35.3–52.1) (22.5–37.3) (24.7–40.6) (12.4–33.9) (22.5–37.3) (27.6–43.0)
n (%) 95% CI (%) n (%) 95% CI (%)
675 1,079 38.9 5.5b 1,033 (95.7)a 649 (96.2)b 1,008 (1.5 0.6)b 426 (42.3)c 295 (43.7) 295 (45.5) 235 (34.9) 235 (36.2) 56 (18.9) 235 (34.8) 274 (40.6)a,b 1,725 3,491 37.4 5.4a 3,289 (94.2)a 1,685 (97.6)b 3,057 (1.8 0.6)a 1,058 (34.6)b 708 (41.0) 708 (42.0) 571 (33.1) 571 (33.9) 123 (17.3) 570 (33.0) 677 (39.2)a
n (%) 95% CI (%) n (%)
147 497 36.1 4.0a 472 (94.9)a 135 (91.8)a 280 (1.9 0.8)a 76 (27.2)a 59 (40.1) 59 (43.7) 44 (29.9) 44 (32.6) 15 (23.1) 44 (29.9) 52 (35.3)a No. of warming cycles No. of warmed embryos Age (y) Survival rate No. of embryo transfers (%) No. of embryos replaced (mean SD) Implantation rate CPR/cycle CPR/transfer OPR/cycle OPR/transfer Miscarriage rate DR/cycle LBR/cycle
D6 D5 D3 D2
Overall outcome according to developmental stage at vitrification.
TABLE 1
cancellation rate of 3%. A total of 5,722 embryos were transferred (mean 1.6; 95% CI 1.7–1.8); among them 2,031 were able to implant (overall implantation 35.5%; CI 95% 33.5– 38.5). Clinical and ongoing pregnancy rates calculated per warming cycle were 41.7% (95% CI 39.9–43.4) and 32.6% (95% CI 31.0–34.2), respectively. Clinical and ongoing pregnancy rates per transfer were 42.9% (95% CI 41.1–44.7) and 33.6% (95% CI 31.9–35.3). Miscarriage rate was 17.6% (95% CI 17.2–20.4). The DR per warming cycle was 32.5% (95% CI 30.9–34.2), and the LBR per warming cycle was 38.1% (95% CI 36.4–39.8). Table 1 presents the clinical outcome according to embryo developmental stage at vitrification. Patients undergoing early cleavage-stage ET were younger than those undergoing blastocyst transfers (P< .05). The proportion of ETs was statistically lower when embryos were vitrified on day 2 (P< .05). The number of embryos replaced was higher for early cleavage-stage than for blastocyst-stage ETs (P< .05), and implantation rates differed between embryos vitrified on day 2 and day 3 (P< .05). There were no statistical differences between CPR, OPR, and DR (Table 1). LBR was higher on day 3 and day 5 (Table 1). Early cleavage-stage embryos were mostly allowed to develop to further stages after warming to accomplish the ET (Supplemental Table 1, available online at www.fertstert.org). No statistical differences (NS) were detected regarding clinical outcome, and more ETs were accomplished when the embryos were vitrified and replaced on day 3 (P< .05). No statistical differences were detected between day 2 and day 3 embryos regarding morphologic parameters before vitrification and after warming (NS). The proportion of intact embryos at warming (100% blastomere survival) was 93% (95% CI 90.1–95.3) for embryos vitrified on day 2 and 95% (95% CI 95.0% 94.3–95.7) for those vitrified on day 3. Figure 1 shows day 3 embryos before and immediately after vitrification (Fig. 1I) and further developed to blastocyst stage (Fig. 1II), a day 5 blastocyst before vitrification, immediately after warming, and at 2 hours when full reexpansion was recorded (Fig. 1III), and a day 6 hatched blastocyst (Fig. 1IV). Survival and DRs were analyzed according to embryo quality at all developmental stages (Table 2). Type A day 3 and day 6 embryos showed a statistically higher survival rate than type B (P< .05). Statistical differences between the DR of type A and B embryos were found on day 5 (P< .05). Table 3 presents the survival rate and LBR according to differentially developed blastocysts (day 5 vs. day 6). Survival was statistically lower among hatching day 5 blastocysts and early blastocysts on day 6 (P< .05). LBR was higher for expanded blastocysts on day 5, but statistical differences were found only when they were compared with early blastocysts (P< .05), whereas LBR was lower when early blastocysts were vitrified and transferred on day 6, although differences were not statistically significant. The linear regression model for embryo developmental stage (odds ratio [OR] 0.983, 95% CI 0.928–1.040), ovum donation/patient's own oocytes (OR 1.014, 95% CI 0.873–1.177), HRT/natural cycle for endometrial preparation (OR 1.102, 95% CI 0.943–1.277), and all confounding variables together (OR 1.179, 95% CI 0.912–1.277) showed no
95% CI (%)
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION
FIGURE 1
(I) Day-3 embryos (a) before vitrification and (b) immediately after warming. (II) (a) Day-3 embryos before vitrification; (b) day-5 blastocysts developed from the day-3 warmed embryos. (III) Day-5 blastocyst (a) before vitrification and (b) 1 hour after vitrification (the reexpansion has not been completed); (c) fully reexpanded 3 hours after warming. (IV) Hatched day-6 blastocyst (a) before vitrification and (b) 1 hour after vitrification (the reexpansion has not been completed); (c) fully reexpanded blastocyst 5 hours after warming. Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
impact of these confounding variables on DR per warming cycle (P< .05).
DISCUSSION The outcome achieved using the vitrification protocol described in the present report is solid evidence of the tremendous 1142
scope of this strategy. This methodology has proved to be very efficient for oocyte storage, whereas other techniques, including protocols for slow cooling and other vitrification procedures, have fared badly. We have extensive experience using the Cryotop device for human oocyte vitrification (19, 21–23) and have established an oocyte bank for ovum donation with >20,000 vitrified donated oocytes (33). Other VOL. 98 NO. 5 / NOVEMBER 2012
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TABLE 2 Effect of embryo quality at vitrification on survival and delivery rates.
Optimum vitrified day 2 embryo Good-quality vitrified day 2 embryo Optimum vitrified day 3 embryo Good-quality vitrified day 3 embryo Optimum vitrified day 5 embryo Good-quality vitrified day 5 embryo Optimum vitrified day 6 embryo Good-quality vitrified day 6 embryo
Survival rate
95% CI (%)
P value
Delivery rate
95% CI (%)
P value
289 (95.7%) 192 (94.1%) 1,691 (96.8%) 1,567 (91.6%) 368 (96.6%) 610 (97.9%) 396 (99.7%) 533 (96.0%)
(93.4–97.9) (90.1–97.3) (95.9–96.3) (90.3–92.9) (94.8–98.4) (96.8–99.0) (99.2–100) (94.4–97.6)
.274
27 (27.6%) 18 (34.0%) 298 (33.8%) 269 (32.4%) 96 (41.6%) 126 (31.5%) 109 (30.4%) 69 (28.3%)
(18.8–36.5) (21.3–46.8) (30.7–36.9) (29.2–35.6) (35.2–47.9) (26.9–35.1) (25.6–31.2) (22.7–33.9)
.458
.001 .141 < .001
.572 .012 .649
Note: Optimum ¼ type A embryo; good quality ¼ type B embryo. Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
authors also have reported the efficiency of this system with the oocytes of infertile patients (34, 35) and in randomized trials comparing the efficiency of slow cooling versus vitrification at early cleavage stages (36, 37). The high efficiency of this technology has also been proved in the vitrification of blastocysts following trophoectoderm biopsy (38). The present study, involving more than 6,000 warmed embryos, confirms the scope of the Cryotop system at all developmental stages. The overall survival rate for early cleavage-stage embryos was slightly lower than that observed for blastocyst-stage embryos, although no impact on delivery or live birth rates was detected. After warming of almost 4,000 early cleavage embryos, a high number (95%) exhibited 100% intact blastomeres after vitrification. Importantly, the practical nonexistence of partially damaged embryos after vitrification represents a significant improvement in our cryopreservation program, which has contributed to a significant increase in the yield of stimulation cycles and cumulative rates. Other authors have evaluated the open vitrification of early-stage embryos, showing survival rate varying from 70% to 95% according to the type of device used (36, 37, 39, 40). Similarly to what happened to early stages, our overall blastocyst survival rate involving almost 2,000 blastocysts was considerably high. Other authors have published similar outcomes using the Cryotop system (38, 41–44). In the past, other devices have been used for blastocyst vitrification. For example, Balaban et al. reported 95% survival (234
blastocysts warmed) with the use of the Cryoloop, and a larger study of 1,129 vitrified blastocysts reported a 85.7% survival rate (45). Similar outcomes were published by others involving 250–700 embryos (46–48). Studies of the use of electron microscope (EM) grid, reporting data from 90–300 warmed blastocysts, have shown survival rates of 50% (49, 50). Open devices for vitrification were used in all of the above-mentioned studies with varying success depending on the device used. Others have addressed the usefulness of closed systems. One study of 839 warmed blastocysts showed a survival rate of 86% with the use of the high security vitrification system (HSVS) (51), and a larger study (1,185 blastocysts) reported an overall survival rate of 77% (52). Our data, based on one of the largest series published so far, indicate that the Cryotop method yields one of the highest outcomes among all the devices available. In the present study, statistical differences in age between patients undergoing early cleavage-stage ET and those undergoing blastocyst transfer, may be due to the latter group being made up mainly of recipients of donor oocytes. In addition, some differences have been found when assessing the survival rate according to embryo developmental stage. Nonetheless, some clarifications need to be made when assessing these data. Survival rates would appear to be higher among optimum (type A) day 3 and day 6 embryos (Table 2). However, these differences could be anecdotal, because the linear regression model revealed that the developmental stage of the embryo had no impact on the delivery rate per warming cycle. This, in turn, indicates that slight differences in the survival
TABLE 3 Effect of blastocyst development on survival and live birth rates. Survival rate Day 5 blastocysts (n ¼ 1,079 warmed embryos) Early blastocyst 432/458 (94.3%) Expanded blastocyst 537/549 (97.9%) Hatching-hatched blastocyst 64/72 (88.8%) Day 6 blastocyst (n ¼ 952 warmed embryos) Early blastocyst 46/47 (97.9%) Expanded blastocyst 556/570 (97.5%) Hatching-hatched blastocyst 327/335 (95.5%) a,b
95% CI (%)
P value
LBR/warming cycle
95% CI (%)
P value
(91.7–96.7) (96.7–99.0)a (81.5–96.6)b
.039
92/285 (32.3%) 155/349 (44.4%) 14/41 (34.1%)
(26.7–37.7) (39.2–49.6)a (19.6–48.6)
.021
(93.8–100) (95.8–98.8) (93.0–96.9)
.107
6/35 (17.1%) 100/292 (34.2%) 96/276 (34.7%)
(4.6–29.6)b (28.8–39.6) (29.1–40.3)
.201
Different superscripts in the same column indicate statistical difference (P< .05).
Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION rates could be neutralized by the high implantation potential inherent to good quality embryos which happen to be intact after vitrification. The absence of variability in the before and after morphologic evaluation of early cleavage stages is another indicator of the safety of the cryopreservation method we employ. The high rate of reexpansion observed for blastocyst-stage embryos and their morphologic appearance after vitrification are also evidence of the lack of damage caused during vitrification. However, it should be pointed out that we observed some differences in survival rates related to blastocyst developmental stage. According to our data, expanded blastocysts on day 5 or day 6 achieved the highest survival rate. This finding contrasts with reports by other groups (52, 53) suggesting that the likelihood of ice formation is higher in expanded blastocysts than in early blastocysts, in which the volume of the blastocoel cavity is much smaller. In fact, this is the rationale for provoking an artificial collapse of blastocysts before vitrification in some cases (53). This is not a strategy we apply routinely, except in isolated cases of hatching blastocysts. It is possible that the high survival rate of our expanded blastocysts was due to the open system we use (Cryotop), because it ensures a very high cooling rate and, most importantly, an extremely high warming rate, which impedes the formation of ice (recrystallization) during warming (18). The warming rates provided by other closed systems may be insufficient. Indeed, the survival rate achieved by expanded blastocysts in the present study, 97.9% at day 5 and 97.7% at day 6, which exceeds that reported in other studies, e.g., 75% (52), supports this theory. Obviously, an extremely high survival rate is an advantage in that the great majority of warming cycles result in embryo replacement, which is why our rate of transfer cancellation was quite low (3%). The implementation of vitrification represented a breakthrough in our center owing to the significant decrease from the previous cancellation rate (30%; data not shown). The statistical differences presented in Table 1, which relate to the number of ETs performed after vitrification at different embryo developmental stages, require careful interpretation, because not all of the embryos vitrified on day 2 or day 3 were subsequently transferred to the uterus at the same stage. Some of them were subjected to further culture and were transferred at a more advanced developmental stage (day 3 or blastocyst). The process of natural selection of embryos during culture may explain the differences in the final count of warming cycles that ended in ET. Additionally it is worth mentioning that despite similar survival rates after warming, lower implantation rates observed on ETs coming from vitrified day 2 embryos show that the vitrification procedures might have a negative effect on this particular stage of development. The relative poor prognosis of day 2 embryos may be related to the embryonic genome activation, which starts at the end of day 2 or beginning of day 3 (54), or to other aspects, such as the sudden interruption by the cryopreservation procedure of the embryonic metabolic activity (measured by oxygen uptake) of day 2 embryos, which is superior compared to day 3 (55). Another explanation might lie with the presence of blastomeres in M-phase during early stages. The presence of the mitotic appa1144
ratus during the vitrification procedure may disturb spindle structures and chromosome segregation, and therefore induce chromosomal abnormalities during the forthcoming divisions, making the embryos less likely to implant. Day 3 embryos also present blastomeres in M-phase; however, the impact on implantation potential could be less. According to the linear regression model, donated ovum/ own oocyte warming cycles, endometrial preparation, and embryo developmental stage at vitrification had no impact on DR, thus confirming that the only variable related to final outcome is related to the quality of the embryos vitrified. Consequently, we have found some differences in survival rates (SR) and LBRs according to embryo quality (type A or B) and the specific stage of development of the blastocyst on day 5, as shown on Table 3. Accordingly, the highest DRSR was achieved by type A day 5 blastocyts and the highest LBR by the group of day 5 expanded blastocysts, which could have been due to the high implantation rate associated with these embryos (56). Lower-quality blastocysts yielded lower clinical outcomes, although their survival rate was high, thereby indicating that vitrification does not affect the potential of embryos to implant and end in live births. Despite their impressive performance, open systems are cause for concern owing to the hypothetical risk of cross-contamination, although no case of infection through LN has ever been documented in ART (57, 58). In this context, the very low risk of virus transmission in seropositive patients has recently been demonstrated (58). Notwithstanding, measures can be taken to improve the tool's safety, such as sterilization of LN (59, 60) and storage in vapor-phase systems (57) or a closed system. In conclusion, highly successful cryopreservation of embryos at all developmental stages is possible with the Cryotop system, because it yields high survival rates and a delivery rate per warming cycle similar to those achieved with fresh embryos. Although subtle differences have been identified regarding the quality and developmental stage of vitrified embryos, there do not seem to be any variables that clearly exert a negative effect on survival and delivery rates.
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SUPPLEMENTAL TABLE 1 Embryo development after warming. Vitrification day 2/embryo transfer day 2
Vitrification day 2/embryo transfer day 3
95% CI N warming cycles N embryo transfers IR/warming cycle CPR/warming cycle OPR/warming cycle DR/warming cycle LBR/warming cycle
11 11 (100) 8/18 (44.4) 6 ( 54.5) 5 (45.5) 5 (45.5) 5 (45.5)
(21.5–67.3) (24.5–83.9) (16.7–74.9) (16.7–74.9) (16.7–74.9)
95% CI 83 83 (100) 46/176 (26.1) 35 (42.2) 25 (24.1) 25 (24.1) 30 (36.1)
Vitrification day 3/embryo transfer day 3
(19.6–32.5) (24.7–45.3) (14.8–33.2) (14.8–33.2) (20.1–39.9)
a
1,525 1,520 (99.7)a 875/2,783 (31.4) 640 (41.9) 515 (33.8) 515 (33.8) 614 (40.2)
(99.4–99.9) (29.6–33.1) (39.4–44.4) (31.4–36.2) (31.4–36.2) (37.7–42.7)
95% CI 53 42 (79.2) 22/86 (25.5) 18 (33.9) 14 (26.4) 14 (26.4) 16 (30.2)
(28.7–55.3) (16.3–34.7) (21.2–46.6) (14.5–38.3) (14.5–38.3) (17.8–42.5)
Vitrification day 3/embryo transfer blastocyst
95% CI N warming cycles N embryo transfers IR/warming cycle CPR/warming cycle OPR/warming cycle DR/warming cycle LBR/warming cycle
Vitrification day 2/embryo transfer blastocyst
95% CI 200 189 (94.5)a 102/274 (37.2) 82 (41.0) 75 (37.5) 73 (36.5) 86 (43.0)
(90.8–97.4) (31.5–42.9) (34.2–47.8) (30.1–44.0) (29.8–43.2) (36.1–49.8)
P< .05.
Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
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Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles de los Santos, Ph.D., Damia Castello , Ph.D., Pilar Ga miz, Ph.D., Ana Cobo, Ph.D., María Jose Remohí, M.D. Pilar Campos, M.L.T., and Jose IVI-Valencia, Institut Universitari IVI, Valencia, Spain
Objective: To assess the outcomes achieved after Cryotop vitrification of both early cleavage and blastocyst-stage embryos and to determine whether the embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer had any effect on the final outcome. Design: Observational study. Setting: Private university-affiliated IVF center. Patient(s): Women undergoing 3,150 warming cycles whose embryos were vitrified due to various reasons. Intervention(s): Vitrification by the Cryotop open device. Main Outcome Measure(s): Delivery rate (DR) per warming cycle. Result(s): Survival rate was 95% (5,722 out of 6,019 embryos). The percentage of intact embryos at warming showing 100% blastomere survival was 93% (95% CI 90.1%–95.3%) for day 2 and 95% (95% CI 94.3%–95.7%) for day 3; 3,057 embryo transfers were performed (3% cancellation rate). The DR/warming cycle was 32.5% (95% CI 30.9%–34.2%). Slight differences in survival rate were found [94.9% (95% CI 93.0%–96.8%) for day 2, 94.2% (95% CI 93.4%–94.9%) for day 3, 95.7% (95% CI 94.5%–96.9%) for day 5, and 97.6% (95% CI 96.9%–98.6%) for day 6]. Overall implantation, clinical pregnancy, ongoing pregnancy, and live birth rates per warming cycle were 35.5% (95% CI 33.5%–38.5%), 41.7% (95% CI 39.9%–43.4%), 32.6% (95% CI 31.0%–34.2%), and 38.1% (95% CI 36.4%–39.8%) respectively. The linear regression model considering embryo developmental stage, ovum donation or patient's own oocytes, and hormonal replacement therapy or natural cycle for endometrial preparation (odds ratio 1.179; 95% CI 0.912–1.277) showed no impact on the DR. Conclusion(s): Highly successful cryopreservation of all embryo developmental stages is possible with the use of the Cryotop system. There are no variables clearly exerting a negative effect Use your smartphone on the survival and delivery rates. (Fertil SterilÒ 2012;98:1138–46. Ó2012 by American Society to scan this QR code for Reproductive Medicine.) and connect to the Key Words: Cryopreservation, embryo vitrification, survival rate, delivery rate Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/coboa-vitrified-embryos-cryopreservation-program/
E
mbryo cryopreservation has become an essential therapeutic strategy in assisted reproduction
technology (ART), because it allows several pregnancies to be achieved from a single controlled ovarian stimu-
Received February 23, 2012; revised July 10, 2012; accepted July 11, 2012; published online August 3, 2012. A.C. has nothing to disclose. M.J.d.l.S. has nothing to disclose. D.C. has nothing to disclose. P.G. has nothing to disclose. P.C. has nothing to disclose. J.R. has nothing to disclose. Reprint requests: Ana Cobo, Ph.D., Embryology laboratory, Plaza de la Policía Local 3, Valencia, Valencia 46015, Spain (E-mail: [email protected]). Fertility and Sterility® Vol. 98, No. 5, November 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.07.1107 1138
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lation (COS) cycle, thus contributing to an increase in cumulative outcome. Furthermore, it allows single-embryo transfer (SET) to take place, thereby minimizing the incidence of multiple pregnancies. The first successful pregnancy resulting from the transfer of a slow-cooled human embryo was reported in 1983 (1). Two years later, Lassalle et al. introduced some modifications to the protocol with the use of VOL. 98 NO. 5 / NOVEMBER 2012
Fertility and Sterility® 1,2-propanediol and sucrose as cryoprotectants (CPAs) (2), which led to the establishment of a standard method that has been applied extensively in IVF centers worldwide. Since then, this protocol has undergone very few modifications, with thousands of infants having been born as a result of the original procedure. Currently, vitrification is being increasingly applied to both early cleavage- and blastocyst-stage embryos. Vitrification was first applied in embryology with the use of mouse embryos almost 30 years ago (3), with successfully vitrification in 1991 by Kono et al. (4). The first human pregnancy and delivery of a baby as a result of blastocyst vitrification was published in 2001 (5). In contrast to slow freezing, vitrification protocols have undergone many changes since their introduction into clinical practice. First adaptations to the procedure were made in an attempt to reduce the CPA concentration necessary to achieve vitrification (6). The accomplishment of the glassy state of vitrification is related to the cooling rate and the viscosity of the sample and is inversely proportional to the volume of the solution containing the vitrified samples (7). In this way, by significantly reducing the volume of the sample the cooling rate is increased and the concentration of cryoprotectant can be reduced (8). This is the principle of the minimum drop size system introduced by Arav (9). A wide variety of vitrification tools and procedures are described in the literature and have been applied with varying degrees of success in several species of mammals, including humans (10–17). Most of these tools require direct contact between samples and liquid nitrogen to ensure high cooling rates, although available evidence suggests that their efficacy depends less on the cooling rate and more on an extremely high warming rate (18). In our institution, we have a vast experience in applying the Cryotop method for oocyte vitrification (19– 23). The present observational study describes the outcome achieved after the routine Cryotop vitrification of both early cleavage- and blastocyst-stage embryos in our IVF program. Additionally we have addressed the impact of embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer (ET) on the final outcome.
MATERIALS AND METHODS Patient Population, Embryo Grading, and Policies for Embryo Transfer Oocyte and embryo vitrification was introduced into our laboratory in 2007. Computerized data from all patients whose vitrified embryos underwent warming cycles from January 2007 to December 2010 were analyzed. Due to the retrospective nature of the study, Institutional Review Board approval was not sought. A total of 3,150 warming cycles were subject to analysis, including those of both infertile patients whose embryos were obtained from their own oocytes (1,685 warming cycles) and recipients of donor oocytes (1,465 warming cycles). Embryo cryopreservation had been performed on day 2, day 3, day 5, or day 6 (147, 1,725, 675, and 603 warming cycles, respectively). VOL. 98 NO. 5 / NOVEMBER 2012
Infertility was due to male factor (n ¼ 660; 39.2%), unexplained infertility (n ¼ 241; 14.3%); polycystic ovary (n ¼ 236; 14%), tubal factor (n ¼ 181; 10.7%), female age (n ¼ 146; 8.7%), endometriosis (n ¼ 145; 8.6%), low response to gonadotropins (n ¼ 68; 4.0%), recurrent miscarriage (n ¼ 6; 0.3%), and immunologic factor (n ¼ 2; 0.1%) in patients whose own oocytes were used. Indications for ovum donation were advanced maternal age (n ¼ 853; 58.2%), low response to godadotropins (n ¼ 309; 21.1%), recurrent IVF failure (n ¼ 147; 10.3%), endometriosis (n ¼ 78; 5.3%), genetic factor (n ¼ 22; 1.5%), premature ovarian failure (n ¼ 21; 1.4%), recurrent miscarriage (n ¼ 20; 1.3%), and poor-quality oocytes (n ¼ 15; 1.2%). The ovarian stimulation protocols used in our center for infertile patients whose own oocytes were used and for ovum donation are described elsewhere (22, 24, 25). Policies for embryo cryopreservation depended on the morphologic quality of surplus embryos after cryopreservation of fresh ETs, or in some cases all good-quality embryos were cryopreserved owing to risk of ovarian hyperstimulation syndrome (OHSS), impaired endometrium pattern, or high progesterone (P) levels. Embryos were classified morphologically according to the criteria of the Asociaci on para el Estudio de la Biología de la Reproducci on (ASEBIR), the Spanish embryology society (26), with slight modifications. A summary of the ASEBIR classification system can be found in the Istanbul Consensus Workshop document on embryo assessment published by Alpha Scientists in Reproductive Medicine (27, 28). In short, a type A embryo (with optimum implantation potential) showed <10% focal fragments, no multinucleation, absence of vacuoles, and no irregularities in the zona pellucida (ZP) and had 4 cells on day 2 and 7–8 cells on day 3 (coming from a 4-cell day-2 embryo). Day 2 type B embryos (with good implantation potential) were defined as those having 4 cells with 11%–25% fragmentation or 2 or 5 cells with <26% fragmentation and no multinucleation, absence of vacuoles, and good appearance of the ZP. Day 3 type B embryos were defined as those having 7–8 cells (from a 4-cell embryo on day-2) with 11%–25% fragmentation or 9 cells (from a 2-cell embryo with <25% fragmentation and a maximum of 1 multinucleated cell) and showing the same parameters for vacuoles and ZP evaluation as described above. Embryos defined as type A or B on day 2 and day 3 were selected for vitrification. Some suboptimal embryos were maintained in extended culture and were vitrified only if they developed into good-quality blastocysts. ASEBIR grading for blastocysts is based on the assessment of inner cell mass (ICM) and trophectoderm appearance as proposed by Gardner et al. (29). Type A ICM was well defined, had a compact appearance, and consisted of many cells (1,900–3,800 mm2 in diameter). Type B ICM was the same size as type A, but showing lower compaction. A Type A trophectoderm was well defined, uniform, and formed of many cells, whereas a type B trophectoderm was formed by fewer cells and irregular in appearance. Type A blastocysts were defined as having both type A ICM and type A trophectoderm. Type B blastocysts were defined as those having a type A ICM and 1139
ORIGINAL ARTICLE: ASSISTED REPRODUCTION type B trophectoderm or both type B ICM and type B trophoectoderm. Type A and B blastocysts were selected for vitrification. Artificial collapsing of blastocysts was occasionally performed in hatching embryos. The number of embryos to be replaced varied with each case, but typically two embryos were transferred on day 2 or day 3 and elective SET was considered for blastocyst stage.
Endometrial Preparation for ET For natural cycles, follicular growing was monitored. When a leading follicle was 18 mm in diameter, ovulation was triggered with 6,500 IU hCG (Ovitrelle; Merck Serono). Micronized P was initiated 3 days later (400 mg/d vaginally). Day 2 and day 3 ETs were conducted 4 and 5 days, respectively, after hCG administration. Day 5 and day 6 blastocyst ETs were scheduled 7 and 8 days, respectively, after hCG. The protocol for hormonal replacement therapy (HRT) was that described previously (30), with slight modifications. Women with functioning ovaries were first down-regulated in the luteal phase with a single dose of GnRH agonist depot (Decapeptyl, 3.75 mg; Ipsen Pharm, or Gonapeptyl, 3.75 mg; Ferring) and received 6 mgs oral estradiol valerate (EV; Progynovaw, Schering Spain) after menses. If E2 levels were >150 pg/mL and a triple layer endometrial pattern was confirmed 10–15 days after initiation of EV, administration of micronized P (800 mg/d vaginally; Progeffik; Effik Laboratories) was initiated. Day 2, day 3, day 5, and day 6 ETs were scheduled 1, 2, 4, or 5 days, respectively, after P administration. The GnRH antagonist protocol (Cetrotide; Merck Serono) was very similar, except for the administration of 0.25 mg/d for the first 7 days of the cycle. EV and P were administered as described above.
Embryo Vitrification Protocol The Cryotop method for embryo vitrification was that described by Kuwayama et al. (16), with slight modifications. Early cleavage- and blastocyst-stage embryos were equilibrated in a single 10–12-minute step at room temperature in 7.5% (v/v) ethylene glycol (EG) þ 7.5% dimethylsulfoxide (DMSO) in TCM199 medium þ 20% synthetic serum substitute (SSS). The equilibration time was defined by the reexpansion of the embryos. Typically, it took 10 minutes for day 2 and day 3 embryos to fully reexpand and 12 minutes for blastocysts. The vitrification step was performed in a solution containing 15% EG þ 15% DMSO þ 0.5 mol/L sucrose. Embryos were ‘‘washed’’ continuously in this solution for 45 seconds, at which point embryo collapsing was checked. Subsequently, embryos were taken up into the pipette and placed at the end of it while making sure it contained the lowest possible volume of vitrification solution ahead of the embryo. Embryos were placed on the Cryotop sheet, and the excess solution was removed by aspiration. After checking the minimum volume, the Cryotop was plunged into liquid nitrogen (LN). This step should not be longer than 10 seconds. The Cryotop was loaded with no more than two day 3 embryos and only one blastocyst. 1140
Although all of a patient's embryos could have been equilibrated at the same time (in separate wells), the vitrification step was always performed strictly for the number of embryos designated to be loaded onto the Cryotop. For warming, the Cryotop was removed from the LN and instantly placed in 1.0 mol/L sucrose in TCM199 þ 20% SSS at 37 C. Special care was taken to avoid any manipulation of the embryos and thus protect them from mechanical stress. After 1 minute, oocytes were placed in 0.5 mol/L sucrose in TCM199 þ 20% SSS at room temperature for 3 minutes and were not subjected to any further manipulation. Finally, oocytes were washed for 5 minutes and then for 1 minute with TCM199 þ 20% SSS at room temperature. The embryos were cultured at 37 C for R2 hours before ET. All vitrification materials were obtained from Kitazato. Immediately after warming, embryo survival was determined according to the appearance of the blastomeres and ZP. For early cleavage, blastomere survival of R50% (with clear cellular boundaries and no fragmentation) identified a live embryo. The proportion of intact embryos (100% of survived cells) and embryo morphology were recorded after warming. Blastocyst survival was evaluated according to morphologic appearance after warming and the ability of the blastocele to reexpand before transfer. None of the embryos were subjected to assisted hatching after warming. If embryos had degenerated by the time of ET they were catalogued as ‘‘dead embryos,’’ which represented a change to their original classification as ‘‘surviving embryos.’’
Definition of Outcomes and Statistics Our main measurement of outcome was the delivery rate (DR) per warming cycle. Secondary outcomes were survival, implantation, clinical pregnancy (CPR), ongoing pregnancy (OPR), and live birth (LBR) rates. Clinical pregnancy was confirmed when an intrauterine gestational sac was revealed by transvaginal ultrasound scan R5 weeks after ET (31). Ongoing pregnancy was defined as a clinical pregnancy with a fetal heartbeat (FHB) at R12 weeks (32) and implantation rate (with FHB) referred to implantation per transferred embryo. Survival, delivery, and live birth rates were determined according to the embryo's developmental stage and morphologic quality at vitrification. One-way analysis of variance with Bonferroni, Tukey, and Jaffe post hoc tests was performed for continuous variables. The chi-square test, Student t test, and Fisher exact test were used as appropriate. Logistic regression analysis was carried out to assess the impact of the following confounding variables on the DR: embryo developmental stage, own oocytes/ovum donation cycle, and type of endometrial preparation (HRT or natural cycle). A P value of < .05 was considered to be significant. Statistical analyses were performed using SPSS software (version 15.0).
RESULTS A total of 6,019 embryos were warmed in 3,150 warming cycles; mean patient age was 37.9 years (95% CI 37.5–38.1); 5,722 embryos survived warming (95% survival rate); 3,057 cryotransfers were performed, which resulted in an ET VOL. 98 NO. 5 / NOVEMBER 2012
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(38.2–39.0) (96.9–98.6) (96.4–98.8) (1.5–1.6) (39.2–45.6) (37.7–45.5) (38.6–46.6) (25.8–33.1) (27.0–34.4) (23.4–34.6) (25.9–33.1) (28.8–36.2) (38.5–39.3) (94.5–96.9) (94.7–97.6) (1.5–1.6) (39.3–45.3) (40.0–47.4) (41.7–49.3) (31.3–38.5) (32.5–39.9) (14.4–23.4) (31.2–38.4) (36.9–44.3)
603 952 38.6 5.5b 929 (97.6)b 589 (97.6)b 919 (1.5 0.6)b 390 (42.4)c 251 (41.6) 251 (42.6) 178 (29.5) 178 (30.7) 73 (29) 178 (29.5) 196 (32.5)a,c
Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
Note: CPR ¼ clinical pregnancy rate; OPR ¼ ongoing pregnancy rate; DR ¼ delivery rate; LBR ¼ live birth rate. a,b,c Different superscripts in the same row indicate statistical difference (P< .05).
(36.7–37.8) (93.4–94.9) (96.9–98.3) (1.8–1.9) (32.9–36.3) (38.7–43.3) (39.6–44.4) (30.1–35.3) (31.6–36.1) (14.5–20.1) (30.9–35.3) (36.9–41.5) (35.6–36.4) (93.0–96.8) (87.4–96.2) (1.9–2.0) (22.0–32.4) (32.2–48.0) (35.3–52.1) (22.5–37.3) (24.7–40.6) (12.4–33.9) (22.5–37.3) (27.6–43.0)
n (%) 95% CI (%) n (%) 95% CI (%)
675 1,079 38.9 5.5b 1,033 (95.7)a 649 (96.2)b 1,008 (1.5 0.6)b 426 (42.3)c 295 (43.7) 295 (45.5) 235 (34.9) 235 (36.2) 56 (18.9) 235 (34.8) 274 (40.6)a,b 1,725 3,491 37.4 5.4a 3,289 (94.2)a 1,685 (97.6)b 3,057 (1.8 0.6)a 1,058 (34.6)b 708 (41.0) 708 (42.0) 571 (33.1) 571 (33.9) 123 (17.3) 570 (33.0) 677 (39.2)a
n (%) 95% CI (%) n (%)
147 497 36.1 4.0a 472 (94.9)a 135 (91.8)a 280 (1.9 0.8)a 76 (27.2)a 59 (40.1) 59 (43.7) 44 (29.9) 44 (32.6) 15 (23.1) 44 (29.9) 52 (35.3)a No. of warming cycles No. of warmed embryos Age (y) Survival rate No. of embryo transfers (%) No. of embryos replaced (mean SD) Implantation rate CPR/cycle CPR/transfer OPR/cycle OPR/transfer Miscarriage rate DR/cycle LBR/cycle
D6 D5 D3 D2
Overall outcome according to developmental stage at vitrification.
TABLE 1
cancellation rate of 3%. A total of 5,722 embryos were transferred (mean 1.6; 95% CI 1.7–1.8); among them 2,031 were able to implant (overall implantation 35.5%; CI 95% 33.5– 38.5). Clinical and ongoing pregnancy rates calculated per warming cycle were 41.7% (95% CI 39.9–43.4) and 32.6% (95% CI 31.0–34.2), respectively. Clinical and ongoing pregnancy rates per transfer were 42.9% (95% CI 41.1–44.7) and 33.6% (95% CI 31.9–35.3). Miscarriage rate was 17.6% (95% CI 17.2–20.4). The DR per warming cycle was 32.5% (95% CI 30.9–34.2), and the LBR per warming cycle was 38.1% (95% CI 36.4–39.8). Table 1 presents the clinical outcome according to embryo developmental stage at vitrification. Patients undergoing early cleavage-stage ET were younger than those undergoing blastocyst transfers (P< .05). The proportion of ETs was statistically lower when embryos were vitrified on day 2 (P< .05). The number of embryos replaced was higher for early cleavage-stage than for blastocyst-stage ETs (P< .05), and implantation rates differed between embryos vitrified on day 2 and day 3 (P< .05). There were no statistical differences between CPR, OPR, and DR (Table 1). LBR was higher on day 3 and day 5 (Table 1). Early cleavage-stage embryos were mostly allowed to develop to further stages after warming to accomplish the ET (Supplemental Table 1, available online at www.fertstert.org). No statistical differences (NS) were detected regarding clinical outcome, and more ETs were accomplished when the embryos were vitrified and replaced on day 3 (P< .05). No statistical differences were detected between day 2 and day 3 embryos regarding morphologic parameters before vitrification and after warming (NS). The proportion of intact embryos at warming (100% blastomere survival) was 93% (95% CI 90.1–95.3) for embryos vitrified on day 2 and 95% (95% CI 95.0% 94.3–95.7) for those vitrified on day 3. Figure 1 shows day 3 embryos before and immediately after vitrification (Fig. 1I) and further developed to blastocyst stage (Fig. 1II), a day 5 blastocyst before vitrification, immediately after warming, and at 2 hours when full reexpansion was recorded (Fig. 1III), and a day 6 hatched blastocyst (Fig. 1IV). Survival and DRs were analyzed according to embryo quality at all developmental stages (Table 2). Type A day 3 and day 6 embryos showed a statistically higher survival rate than type B (P< .05). Statistical differences between the DR of type A and B embryos were found on day 5 (P< .05). Table 3 presents the survival rate and LBR according to differentially developed blastocysts (day 5 vs. day 6). Survival was statistically lower among hatching day 5 blastocysts and early blastocysts on day 6 (P< .05). LBR was higher for expanded blastocysts on day 5, but statistical differences were found only when they were compared with early blastocysts (P< .05), whereas LBR was lower when early blastocysts were vitrified and transferred on day 6, although differences were not statistically significant. The linear regression model for embryo developmental stage (odds ratio [OR] 0.983, 95% CI 0.928–1.040), ovum donation/patient's own oocytes (OR 1.014, 95% CI 0.873–1.177), HRT/natural cycle for endometrial preparation (OR 1.102, 95% CI 0.943–1.277), and all confounding variables together (OR 1.179, 95% CI 0.912–1.277) showed no
95% CI (%)
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FIGURE 1
(I) Day-3 embryos (a) before vitrification and (b) immediately after warming. (II) (a) Day-3 embryos before vitrification; (b) day-5 blastocysts developed from the day-3 warmed embryos. (III) Day-5 blastocyst (a) before vitrification and (b) 1 hour after vitrification (the reexpansion has not been completed); (c) fully reexpanded 3 hours after warming. (IV) Hatched day-6 blastocyst (a) before vitrification and (b) 1 hour after vitrification (the reexpansion has not been completed); (c) fully reexpanded blastocyst 5 hours after warming. Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
impact of these confounding variables on DR per warming cycle (P< .05).
DISCUSSION The outcome achieved using the vitrification protocol described in the present report is solid evidence of the tremendous 1142
scope of this strategy. This methodology has proved to be very efficient for oocyte storage, whereas other techniques, including protocols for slow cooling and other vitrification procedures, have fared badly. We have extensive experience using the Cryotop device for human oocyte vitrification (19, 21–23) and have established an oocyte bank for ovum donation with >20,000 vitrified donated oocytes (33). Other VOL. 98 NO. 5 / NOVEMBER 2012
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TABLE 2 Effect of embryo quality at vitrification on survival and delivery rates.
Optimum vitrified day 2 embryo Good-quality vitrified day 2 embryo Optimum vitrified day 3 embryo Good-quality vitrified day 3 embryo Optimum vitrified day 5 embryo Good-quality vitrified day 5 embryo Optimum vitrified day 6 embryo Good-quality vitrified day 6 embryo
Survival rate
95% CI (%)
P value
Delivery rate
95% CI (%)
P value
289 (95.7%) 192 (94.1%) 1,691 (96.8%) 1,567 (91.6%) 368 (96.6%) 610 (97.9%) 396 (99.7%) 533 (96.0%)
(93.4–97.9) (90.1–97.3) (95.9–96.3) (90.3–92.9) (94.8–98.4) (96.8–99.0) (99.2–100) (94.4–97.6)
.274
27 (27.6%) 18 (34.0%) 298 (33.8%) 269 (32.4%) 96 (41.6%) 126 (31.5%) 109 (30.4%) 69 (28.3%)
(18.8–36.5) (21.3–46.8) (30.7–36.9) (29.2–35.6) (35.2–47.9) (26.9–35.1) (25.6–31.2) (22.7–33.9)
.458
.001 .141 < .001
.572 .012 .649
Note: Optimum ¼ type A embryo; good quality ¼ type B embryo. Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
authors also have reported the efficiency of this system with the oocytes of infertile patients (34, 35) and in randomized trials comparing the efficiency of slow cooling versus vitrification at early cleavage stages (36, 37). The high efficiency of this technology has also been proved in the vitrification of blastocysts following trophoectoderm biopsy (38). The present study, involving more than 6,000 warmed embryos, confirms the scope of the Cryotop system at all developmental stages. The overall survival rate for early cleavage-stage embryos was slightly lower than that observed for blastocyst-stage embryos, although no impact on delivery or live birth rates was detected. After warming of almost 4,000 early cleavage embryos, a high number (95%) exhibited 100% intact blastomeres after vitrification. Importantly, the practical nonexistence of partially damaged embryos after vitrification represents a significant improvement in our cryopreservation program, which has contributed to a significant increase in the yield of stimulation cycles and cumulative rates. Other authors have evaluated the open vitrification of early-stage embryos, showing survival rate varying from 70% to 95% according to the type of device used (36, 37, 39, 40). Similarly to what happened to early stages, our overall blastocyst survival rate involving almost 2,000 blastocysts was considerably high. Other authors have published similar outcomes using the Cryotop system (38, 41–44). In the past, other devices have been used for blastocyst vitrification. For example, Balaban et al. reported 95% survival (234
blastocysts warmed) with the use of the Cryoloop, and a larger study of 1,129 vitrified blastocysts reported a 85.7% survival rate (45). Similar outcomes were published by others involving 250–700 embryos (46–48). Studies of the use of electron microscope (EM) grid, reporting data from 90–300 warmed blastocysts, have shown survival rates of 50% (49, 50). Open devices for vitrification were used in all of the above-mentioned studies with varying success depending on the device used. Others have addressed the usefulness of closed systems. One study of 839 warmed blastocysts showed a survival rate of 86% with the use of the high security vitrification system (HSVS) (51), and a larger study (1,185 blastocysts) reported an overall survival rate of 77% (52). Our data, based on one of the largest series published so far, indicate that the Cryotop method yields one of the highest outcomes among all the devices available. In the present study, statistical differences in age between patients undergoing early cleavage-stage ET and those undergoing blastocyst transfer, may be due to the latter group being made up mainly of recipients of donor oocytes. In addition, some differences have been found when assessing the survival rate according to embryo developmental stage. Nonetheless, some clarifications need to be made when assessing these data. Survival rates would appear to be higher among optimum (type A) day 3 and day 6 embryos (Table 2). However, these differences could be anecdotal, because the linear regression model revealed that the developmental stage of the embryo had no impact on the delivery rate per warming cycle. This, in turn, indicates that slight differences in the survival
TABLE 3 Effect of blastocyst development on survival and live birth rates. Survival rate Day 5 blastocysts (n ¼ 1,079 warmed embryos) Early blastocyst 432/458 (94.3%) Expanded blastocyst 537/549 (97.9%) Hatching-hatched blastocyst 64/72 (88.8%) Day 6 blastocyst (n ¼ 952 warmed embryos) Early blastocyst 46/47 (97.9%) Expanded blastocyst 556/570 (97.5%) Hatching-hatched blastocyst 327/335 (95.5%) a,b
95% CI (%)
P value
LBR/warming cycle
95% CI (%)
P value
(91.7–96.7) (96.7–99.0)a (81.5–96.6)b
.039
92/285 (32.3%) 155/349 (44.4%) 14/41 (34.1%)
(26.7–37.7) (39.2–49.6)a (19.6–48.6)
.021
(93.8–100) (95.8–98.8) (93.0–96.9)
.107
6/35 (17.1%) 100/292 (34.2%) 96/276 (34.7%)
(4.6–29.6)b (28.8–39.6) (29.1–40.3)
.201
Different superscripts in the same column indicate statistical difference (P< .05).
Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION rates could be neutralized by the high implantation potential inherent to good quality embryos which happen to be intact after vitrification. The absence of variability in the before and after morphologic evaluation of early cleavage stages is another indicator of the safety of the cryopreservation method we employ. The high rate of reexpansion observed for blastocyst-stage embryos and their morphologic appearance after vitrification are also evidence of the lack of damage caused during vitrification. However, it should be pointed out that we observed some differences in survival rates related to blastocyst developmental stage. According to our data, expanded blastocysts on day 5 or day 6 achieved the highest survival rate. This finding contrasts with reports by other groups (52, 53) suggesting that the likelihood of ice formation is higher in expanded blastocysts than in early blastocysts, in which the volume of the blastocoel cavity is much smaller. In fact, this is the rationale for provoking an artificial collapse of blastocysts before vitrification in some cases (53). This is not a strategy we apply routinely, except in isolated cases of hatching blastocysts. It is possible that the high survival rate of our expanded blastocysts was due to the open system we use (Cryotop), because it ensures a very high cooling rate and, most importantly, an extremely high warming rate, which impedes the formation of ice (recrystallization) during warming (18). The warming rates provided by other closed systems may be insufficient. Indeed, the survival rate achieved by expanded blastocysts in the present study, 97.9% at day 5 and 97.7% at day 6, which exceeds that reported in other studies, e.g., 75% (52), supports this theory. Obviously, an extremely high survival rate is an advantage in that the great majority of warming cycles result in embryo replacement, which is why our rate of transfer cancellation was quite low (3%). The implementation of vitrification represented a breakthrough in our center owing to the significant decrease from the previous cancellation rate (30%; data not shown). The statistical differences presented in Table 1, which relate to the number of ETs performed after vitrification at different embryo developmental stages, require careful interpretation, because not all of the embryos vitrified on day 2 or day 3 were subsequently transferred to the uterus at the same stage. Some of them were subjected to further culture and were transferred at a more advanced developmental stage (day 3 or blastocyst). The process of natural selection of embryos during culture may explain the differences in the final count of warming cycles that ended in ET. Additionally it is worth mentioning that despite similar survival rates after warming, lower implantation rates observed on ETs coming from vitrified day 2 embryos show that the vitrification procedures might have a negative effect on this particular stage of development. The relative poor prognosis of day 2 embryos may be related to the embryonic genome activation, which starts at the end of day 2 or beginning of day 3 (54), or to other aspects, such as the sudden interruption by the cryopreservation procedure of the embryonic metabolic activity (measured by oxygen uptake) of day 2 embryos, which is superior compared to day 3 (55). Another explanation might lie with the presence of blastomeres in M-phase during early stages. The presence of the mitotic appa1144
ratus during the vitrification procedure may disturb spindle structures and chromosome segregation, and therefore induce chromosomal abnormalities during the forthcoming divisions, making the embryos less likely to implant. Day 3 embryos also present blastomeres in M-phase; however, the impact on implantation potential could be less. According to the linear regression model, donated ovum/ own oocyte warming cycles, endometrial preparation, and embryo developmental stage at vitrification had no impact on DR, thus confirming that the only variable related to final outcome is related to the quality of the embryos vitrified. Consequently, we have found some differences in survival rates (SR) and LBRs according to embryo quality (type A or B) and the specific stage of development of the blastocyst on day 5, as shown on Table 3. Accordingly, the highest DRSR was achieved by type A day 5 blastocyts and the highest LBR by the group of day 5 expanded blastocysts, which could have been due to the high implantation rate associated with these embryos (56). Lower-quality blastocysts yielded lower clinical outcomes, although their survival rate was high, thereby indicating that vitrification does not affect the potential of embryos to implant and end in live births. Despite their impressive performance, open systems are cause for concern owing to the hypothetical risk of cross-contamination, although no case of infection through LN has ever been documented in ART (57, 58). In this context, the very low risk of virus transmission in seropositive patients has recently been demonstrated (58). Notwithstanding, measures can be taken to improve the tool's safety, such as sterilization of LN (59, 60) and storage in vapor-phase systems (57) or a closed system. In conclusion, highly successful cryopreservation of embryos at all developmental stages is possible with the Cryotop system, because it yields high survival rates and a delivery rate per warming cycle similar to those achieved with fresh embryos. Although subtle differences have been identified regarding the quality and developmental stage of vitrified embryos, there do not seem to be any variables that clearly exert a negative effect on survival and delivery rates.
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SUPPLEMENTAL TABLE 1 Embryo development after warming. Vitrification day 2/embryo transfer day 2
Vitrification day 2/embryo transfer day 3
95% CI N warming cycles N embryo transfers IR/warming cycle CPR/warming cycle OPR/warming cycle DR/warming cycle LBR/warming cycle
11 11 (100) 8/18 (44.4) 6 ( 54.5) 5 (45.5) 5 (45.5) 5 (45.5)
(21.5–67.3) (24.5–83.9) (16.7–74.9) (16.7–74.9) (16.7–74.9)
95% CI 83 83 (100) 46/176 (26.1) 35 (42.2) 25 (24.1) 25 (24.1) 30 (36.1)
Vitrification day 3/embryo transfer day 3
(19.6–32.5) (24.7–45.3) (14.8–33.2) (14.8–33.2) (20.1–39.9)
a
1,525 1,520 (99.7)a 875/2,783 (31.4) 640 (41.9) 515 (33.8) 515 (33.8) 614 (40.2)
(99.4–99.9) (29.6–33.1) (39.4–44.4) (31.4–36.2) (31.4–36.2) (37.7–42.7)
95% CI 53 42 (79.2) 22/86 (25.5) 18 (33.9) 14 (26.4) 14 (26.4) 16 (30.2)
(28.7–55.3) (16.3–34.7) (21.2–46.6) (14.5–38.3) (14.5–38.3) (17.8–42.5)
Vitrification day 3/embryo transfer blastocyst
95% CI N warming cycles N embryo transfers IR/warming cycle CPR/warming cycle OPR/warming cycle DR/warming cycle LBR/warming cycle
Vitrification day 2/embryo transfer blastocyst
95% CI 200 189 (94.5)a 102/274 (37.2) 82 (41.0) 75 (37.5) 73 (36.5) 86 (43.0)
(90.8–97.4) (31.5–42.9) (34.2–47.8) (30.1–44.0) (29.8–43.2) (36.1–49.8)
P< .05.
Cobo. Clinical outcomes of vitrified embryos. Fertil Steril 2012.
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