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Utilization of high-security straws for embryo freezing in an in vitro fertilization program: a prospective, randomized study
TECHNIQUES AND INSTRUMENTATION Utilization of high-security straws for embryo freezing in an in vitro fertilization program: a prospective, randomized study Basak Balaban, B.Sc., Kayhan Yakin, M.D., Aycan Isiklar, M.Sc., and Bulent Urman, M.D. Assisted Reproduction Unit, Vehbi Koc Vakfi American Hospital, Istanbul, Turkey
Objective: To compare the outcome of frozen-thawed ET cycles where embryos had been stored in conventional versus ionomeric resin-based, high-security straws (HSSs). Design: Prospective, randomized study. Setting: Private assisted-reproduction unit. Patient(s): Three hundred and six freeze cycles, and 197 thaw cycles. Intervention(s): Day 3 embryos (n ⫽ 1,268) were frozen, and 517 were thawed using HSSs. Alternately, day 3 embryos (n ⫽ 1,228) were frozen, and 505 were thawed using conventional straws. Main Outcome Measure(s): Cryosurvival, cleavage and morulae formation rates, and clinical pregnancy, implantation, and multiple pregnancy rates. Result(s): Although cycle characteristics did not show any differences, the cryosurvival rate was higher in the HSS group (94.7%) than in the conventional straw group (86%), as was the morulae formation rate (58.7% versus 42.7%). Despite a similar number of embryos being transferred, the clinical pregnancy rate (PR) was higher in the HSS group, but the difference lacked statistical significance (42.5% versus 31.2). Implantation rates (19.4% versus 11.4%) and multiple PRs (41.8% versus 16.6%) were significantly higher in the HSS group than in the conventional straw group. Conclusion(s): High-security straws are high effective in human embryo cryopreservation, because they provide higher cryosurvival and implantation rates, as well as a lower risk of cross-contamination compared to conventional straws. (Fertil Steril威 2007;87:691– 6. ©2007 by American Society for Reproductive Medicine.) Key Words: Human embryo cryopreservation, freezing, thawing, high-security straw
Assisted reproduction generally results in surplus embryos that can be cryopreserved for later use. A cryopreservation program will undoubtedly increase the cumulative conception rates attained in IVF and intracytoplasmic sperm injection (ICSI). A cryopreservation program has been performed in our center since its legalization in Turkey in 1998. Since then, cleavage-stage embryos have been frozen and stored in conventional straws within liquid nitrogen tanks. For cryopreservation purposes, samples from numerous couples are preserved in the same tank for a long period of time. This situation raises the concern of cross-contamination of cells or tissues with viral pathogens. By 2000, a different type of straw (high-security) was introduced into the market to prevent any risk of cross-contamination of frozen embryos or spermatozoa in the tank (1). High-security straws (HSSs) are composed of a different material than conventional straws. Many reports demonstrated the benefits
Received January 17, 2006; revised and accepted July 20, 2006. Reprint requests: Kayhan Yakin, M.D., Assisted Reproduction Unit, Vehbi Koc Vakfi American Hospital, Guzelbahce Sokak no. 20, Nisantasi 34365, Istanbul, Turkey (FAX: 90-212-3112339; E-mail: [email protected]).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.07.1504
of these new straws against hepatitis C virus (HCV) or HIV contamination (1–3). In addition to their different composition, there are differences in size, and in plugging and sealing properties, between HSSs and conventional straws. Although many studies were published showing the efficacy of HSSs against viral contamination, there are limited data showing the effect of HSSs on the clinical outcome of frozen-thawed ET cycles. This prospective, randomized study was designed to compare the outcome of frozen-thawed ET cycles where embryos had been stored in conventional straws versus HSSs. MATERIALS AND METHODS Study Design The study group consisted of 396 freeze cycles and 197 thaw cycles which had been performed between March 2004 – May 2005. During 14 months of the study period, 1,448 fresh cycles were performed, and the cryopreservation rate was 27.3%. Testicular sperm extraction and percutaneous epididymal aspiration cases were excluded from the study group. Good-quality supernumerary embryos were frozen on day 3 of cleavage. These 396 freeze cycles were randomly
Fertility and Sterility姞 Vol. 87, No. 3, March 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
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divided into two groups, according to a computer-generated randomization list: 198 freeze cycles were assigned to the HSS group, and 198 freeze cycles to the conventional straws group. The randomization sequence was concealed from researchers and patients until freezing had been assigned. Approval was obtained from the Institutional Review Board of the American Hospital, Istanbul, Turkey. In total, 1,268 day 3 embryos were cryopreserved in HSSs, and 1,228 in conventional straws. Thawing was performed for 101 cycles (51%) in the HSS group, and for 96 cycles (48.4%) in the conventional straw group. Five hundred and seventeen embryos were thawed from HSSs, and 505 embryos were thawed from conventional straws. Laboratory environment, culture media, stimulation protocols, and ET policy were unchanged during the study period. The outcomes of fresh cycles performed in the HSS group were as follows: 92 cycles (91.0%) failed to achieve pregnancy, whereas out of nine cycles with positive pregnancy results, two ended with biochemical abortions, and seven with clinical abortions. Out of 96 thawing cycles in the conventional straw group, 86 (89.5%) fresh ET cycles failed, whereas three had biochemical abortions, and seven had clinical abortions. Hence, there were no ongoing pregnancies following fresh ET cycles in both study groups. Embryo-Freezing With Conventional Straws A method modified from that of Testart et al. (4) was used for early embryo freezing, whereby 1,2 propanediol (PrOH) was used as a permeating cryoprotectant, and sucrose was used as a nonpermeating cryoprotectant. A phosphate-buffered solution was also used, so that all steps could be performed outside the incubator at ambient temperature. Freeze Kit-1 (refrence no. 10012; Vitrolife, Gothenburg, Sweden) was used throughout the freezing procedure. Only good-quality embryos with equal, homogeneous blastomeres and ⬍20% fragmentation (grades 1–2), which reached at least cell stage 5 on day 3, were cryopreserved (5). All solutions used for the procedure should be equilibrated to ambient temperature before usage. Embryos were first rinsed for approximately 2 minutes in Cryo-PBS (Vitrolife), a phosphate buffer solution with 25 mg/mL human serum albumin (HSA). They were then gently replaced into Freezing Solution-1 (FS1) (1.5 M PrOH in Cryo-PBS) for 10 minutes. The cells of the embryo were shrunken and then reequilibrated in this solution. The embryos were moved across to FS2 (1.5 M PrOH ⫹ 0.1 M sucrose in Cryo-PBS) and loaded into straws (reference no. 014103; Cryo Bio System Groupe, I.M.V. Technologies, Normandie, France) by attaching each straw to a 1-mL syringe, which was connected to the straw by 1-cm silastic tubing. Straws were rinsed with the storage medium before loading, to remove any traces of fiber or powder constituents of the plug. Straws were then attached 692
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to a sterile plug (reference no. 007442; Cryo Bio System Groupe, I.M.V. Technologies) to avoid leaking inside the straw and into the liquid nitrogen (LN2) tank during storage. A maximum of two embryos was placed in each straw. Straws were then placed into the freezing chamber at ambient temperature, and the program began. Planer Kryo 10 Series III (Planer Products Ltd., Sunbury on Thames, United Kingdom) was used for cryopreservation. The freezing program used was as follows: Starting temperature: 18 –25°C. Step 1: ⫺2.0°C/min to ⫺7.0°C. Step 2: Hold at ⫺7.0°C for 10 minutes. Seed after 2 minutes. Straws were manually seeded at ⫺7.0°C with LN2-cooled forceps close to the cotton plug. Step 3: ⫺0.3 C°/min to ⫺30.0°C. Step 4: ⫺30.0°C to below ⫺80.0°C (at least 10°C/min). Straws were then removed and plunged into the LN2 storage tank immediately. Embryo-Freezing With High-Security Straws All procedures were the same as for freezing with conventional straws, until embryos were loaded into CBS HSSs (reference no. 010286; Cryo Bio System Groupe, I.M.V. Technologies). The straw was attached to a 1-mL syringe which was connected to the straw by 1-cm silastic tubing. Loading of the straw was as follows: a medium column of 0.02 mL was aspirated first, then an air bubble of 0.03 mL (approximately 7 mm), medium of 0.05 mL (10 mm) with the embryos (embryos aspirated at the last point), air bubble of 0.03 mL, medium of 0.02 mL, and finally an air bubble of approximately 0.02 mL were aspirated. The seeding procedure was performed on the medium column where the embryos were placed, but on the opposite side from where the embryos were aspirated. After the embryos were loaded, straws were heat-sealed on both ends with the SYMS unit especially designed by the Cryo Bio System Group (CBS) for this purpose. A maximum of two embryos was placed in each straw. Straws were then placed into the freezing chamber at ambient temperature, and the program began. Planer Kryo 10 Series III (Planer Products Ltd.) was used for cryopreservation. The freezing program and tank storage were the same as for conventional straws. Embryo-Thawing in Conventional and High-Security Straws Straws were thawed one at a time, and all steps were performed at ambient temperature. Thaw-kit 1 (reference no. 10013; Vitrolife) was used for the thawing procedure. All solutions were preequilibrated to ambient temperature before use. The manual for recommended use of Vitrolife Fertility Systems was followed throughout the procedure. Each straw was removed from the LN2 tank and airthawed for 30 seconds. During this time, the straws were handled carefully and examined for air bubbles, cracks on
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TABLE 1 Comparison of laboratory parameters between groups using either high-security or conventional straws. Laboratory parameters Freezing cycles Mean female age (y) ⫾ SD Embryos frozen (mean ⫾ SD) Eight-cell embryos frozen (%) Thaw cycles (%) Embryos thawed (%) Cryosurvival (%) Morulae on day 4 (%)
HSSs
Conventional straws
P value
198 31.8 ⫾ 3.6 1,268 (6.4 ⫾ 1.8) 446 (35.1) 101 (51) 517 (5.1) 490 (94.7) 288 (58.7)
198 32.1 ⫾ 3.3 1,228 (6.2 ⫾ 1.7) 455 (37.0) 96 (48.4) 505 (5.2) 430 (86) 184 (42.7)
NS NS NS NS NS ⬍.001 ⬍.001
Note: NS ⫽ no significance. Balaban. Embryo cryopreservation in high-security straws. Fertil Steril 2007.
the plug, and any leakage of LN2. Each straw was then placed in a 30°C water bath for 30 seconds. After the straw was removed from the bath, it was carefully wiped, and the plug at the end of the straw was cut with sterile scissors and attached to a 1-mL syringe. The other end was cut carefully without shaking the straw or making any air bubbles. The presence of embryos was checked under a stereo microscope, and they were immediately expelled into Thawing Solution-1 (TS1) (1.0 M PrOH ⫹ 0.2 M sucrose in CryoPBS) for an incubation of 5 minutes. Embryos were transferred to TS2 (0.5 M PrOH ⫹ 0.2 M sucrose in Cryo-PBS) and incubated for a further 5 minutes. They were then transferred to TS3 (0.2 M sucrose in Cryo-PBS) for 5–10 minutes. Embryos were placed in Cryo-PBS at ambient temperature for 5–10 minutes. They were rinsed in preequilibrated and supplemented G2 culture media (GIII series; Vitrolife) a few times, and transferred individually to a preequilibrated fresh culture dish containing 10 L of supplemented G2 culture media covered with oil. The embryo-thawing procedure was performed 24 hours before the transfer to examine cleavage. Assessment of cryosurvival was performed as described by Rienzi et al (6). Frozen-thawed embryos were considered to have survived if ⱖ50% of the blastomeres were intact, or had at least three viable cells present at thawing and showed at least one blastomere divided by 18 hours of postthaw culture. Embryo Transfer To prepare the endometrium for transfer, patients received increasing doses of E2 valerate tablets following downregulation with a GnRH agonist. When the endometrium reached or exceeded 8 mm and a triple-line echo was evident, adminsitration of P (Crinone vaginal gel; Serono, Geneva, Switzerland) began, and the patient was asked to come for ET on the morning of day 4. Embryos were thawed on day 3, and were kept in culture overnight. Embryos that cleaved or reached the morula stage on day 4 were selected for transfer. Fertility and Sterility姞
Laser-assisted hatching was performed prior to transfer. A 1,480-nm diode laser in a computer-controlled noncontact mode was used for laser hatching (IVF Workstation and Zona Laser Treatment System; Hamilton Thorne Instruments, Beverly, MA). Embryo transfer was performed under ultrasound guidance with the use of Wallace (Sims Portex Ltd., Hythe, Kent, UK) or Frydman (Laboratorie CCD, Paris, France) catheters, with the catheter of choice determined in a trial transfer. Cleavage-stage embryos were graded according to Hardarson et al. (7). Pregnancy was confirmed by measuring -human chorionic gonadotropin (hCG) 12 days after day 3, and 10 days after blastocyst transfers. Clinical pregnancy was defined as the presence of a gestational sac(s), documented by vaginal ultrasonography 2 weeks after a positive pregnancy test. Viable excess thawed embryos were discarded with the consent of the couples. Refreezing of cryothawed embryos is not performed at our center. Statistics Normal distribution of data was verified prior to selecting statistical tests. Numerical variables were analyzed using a paired Student’s t-test. Categorical variables were analyzed using 2 and Fisher’s exact tests when applicable. P⬍.05 was accepted as significant. RESULTS The characteristics of 396 cryopreservation cycles were as follows. In the HSS group, the main cause of indication for treatment was male-factor infertility (135 cycles; 68.1%). Other indications were unexplained infertility (10 cycles; 5%) and combined causes (53 cycles; 26.7%). In the conventional straw group, the numbers and percentages of cycles according to causes of infertility were male factor infertility (138 cycles; 69.6%), unexplained infertility (6 693
TABLE 2 Comparison of clinical outcome between groups using either high-security or conventional straws. Clinical parameters
HSSs
Embryos transferred (mean ⫾ SD) Clinical pregnancies/ET (%) Implantation rate Multiple pregnancies (%) Abortions (%)
313 (3.09 ⫾ 0.6) 43/101 (42.5) 61/313 (19.4) 18 twins (41.8) 4 (9.3)
Conventional straws
P value
307 (3.19 ⫾ 0.6) 30/96 (31.2) 36/307 (11.4) 5 twins (16.6) 3 (10)
NS NS ⬍.05 ⬍.05 NS
Note: NS ⫽ no significance. Balaban. Embryo cryopreservation in high-security straws. Fertil Steril 2007.
cycles; 3%), and combined causes (54 cycles; 27.2%). Therefore, the HSS and conventional straw groups were similar in terms of causes and duration of infertility. The mean duration of infertility was 7.2 years for the HSS group, and 7.4 years for the conventional straw group. Cycle characteristics did not show any statistical difference between the two groups (P⬎.05). Laboratory parameters of frozen and thawed embryos are summarized in Table 1. There was no significant difference between the two groups in terms of mean female age. A mean number of 6.4 embryos were frozen in the HSS group. The mean number was 6.2 for the conventional straw group. Embryo quality was similar in both groups. There was no statistically significant difference between groups in terms of the number and rate of eight-cell embryos that were frozen (P⬎.05). Thawing was performed in 51% of HSS cycles, and in 48.4% of conventional straw frozen cycles. In the HSS group, of the 1,268 frozen embryos, 517 (40.8%) were thawed. In the conventional straw group, of the 1,228 frozen embryos, 505 (41.1%) were thawed. There was no statistically significant difference between the two groups in terms of mean number of thawed embryos (5.1 versus 5.2, respectively; P⬎.05). However, the cryosurvival rate was higher in the HSS group than in the conventional straw group (94.7% versus 86%, respectively; P⬍.001). Also, the rate of morula formation after thawing was significantly higher in the HSS group (58.7% versus 42.7%; P⬍.001). A comparison of clinical outcomes between the two groups is presented in Table 2. The mean numbers of transferred embryos for the HSS and conventional straw groups were 3.1 and 3.2, respectively (P⬎.05). Despite a similar number of embryos being transferred, clinical pregnancy rates (PRs) were higher in the HSS group (42.5% versus 31.2%), but the difference lacked statistical significance (P⬎.05). However, implantation rates (19.4% versus 11.4%; P⬍.05) and multiple PRs (41.8% versus 16.6%; P⬍.05) were significantly higher in the HSS group. There was no significant difference between groups in terms of miscarriages (9.3% versus 10%; P⬎.05). 694
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DISCUSSION In a group of patients with similar cycle characteristics, the use of HSSs was associated with significantly higher cryosurvival, embryo viability, and implantation rates. Although the clinical PR was also higher, this did not reach statistical significance. Several points need to be discussed, to explain the differences when embryos are frozen using HSSs. Different factors may affect the outcome of human embryo cryopreservation. These factors can be divided into four groups: clinical, technical, embryological, and genetic. Embryo quality and the outcome of the fresh cycle are the leading factors affecting the efficiency of cryopreservation (8 –11). When good-quality embryos are frozen and thawed, higher implantation rates are obtained (5,12). The etiology of infertility, the presence of a leading high-quality embryo, the developmental stage of frozen embryos, the number of intact blastomeres after thawing, and their further cleavage potential are other factors that may affect the outcome of frozenthawed ETs (11). Female age will also be an important clinical and genetic factor (5,11,13). The limited number of oocytes and embryos developed in women of advanced age, and the chromosomal errors in the resultant embryos, will undoubtedly affect the outcome. Besides these clinical, embryological, and genetic factors, technical factors such as freezing protocols (slow freezing or vitrification), fertilization by IVF or ICSI, and the experience of the laboratory personnel may all affect the outcome (14 –20). Changes in embryo culture and cryopreservation medium may have a role in explaining the difference between the two groups. However, in this prospective study, the same media were used in both groups. Differences in the quality of embryos that were frozen may also be an issue, if better embryos were cryopreserved in one particular group, which would inevitably lead to a better clinical outcome. However, neither the mean number of embryos nor the embryo quality on day 3 was different between the two groups. Before 2003, conventional straws were used for storing the embryos in our program. These were high-quality, nontoxic straws with a thickness of 0.17 mm and an inner diameter of 1.55 mm (reference no. 014103; Cryo Bio Sys-
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TABLE 3 Technical differences in straws used for cryopreservation. Technical properties
Conventional straws
HSSs No plasticizing agent (ionomeric resin) 0.33 mm 2.55 mm (lower flexibility) 0.3 mL 133 mm Hydrophobic safety plug No need for rinsing
Tip properties
Plasticizing agent 0.17 mm 1.55 mm 0.3 mL 130 mm Cotton plug Preliminary rinsing with medium is necessary to avoid any risk of toxicity –
Sealing properties
With sterile plug
Material Thickness Inner diameter Volume Length Sections Rinsing
Disposable transparent filling nozzle avoids contamination of sealable end Heat-sealing with SYMS unit (no risk of leakage during storage)
Balaban. Embryo cryopreservation in high-security straws. Fertil Steril 2007.
tem Group, I.M.V. Technologies). These should be rinsed with storage medium to flush out any traces of fiber or powder from the sterile plug that is used to avoid leaking (reference no. 007442; Cryo Bio System Group, I.M.V. Technologies). Since the beginning of 2003, CBS HSSs (reference no. 010286; Cryo Bio System Group, I.M.V. Technologies) have been used for embryo storage. Although the material of the previously used straws contained some plasticizing agents, the CBS HSSs are made of ionomeric resin, improving the safety of the straws with respect to the biological materials that they may harbor. Because the flexibility of the material is lower than that of the previously used straws, they are thicker (0.32 mm), with a larger inner diameter (2.55 mm), resulting in higher rigidity and mechanical resistance. These straws are composed of two distinct sections, separated by a unique hydrophobic safety plug that guarantees sterility of the embryo when placing it into the straw and when removing it after thawing. This hydrophobic safety plug was designed for storage in straws without the risk of losing biological material by absorption. The use of this plug in the cryopreservation of embryos also eliminates the need for preliminary rinsing of the straw with the storage medium. Although loading of the embryos in these straws is almost the same as with the previously used straws, the physical properties and the method of filling and sealing are specially designed to ensure a leakproof container for the specimen, even when immersed in liquid nitrogen. After the loading of the embryos, these straws are heat-sealed on both ends with the SYMS unit specially designed by CBS for this purpose, instead of closing the tip of the straw with a sterile plug, as described in the previous system. The other main difference in these HSSs involves the embryo being loaded into the straw by using a disposable transparent filling nozzle already mounted on the straw, hence avoiding contamination of the sealable end. Fertility and Sterility姞
The differences between the two kinds of straws are summarized in Table 3. To date, conventional straws were reported to be effective only for protection of cryopreserved samples against crosscontamination. There are very limited data in the literature comparing the efficacy of HSSs to conventional straws in terms of clinical outcome. The manufacturer’s manual reported that, in comparison to conventional straws, no significant differences were found in rabbit embryo development or sheep embryo survival rates and PRs (21). Another study compared conventional straws and HSSs in terms of cryosurvival of triploid and diploid human zygotes (22). Cryosurvival rates were found to be higher using HSSs. The only well-designed study which compared HSSs with conventional straws were in an animal study by Walker et al. in 2003 (23). This was a prospective comparison of blastocyst development rates in 278 murine embryos after refreezing and thawing at the two-cell stage against the standard Instruments-Medicine-Veterinarian (IMV) straws used in their cryopreservation program. When the manufacturer’s filling and loading protocol was used for the CBS straw, there was no significant difference in the blastocyst development rate between CBS (75.0%) and IMV (76.4%) straws. An interesting finding was the dramatic effect of filling and loading procedures of HSSs. When only the conventional straw was replaced with the HSS without changing the filling and loading protocols, the authors failed to reproduce their previous success. Only after adopting the manufacturer’s filling and loading instructions for HSSs, and freezing fewer embryos per straw, were the results achieved between the two straws nearly identical. This finding was attributed to the difference in overall dimensions of the two straw types (23). 695
It is not clear how HSSs provide higher implantation rates in cryopreservation cycles. Possible mechanisms can be suggested for the improvement in cryosurvival and implantation rates. First of all, the difference may arise from the freezing and thawing dynamics of the medium within the straws, because of physical differences such as different volumes and surface areas. If this is so, a better preservation of embryo viability by better preserving the properties of the cryo-thaw media could affect cryosurvival rates. Another reason may be a difference in the seeding characteristics of the straws. It is possible that a more effective seeding can be achieved in HSSs. In addition, the ionomeric-resin composition could be a better conductor for the formation of ice crystals. They may provide better temperature control during freezing. Better preservation of embryo viability through maintenance of the temperature of the cryomedia in which the embryos are placed may be responsible for the improvement of cryosurvival and implantation rates. The loading technique for both types of straws was not different. The only difference in loading could be the amount of medium aspirated in each column, because of differences in the thickness of straws. More studies comparing the physical and technical properties of these two different types of straws are needed, to clarify the mechanism(s) underlying the better cryosurvival and implantation rates. Cryopreservation offers clear benefits in the efficiency of assisted reproductive techniques. Parallel to the improvement in in vitro culture conditions and implantation rates, the number of embryos transferred in utero can be safely limited, to avoid multiple pregnancies. Many of the surplus embryos generated through assisted reproduction can be cryopreserved. Thus, cryopreservation will lead to an increase in cumulative PRs with a single cycle of ovarian stimulation and assisted reproduction (24). In addition to prevention of cross-contamination, HSSs provide improved outcomes in frozen-thawed ET cycles in terms of higher implantation rates. Taking into account the improved implantation rate, as well as the higher number of multiple pregnancies, the number of transferred embryos in cryo-thawed cycles can be safely reduced. In conclusion, given the advantages of higher cryosurvival and implantation rates, as well as the lower risk of crosscontamination of cryopreserved tissues, the use of HSSs is preferred in frozen-thawed embryo cryopreservation cycles. REFERENCES 1. Benifla JL, Letur-Konirsch H, Collin G, Devaux A, Kuttenn F, Madelenat P, et al. Safety of cryopreservation straws for human gametes or embryos: a preliminary study with human immunodeficiency virus-1. Hum Reprod 2000;15:2186 –9. 2. Letur-Konirsch H, Collin G, Sifer C, Devaux A, Kuttenn F, Madelenat P, et al. Safety of cryopreservation straws for human gametes or embryos: a study with human immunodeficiency virus-1 under cryopreservation conditions. Hum Reprod 2003;18:140 – 4. 3. Maertens A, Bourlet T, Plotton N, Pozzetto B, Levy R. Validation of safety procedures for the cryopreservation of semen contaminated with hepatitis C virus in assisted reproductive technology. Hum Reprod 2004;19:1554 –7.
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4. Testart J, Laselle B, Belaissch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268 –72. 5. Karlstrom PO, Bergh T, Forsberg AS, Sandkvist V, Wikland M. Prognostic factors for the success rate of embryo freezing. Hum Reprod 1997;12:1263– 6. 6. Rienzi L, Nagy ZP, Ubaldi P, Iacobelli M, Anniballo R, Tesarik J, et al. Laser-assisted removal of necrotic blastomeres from the cryopreserved embryos that were partially damaged. Fertil Steril 2002;77:1196 –201. 7. Hardarson T, Hanson C, Sjögren A, Lundin K. Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation. Hum Reprod 2001;16:313– 8. 8. Toner JP, Veeck LL, Acosta AA, Muasher SJ. Predictive value of pregnancy during original in vitro fertilization cycle on implantation and pregnancy in subsequent cryothaw cycles. Fertil Steril 1991;56: 505– 8. 9. Schalkoff ME, Oskowitz SP, Powers RD. A multifactorial analysis of the pregnancy outcome in a successful embryo cryopreservation program. Fertil Steril 1993;59:1070 – 4. 10. Lin YP, Cassidenti DL, Chacon RR, Soubra SS, Rosen GF, Yee B. Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they are derived. Fertil Steril 1995; 63:262–7. 11. Wang JX, Yap YY, Matthews CD. Frozen-thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Hum Reprod 2001;16:2316 –9. 12. Behr B, Gebhardt J, Lyon J, Milki AA. Factors relating to a successful cryopreserved blastocyst transfer program. Fertil Steril 2002;77:697–9. 13. Damario MA, Hammitt DG, Session DR, Dumesic DA. Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil Steril 2000;73:767–73. 14. Macas E, Imthurn B, Borsos M, Rosselli M, Maurer-Major E, Keller PJ. Impairment of the developmental potential of frozen-thawed human zygotes obtained after intracytoplasmic sperm injection. Fertil Steril 1998;69:630 –5. 15. Damario MA, Hammitt DG, Galanits TM, Session DR, Dumesic DA. Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Fertil Steril 1999;72:1049 –54. 16. Oehninger S, Mayer J, Muasher S. Impact of different clinical variables on pregnancy outcome following embryo cryopreservation. Mol Cell Endocrinol 2000;27:73–7. 17. Senn A, Vozzi C, Chanson A, De Grandi P, Germond M. Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage. Fertil Steril 2000;74:946 –52. 18. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002;78:449 –54. 19. Veeck LL. Does the developmental stage at freeze impact on clinical results post-thaw? Reprod Biomed Online 2003;6:367–74. 20. Boone WR, Crane IV MM, Johnson JE, Higdon HL III, Blackhurst DW. Changes in the freezing protocol for human zygotes alter embryonic development and pregnancy rates. Fertil Steril 2005;83:182– 8. 21. Cryo Bio System ITG. CBS brochure and instruction manual. L’Aigle, France: IMV Technologies Group, 2001. 22. Schiewe MC, Hubert G, Buyalos R. Human zygote cryopreservation using CBS™cryobiostraws: a clinical trial. Fertil Steril 2002;77(Suppl 3):22. 23. Walker DL, Hammitt DG, Dumesic PA, Thornhill AR. Equivalent blastocyst rates after freezing murine embryos in Cryo Bio System high security or standard Instruments-Medicine-Veterinarian straws. Fertil Steril 2003;80(Suppl 2):743– 6. 24. Van Voorhis BJ, Syrop CH, Allen BD, Sparks AE, Stovall DW. The efficacy and cost effectiveness of embryo cryopreservation compared with other assisted reproductive techniques. Fertil Steril 1995;64: 647–50.
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Objective: To compare the outcome of frozen-thawed ET cycles where embryos had been stored in conventional versus ionomeric resin-based, high-security straws (HSSs). Design: Prospective, randomized study. Setting: Private assisted-reproduction unit. Patient(s): Three hundred and six freeze cycles, and 197 thaw cycles. Intervention(s): Day 3 embryos (n ⫽ 1,268) were frozen, and 517 were thawed using HSSs. Alternately, day 3 embryos (n ⫽ 1,228) were frozen, and 505 were thawed using conventional straws. Main Outcome Measure(s): Cryosurvival, cleavage and morulae formation rates, and clinical pregnancy, implantation, and multiple pregnancy rates. Result(s): Although cycle characteristics did not show any differences, the cryosurvival rate was higher in the HSS group (94.7%) than in the conventional straw group (86%), as was the morulae formation rate (58.7% versus 42.7%). Despite a similar number of embryos being transferred, the clinical pregnancy rate (PR) was higher in the HSS group, but the difference lacked statistical significance (42.5% versus 31.2). Implantation rates (19.4% versus 11.4%) and multiple PRs (41.8% versus 16.6%) were significantly higher in the HSS group than in the conventional straw group. Conclusion(s): High-security straws are high effective in human embryo cryopreservation, because they provide higher cryosurvival and implantation rates, as well as a lower risk of cross-contamination compared to conventional straws. (Fertil Steril威 2007;87:691– 6. ©2007 by American Society for Reproductive Medicine.) Key Words: Human embryo cryopreservation, freezing, thawing, high-security straw
Assisted reproduction generally results in surplus embryos that can be cryopreserved for later use. A cryopreservation program will undoubtedly increase the cumulative conception rates attained in IVF and intracytoplasmic sperm injection (ICSI). A cryopreservation program has been performed in our center since its legalization in Turkey in 1998. Since then, cleavage-stage embryos have been frozen and stored in conventional straws within liquid nitrogen tanks. For cryopreservation purposes, samples from numerous couples are preserved in the same tank for a long period of time. This situation raises the concern of cross-contamination of cells or tissues with viral pathogens. By 2000, a different type of straw (high-security) was introduced into the market to prevent any risk of cross-contamination of frozen embryos or spermatozoa in the tank (1). High-security straws (HSSs) are composed of a different material than conventional straws. Many reports demonstrated the benefits
Received January 17, 2006; revised and accepted July 20, 2006. Reprint requests: Kayhan Yakin, M.D., Assisted Reproduction Unit, Vehbi Koc Vakfi American Hospital, Guzelbahce Sokak no. 20, Nisantasi 34365, Istanbul, Turkey (FAX: 90-212-3112339; E-mail: [email protected]).
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.07.1504
of these new straws against hepatitis C virus (HCV) or HIV contamination (1–3). In addition to their different composition, there are differences in size, and in plugging and sealing properties, between HSSs and conventional straws. Although many studies were published showing the efficacy of HSSs against viral contamination, there are limited data showing the effect of HSSs on the clinical outcome of frozen-thawed ET cycles. This prospective, randomized study was designed to compare the outcome of frozen-thawed ET cycles where embryos had been stored in conventional straws versus HSSs. MATERIALS AND METHODS Study Design The study group consisted of 396 freeze cycles and 197 thaw cycles which had been performed between March 2004 – May 2005. During 14 months of the study period, 1,448 fresh cycles were performed, and the cryopreservation rate was 27.3%. Testicular sperm extraction and percutaneous epididymal aspiration cases were excluded from the study group. Good-quality supernumerary embryos were frozen on day 3 of cleavage. These 396 freeze cycles were randomly
Fertility and Sterility姞 Vol. 87, No. 3, March 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
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divided into two groups, according to a computer-generated randomization list: 198 freeze cycles were assigned to the HSS group, and 198 freeze cycles to the conventional straws group. The randomization sequence was concealed from researchers and patients until freezing had been assigned. Approval was obtained from the Institutional Review Board of the American Hospital, Istanbul, Turkey. In total, 1,268 day 3 embryos were cryopreserved in HSSs, and 1,228 in conventional straws. Thawing was performed for 101 cycles (51%) in the HSS group, and for 96 cycles (48.4%) in the conventional straw group. Five hundred and seventeen embryos were thawed from HSSs, and 505 embryos were thawed from conventional straws. Laboratory environment, culture media, stimulation protocols, and ET policy were unchanged during the study period. The outcomes of fresh cycles performed in the HSS group were as follows: 92 cycles (91.0%) failed to achieve pregnancy, whereas out of nine cycles with positive pregnancy results, two ended with biochemical abortions, and seven with clinical abortions. Out of 96 thawing cycles in the conventional straw group, 86 (89.5%) fresh ET cycles failed, whereas three had biochemical abortions, and seven had clinical abortions. Hence, there were no ongoing pregnancies following fresh ET cycles in both study groups. Embryo-Freezing With Conventional Straws A method modified from that of Testart et al. (4) was used for early embryo freezing, whereby 1,2 propanediol (PrOH) was used as a permeating cryoprotectant, and sucrose was used as a nonpermeating cryoprotectant. A phosphate-buffered solution was also used, so that all steps could be performed outside the incubator at ambient temperature. Freeze Kit-1 (refrence no. 10012; Vitrolife, Gothenburg, Sweden) was used throughout the freezing procedure. Only good-quality embryos with equal, homogeneous blastomeres and ⬍20% fragmentation (grades 1–2), which reached at least cell stage 5 on day 3, were cryopreserved (5). All solutions used for the procedure should be equilibrated to ambient temperature before usage. Embryos were first rinsed for approximately 2 minutes in Cryo-PBS (Vitrolife), a phosphate buffer solution with 25 mg/mL human serum albumin (HSA). They were then gently replaced into Freezing Solution-1 (FS1) (1.5 M PrOH in Cryo-PBS) for 10 minutes. The cells of the embryo were shrunken and then reequilibrated in this solution. The embryos were moved across to FS2 (1.5 M PrOH ⫹ 0.1 M sucrose in Cryo-PBS) and loaded into straws (reference no. 014103; Cryo Bio System Groupe, I.M.V. Technologies, Normandie, France) by attaching each straw to a 1-mL syringe, which was connected to the straw by 1-cm silastic tubing. Straws were rinsed with the storage medium before loading, to remove any traces of fiber or powder constituents of the plug. Straws were then attached 692
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to a sterile plug (reference no. 007442; Cryo Bio System Groupe, I.M.V. Technologies) to avoid leaking inside the straw and into the liquid nitrogen (LN2) tank during storage. A maximum of two embryos was placed in each straw. Straws were then placed into the freezing chamber at ambient temperature, and the program began. Planer Kryo 10 Series III (Planer Products Ltd., Sunbury on Thames, United Kingdom) was used for cryopreservation. The freezing program used was as follows: Starting temperature: 18 –25°C. Step 1: ⫺2.0°C/min to ⫺7.0°C. Step 2: Hold at ⫺7.0°C for 10 minutes. Seed after 2 minutes. Straws were manually seeded at ⫺7.0°C with LN2-cooled forceps close to the cotton plug. Step 3: ⫺0.3 C°/min to ⫺30.0°C. Step 4: ⫺30.0°C to below ⫺80.0°C (at least 10°C/min). Straws were then removed and plunged into the LN2 storage tank immediately. Embryo-Freezing With High-Security Straws All procedures were the same as for freezing with conventional straws, until embryos were loaded into CBS HSSs (reference no. 010286; Cryo Bio System Groupe, I.M.V. Technologies). The straw was attached to a 1-mL syringe which was connected to the straw by 1-cm silastic tubing. Loading of the straw was as follows: a medium column of 0.02 mL was aspirated first, then an air bubble of 0.03 mL (approximately 7 mm), medium of 0.05 mL (10 mm) with the embryos (embryos aspirated at the last point), air bubble of 0.03 mL, medium of 0.02 mL, and finally an air bubble of approximately 0.02 mL were aspirated. The seeding procedure was performed on the medium column where the embryos were placed, but on the opposite side from where the embryos were aspirated. After the embryos were loaded, straws were heat-sealed on both ends with the SYMS unit especially designed by the Cryo Bio System Group (CBS) for this purpose. A maximum of two embryos was placed in each straw. Straws were then placed into the freezing chamber at ambient temperature, and the program began. Planer Kryo 10 Series III (Planer Products Ltd.) was used for cryopreservation. The freezing program and tank storage were the same as for conventional straws. Embryo-Thawing in Conventional and High-Security Straws Straws were thawed one at a time, and all steps were performed at ambient temperature. Thaw-kit 1 (reference no. 10013; Vitrolife) was used for the thawing procedure. All solutions were preequilibrated to ambient temperature before use. The manual for recommended use of Vitrolife Fertility Systems was followed throughout the procedure. Each straw was removed from the LN2 tank and airthawed for 30 seconds. During this time, the straws were handled carefully and examined for air bubbles, cracks on
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TABLE 1 Comparison of laboratory parameters between groups using either high-security or conventional straws. Laboratory parameters Freezing cycles Mean female age (y) ⫾ SD Embryos frozen (mean ⫾ SD) Eight-cell embryos frozen (%) Thaw cycles (%) Embryos thawed (%) Cryosurvival (%) Morulae on day 4 (%)
HSSs
Conventional straws
P value
198 31.8 ⫾ 3.6 1,268 (6.4 ⫾ 1.8) 446 (35.1) 101 (51) 517 (5.1) 490 (94.7) 288 (58.7)
198 32.1 ⫾ 3.3 1,228 (6.2 ⫾ 1.7) 455 (37.0) 96 (48.4) 505 (5.2) 430 (86) 184 (42.7)
NS NS NS NS NS ⬍.001 ⬍.001
Note: NS ⫽ no significance. Balaban. Embryo cryopreservation in high-security straws. Fertil Steril 2007.
the plug, and any leakage of LN2. Each straw was then placed in a 30°C water bath for 30 seconds. After the straw was removed from the bath, it was carefully wiped, and the plug at the end of the straw was cut with sterile scissors and attached to a 1-mL syringe. The other end was cut carefully without shaking the straw or making any air bubbles. The presence of embryos was checked under a stereo microscope, and they were immediately expelled into Thawing Solution-1 (TS1) (1.0 M PrOH ⫹ 0.2 M sucrose in CryoPBS) for an incubation of 5 minutes. Embryos were transferred to TS2 (0.5 M PrOH ⫹ 0.2 M sucrose in Cryo-PBS) and incubated for a further 5 minutes. They were then transferred to TS3 (0.2 M sucrose in Cryo-PBS) for 5–10 minutes. Embryos were placed in Cryo-PBS at ambient temperature for 5–10 minutes. They were rinsed in preequilibrated and supplemented G2 culture media (GIII series; Vitrolife) a few times, and transferred individually to a preequilibrated fresh culture dish containing 10 L of supplemented G2 culture media covered with oil. The embryo-thawing procedure was performed 24 hours before the transfer to examine cleavage. Assessment of cryosurvival was performed as described by Rienzi et al (6). Frozen-thawed embryos were considered to have survived if ⱖ50% of the blastomeres were intact, or had at least three viable cells present at thawing and showed at least one blastomere divided by 18 hours of postthaw culture. Embryo Transfer To prepare the endometrium for transfer, patients received increasing doses of E2 valerate tablets following downregulation with a GnRH agonist. When the endometrium reached or exceeded 8 mm and a triple-line echo was evident, adminsitration of P (Crinone vaginal gel; Serono, Geneva, Switzerland) began, and the patient was asked to come for ET on the morning of day 4. Embryos were thawed on day 3, and were kept in culture overnight. Embryos that cleaved or reached the morula stage on day 4 were selected for transfer. Fertility and Sterility姞
Laser-assisted hatching was performed prior to transfer. A 1,480-nm diode laser in a computer-controlled noncontact mode was used for laser hatching (IVF Workstation and Zona Laser Treatment System; Hamilton Thorne Instruments, Beverly, MA). Embryo transfer was performed under ultrasound guidance with the use of Wallace (Sims Portex Ltd., Hythe, Kent, UK) or Frydman (Laboratorie CCD, Paris, France) catheters, with the catheter of choice determined in a trial transfer. Cleavage-stage embryos were graded according to Hardarson et al. (7). Pregnancy was confirmed by measuring -human chorionic gonadotropin (hCG) 12 days after day 3, and 10 days after blastocyst transfers. Clinical pregnancy was defined as the presence of a gestational sac(s), documented by vaginal ultrasonography 2 weeks after a positive pregnancy test. Viable excess thawed embryos were discarded with the consent of the couples. Refreezing of cryothawed embryos is not performed at our center. Statistics Normal distribution of data was verified prior to selecting statistical tests. Numerical variables were analyzed using a paired Student’s t-test. Categorical variables were analyzed using 2 and Fisher’s exact tests when applicable. P⬍.05 was accepted as significant. RESULTS The characteristics of 396 cryopreservation cycles were as follows. In the HSS group, the main cause of indication for treatment was male-factor infertility (135 cycles; 68.1%). Other indications were unexplained infertility (10 cycles; 5%) and combined causes (53 cycles; 26.7%). In the conventional straw group, the numbers and percentages of cycles according to causes of infertility were male factor infertility (138 cycles; 69.6%), unexplained infertility (6 693
TABLE 2 Comparison of clinical outcome between groups using either high-security or conventional straws. Clinical parameters
HSSs
Embryos transferred (mean ⫾ SD) Clinical pregnancies/ET (%) Implantation rate Multiple pregnancies (%) Abortions (%)
313 (3.09 ⫾ 0.6) 43/101 (42.5) 61/313 (19.4) 18 twins (41.8) 4 (9.3)
Conventional straws
P value
307 (3.19 ⫾ 0.6) 30/96 (31.2) 36/307 (11.4) 5 twins (16.6) 3 (10)
NS NS ⬍.05 ⬍.05 NS
Note: NS ⫽ no significance. Balaban. Embryo cryopreservation in high-security straws. Fertil Steril 2007.
cycles; 3%), and combined causes (54 cycles; 27.2%). Therefore, the HSS and conventional straw groups were similar in terms of causes and duration of infertility. The mean duration of infertility was 7.2 years for the HSS group, and 7.4 years for the conventional straw group. Cycle characteristics did not show any statistical difference between the two groups (P⬎.05). Laboratory parameters of frozen and thawed embryos are summarized in Table 1. There was no significant difference between the two groups in terms of mean female age. A mean number of 6.4 embryos were frozen in the HSS group. The mean number was 6.2 for the conventional straw group. Embryo quality was similar in both groups. There was no statistically significant difference between groups in terms of the number and rate of eight-cell embryos that were frozen (P⬎.05). Thawing was performed in 51% of HSS cycles, and in 48.4% of conventional straw frozen cycles. In the HSS group, of the 1,268 frozen embryos, 517 (40.8%) were thawed. In the conventional straw group, of the 1,228 frozen embryos, 505 (41.1%) were thawed. There was no statistically significant difference between the two groups in terms of mean number of thawed embryos (5.1 versus 5.2, respectively; P⬎.05). However, the cryosurvival rate was higher in the HSS group than in the conventional straw group (94.7% versus 86%, respectively; P⬍.001). Also, the rate of morula formation after thawing was significantly higher in the HSS group (58.7% versus 42.7%; P⬍.001). A comparison of clinical outcomes between the two groups is presented in Table 2. The mean numbers of transferred embryos for the HSS and conventional straw groups were 3.1 and 3.2, respectively (P⬎.05). Despite a similar number of embryos being transferred, clinical pregnancy rates (PRs) were higher in the HSS group (42.5% versus 31.2%), but the difference lacked statistical significance (P⬎.05). However, implantation rates (19.4% versus 11.4%; P⬍.05) and multiple PRs (41.8% versus 16.6%; P⬍.05) were significantly higher in the HSS group. There was no significant difference between groups in terms of miscarriages (9.3% versus 10%; P⬎.05). 694
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DISCUSSION In a group of patients with similar cycle characteristics, the use of HSSs was associated with significantly higher cryosurvival, embryo viability, and implantation rates. Although the clinical PR was also higher, this did not reach statistical significance. Several points need to be discussed, to explain the differences when embryos are frozen using HSSs. Different factors may affect the outcome of human embryo cryopreservation. These factors can be divided into four groups: clinical, technical, embryological, and genetic. Embryo quality and the outcome of the fresh cycle are the leading factors affecting the efficiency of cryopreservation (8 –11). When good-quality embryos are frozen and thawed, higher implantation rates are obtained (5,12). The etiology of infertility, the presence of a leading high-quality embryo, the developmental stage of frozen embryos, the number of intact blastomeres after thawing, and their further cleavage potential are other factors that may affect the outcome of frozenthawed ETs (11). Female age will also be an important clinical and genetic factor (5,11,13). The limited number of oocytes and embryos developed in women of advanced age, and the chromosomal errors in the resultant embryos, will undoubtedly affect the outcome. Besides these clinical, embryological, and genetic factors, technical factors such as freezing protocols (slow freezing or vitrification), fertilization by IVF or ICSI, and the experience of the laboratory personnel may all affect the outcome (14 –20). Changes in embryo culture and cryopreservation medium may have a role in explaining the difference between the two groups. However, in this prospective study, the same media were used in both groups. Differences in the quality of embryos that were frozen may also be an issue, if better embryos were cryopreserved in one particular group, which would inevitably lead to a better clinical outcome. However, neither the mean number of embryos nor the embryo quality on day 3 was different between the two groups. Before 2003, conventional straws were used for storing the embryos in our program. These were high-quality, nontoxic straws with a thickness of 0.17 mm and an inner diameter of 1.55 mm (reference no. 014103; Cryo Bio Sys-
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TABLE 3 Technical differences in straws used for cryopreservation. Technical properties
Conventional straws
HSSs No plasticizing agent (ionomeric resin) 0.33 mm 2.55 mm (lower flexibility) 0.3 mL 133 mm Hydrophobic safety plug No need for rinsing
Tip properties
Plasticizing agent 0.17 mm 1.55 mm 0.3 mL 130 mm Cotton plug Preliminary rinsing with medium is necessary to avoid any risk of toxicity –
Sealing properties
With sterile plug
Material Thickness Inner diameter Volume Length Sections Rinsing
Disposable transparent filling nozzle avoids contamination of sealable end Heat-sealing with SYMS unit (no risk of leakage during storage)
Balaban. Embryo cryopreservation in high-security straws. Fertil Steril 2007.
tem Group, I.M.V. Technologies). These should be rinsed with storage medium to flush out any traces of fiber or powder from the sterile plug that is used to avoid leaking (reference no. 007442; Cryo Bio System Group, I.M.V. Technologies). Since the beginning of 2003, CBS HSSs (reference no. 010286; Cryo Bio System Group, I.M.V. Technologies) have been used for embryo storage. Although the material of the previously used straws contained some plasticizing agents, the CBS HSSs are made of ionomeric resin, improving the safety of the straws with respect to the biological materials that they may harbor. Because the flexibility of the material is lower than that of the previously used straws, they are thicker (0.32 mm), with a larger inner diameter (2.55 mm), resulting in higher rigidity and mechanical resistance. These straws are composed of two distinct sections, separated by a unique hydrophobic safety plug that guarantees sterility of the embryo when placing it into the straw and when removing it after thawing. This hydrophobic safety plug was designed for storage in straws without the risk of losing biological material by absorption. The use of this plug in the cryopreservation of embryos also eliminates the need for preliminary rinsing of the straw with the storage medium. Although loading of the embryos in these straws is almost the same as with the previously used straws, the physical properties and the method of filling and sealing are specially designed to ensure a leakproof container for the specimen, even when immersed in liquid nitrogen. After the loading of the embryos, these straws are heat-sealed on both ends with the SYMS unit specially designed by CBS for this purpose, instead of closing the tip of the straw with a sterile plug, as described in the previous system. The other main difference in these HSSs involves the embryo being loaded into the straw by using a disposable transparent filling nozzle already mounted on the straw, hence avoiding contamination of the sealable end. Fertility and Sterility姞
The differences between the two kinds of straws are summarized in Table 3. To date, conventional straws were reported to be effective only for protection of cryopreserved samples against crosscontamination. There are very limited data in the literature comparing the efficacy of HSSs to conventional straws in terms of clinical outcome. The manufacturer’s manual reported that, in comparison to conventional straws, no significant differences were found in rabbit embryo development or sheep embryo survival rates and PRs (21). Another study compared conventional straws and HSSs in terms of cryosurvival of triploid and diploid human zygotes (22). Cryosurvival rates were found to be higher using HSSs. The only well-designed study which compared HSSs with conventional straws were in an animal study by Walker et al. in 2003 (23). This was a prospective comparison of blastocyst development rates in 278 murine embryos after refreezing and thawing at the two-cell stage against the standard Instruments-Medicine-Veterinarian (IMV) straws used in their cryopreservation program. When the manufacturer’s filling and loading protocol was used for the CBS straw, there was no significant difference in the blastocyst development rate between CBS (75.0%) and IMV (76.4%) straws. An interesting finding was the dramatic effect of filling and loading procedures of HSSs. When only the conventional straw was replaced with the HSS without changing the filling and loading protocols, the authors failed to reproduce their previous success. Only after adopting the manufacturer’s filling and loading instructions for HSSs, and freezing fewer embryos per straw, were the results achieved between the two straws nearly identical. This finding was attributed to the difference in overall dimensions of the two straw types (23). 695
It is not clear how HSSs provide higher implantation rates in cryopreservation cycles. Possible mechanisms can be suggested for the improvement in cryosurvival and implantation rates. First of all, the difference may arise from the freezing and thawing dynamics of the medium within the straws, because of physical differences such as different volumes and surface areas. If this is so, a better preservation of embryo viability by better preserving the properties of the cryo-thaw media could affect cryosurvival rates. Another reason may be a difference in the seeding characteristics of the straws. It is possible that a more effective seeding can be achieved in HSSs. In addition, the ionomeric-resin composition could be a better conductor for the formation of ice crystals. They may provide better temperature control during freezing. Better preservation of embryo viability through maintenance of the temperature of the cryomedia in which the embryos are placed may be responsible for the improvement of cryosurvival and implantation rates. The loading technique for both types of straws was not different. The only difference in loading could be the amount of medium aspirated in each column, because of differences in the thickness of straws. More studies comparing the physical and technical properties of these two different types of straws are needed, to clarify the mechanism(s) underlying the better cryosurvival and implantation rates. Cryopreservation offers clear benefits in the efficiency of assisted reproductive techniques. Parallel to the improvement in in vitro culture conditions and implantation rates, the number of embryos transferred in utero can be safely limited, to avoid multiple pregnancies. Many of the surplus embryos generated through assisted reproduction can be cryopreserved. Thus, cryopreservation will lead to an increase in cumulative PRs with a single cycle of ovarian stimulation and assisted reproduction (24). In addition to prevention of cross-contamination, HSSs provide improved outcomes in frozen-thawed ET cycles in terms of higher implantation rates. Taking into account the improved implantation rate, as well as the higher number of multiple pregnancies, the number of transferred embryos in cryo-thawed cycles can be safely reduced. In conclusion, given the advantages of higher cryosurvival and implantation rates, as well as the lower risk of crosscontamination of cryopreserved tissues, the use of HSSs is preferred in frozen-thawed embryo cryopreservation cycles. REFERENCES 1. Benifla JL, Letur-Konirsch H, Collin G, Devaux A, Kuttenn F, Madelenat P, et al. Safety of cryopreservation straws for human gametes or embryos: a preliminary study with human immunodeficiency virus-1. Hum Reprod 2000;15:2186 –9. 2. Letur-Konirsch H, Collin G, Sifer C, Devaux A, Kuttenn F, Madelenat P, et al. Safety of cryopreservation straws for human gametes or embryos: a study with human immunodeficiency virus-1 under cryopreservation conditions. Hum Reprod 2003;18:140 – 4. 3. Maertens A, Bourlet T, Plotton N, Pozzetto B, Levy R. Validation of safety procedures for the cryopreservation of semen contaminated with hepatitis C virus in assisted reproductive technology. Hum Reprod 2004;19:1554 –7.
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4. Testart J, Laselle B, Belaissch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268 –72. 5. Karlstrom PO, Bergh T, Forsberg AS, Sandkvist V, Wikland M. Prognostic factors for the success rate of embryo freezing. Hum Reprod 1997;12:1263– 6. 6. Rienzi L, Nagy ZP, Ubaldi P, Iacobelli M, Anniballo R, Tesarik J, et al. Laser-assisted removal of necrotic blastomeres from the cryopreserved embryos that were partially damaged. Fertil Steril 2002;77:1196 –201. 7. Hardarson T, Hanson C, Sjögren A, Lundin K. Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation. Hum Reprod 2001;16:313– 8. 8. Toner JP, Veeck LL, Acosta AA, Muasher SJ. Predictive value of pregnancy during original in vitro fertilization cycle on implantation and pregnancy in subsequent cryothaw cycles. Fertil Steril 1991;56: 505– 8. 9. Schalkoff ME, Oskowitz SP, Powers RD. A multifactorial analysis of the pregnancy outcome in a successful embryo cryopreservation program. Fertil Steril 1993;59:1070 – 4. 10. Lin YP, Cassidenti DL, Chacon RR, Soubra SS, Rosen GF, Yee B. Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they are derived. Fertil Steril 1995; 63:262–7. 11. Wang JX, Yap YY, Matthews CD. Frozen-thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Hum Reprod 2001;16:2316 –9. 12. Behr B, Gebhardt J, Lyon J, Milki AA. Factors relating to a successful cryopreserved blastocyst transfer program. Fertil Steril 2002;77:697–9. 13. Damario MA, Hammitt DG, Session DR, Dumesic DA. Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil Steril 2000;73:767–73. 14. Macas E, Imthurn B, Borsos M, Rosselli M, Maurer-Major E, Keller PJ. Impairment of the developmental potential of frozen-thawed human zygotes obtained after intracytoplasmic sperm injection. Fertil Steril 1998;69:630 –5. 15. Damario MA, Hammitt DG, Galanits TM, Session DR, Dumesic DA. Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Fertil Steril 1999;72:1049 –54. 16. Oehninger S, Mayer J, Muasher S. Impact of different clinical variables on pregnancy outcome following embryo cryopreservation. Mol Cell Endocrinol 2000;27:73–7. 17. Senn A, Vozzi C, Chanson A, De Grandi P, Germond M. Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage. Fertil Steril 2000;74:946 –52. 18. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002;78:449 –54. 19. Veeck LL. Does the developmental stage at freeze impact on clinical results post-thaw? Reprod Biomed Online 2003;6:367–74. 20. Boone WR, Crane IV MM, Johnson JE, Higdon HL III, Blackhurst DW. Changes in the freezing protocol for human zygotes alter embryonic development and pregnancy rates. Fertil Steril 2005;83:182– 8. 21. Cryo Bio System ITG. CBS brochure and instruction manual. L’Aigle, France: IMV Technologies Group, 2001. 22. Schiewe MC, Hubert G, Buyalos R. Human zygote cryopreservation using CBS™cryobiostraws: a clinical trial. Fertil Steril 2002;77(Suppl 3):22. 23. Walker DL, Hammitt DG, Dumesic PA, Thornhill AR. Equivalent blastocyst rates after freezing murine embryos in Cryo Bio System high security or standard Instruments-Medicine-Veterinarian straws. Fertil Steril 2003;80(Suppl 2):743– 6. 24. Van Voorhis BJ, Syrop CH, Allen BD, Sparks AE, Stovall DW. The efficacy and cost effectiveness of embryo cryopreservation compared with other assisted reproductive techniques. Fertil Steril 1995;64: 647–50.
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