Author(s):Dr Suresh Kattera and Prof Christopher Chen
Aiming to achieve maximum post-thaw survival of frozen embryos, Dr Kattera and Prof Chen designed an historical controlled study in a hospital-based fertility center using 145 patients whose embryos were frozen and thawed according to the standard method and 56 patients whose embryos were frozen and thawed according to a modified method, where various steps of cryopreservation were changed. The main Outcome Measures were post-thaw survival, implantation, and pregnancy rates and the results showed that with the modified method, 138 (93%) of the 149 embryos thawed for the 56 patients survived freezing, and 79.8% had all their blastomeres intact. This was almost double the result obtained (41.8%) for patients whose embryos were thawed with the standard method.
The implantation and pregnancy rates were also significantly higher with the modified method compared with the standard method and it was concluded that greater post-thaw embryo survival was achieved, with a concomitant increase in implantation and pregnancy rates, by modifying the various steps in the standard cryopreservation methodology. This has important implications in IVF practice. SUMMARY: Embryo cryopreservation has been a routine component of clinical IVF programs for more than two decades but has a relatively poor outcome in terms of post-thaw survival and pregnancy rates in a majority of IVF programs. Efficient embryo cryopreservation has several advantages. It helps to reduce costs and increases cumulative pregnancy rates. It can also help in cases of cancelled ET in fresh cycles caused by ovarian hyperstimulation syndrome and difficulties in ET. One of the major concerns in IVF is high-order multiple pregnancies, which result from the transfer of multiple embryos in a given cycle. In the last 5 years, new fertility drugs for better clinical management, improved culture and laboratory systems, and better identification of viable embryos have enhanced the success rates of IVF. During the same period, some clinics have started the practice of transferring two embryos to reduce multiple pregnancy rates without compromising overall pregnancy rates (2–5). Furthermore, in recent years some European countries, particularly the Scandinavian countries, have taken a lead in performing elective single-embryo transfers and have achieved acceptable pregnancy rates (6–9).
This trend is spreading to other countries. This can result in embryos being available for freezing for almost every IVF/intracytoplasmic sperm injection (ICSI) patient aged 40 years and has created the necessity to have an excellent freezing program in routine clinical IVF practice. Review of the current literature reveals unsatisfactory post-thaw embryo survival, implantation, and pregnancy rates. Post-thaw survival rates vary from 50% to 80% for different embryo stages. Implantation and pregnancy rates have varied from 3% to 15% and 15% to 25% respectively, which is approximately half of the rates achieved for fresh Embryo Transfers.
To improve the outcome of frozen ET cycles, the authors attempted to modify the various steps of the cryopreservation protocol. In a pilot study on arrested and fragmented embryos (grades 3 and 4), they achieved post-thaw survival rates of 80% and 65%, respectively, having all blastomeres intact after making modifications to the various steps of the standard method. Encouraged by the post-thaw survival outcomes of these embryos, they used this modified method in a routine clinical frozen ET program. The report describes the improvements of the modified method, in comparison with the standard method, and the results therefrom. Extract from paper “Kattera and Chen Increased post-thaw survival of embryos Vol. 84, No. 5, November 2005” “DISCUSSION: There are several reports on frozen ET (10–18). Most of these studies report an overall post-thaw survival rate of 50%–80%, with 43%–55% having all their blastomeres intact. Thus, on average, only 50% of the embryos that are frozen have all their blastomeres intact. Only 25% of the embryos have 50%–99% of their blastomeres intact. It has been shown that implantation and pregnancy rates of embryos with 100% survival of blastomeres are almost double those of embryos with 50%–99% survival of blastomeres, and embryos with 50% survival of blastomeres do not implant at all (14). Thus, to increase the implantation rate, it is important to improve the cryopreservation method to achieve 100% survival of blastomeres in the maximum number of embryos. In an effort to improve these results, several aspects of the cryopreservation method were modified in this laboratory. In the modified method, we achieved a post-thaw survival rate of 93%, and 80% of the total embryos survived with all their blastomeres intact, with a concomitant increase in implantation rates. This is double the result reported for the standard method of ours and also reported in the literature. This could be attributed to changes made to the various steps of the cryopreservation protocol. First, HEPES-based cleavage medium containing cryoprotectants was used in the modified method, as opposed to PBS as basal medium in the standard method. Some of the components in the HEPES cleavage medium, particularly certain salts, amino acids, and human serum albumin, might have some cryoprotective properties. Furthermore, exposure of embryos to cryoprotectants in PBS might compromise their viability, owing to lack of amino acids and other nutrients that are present in cleavage medium. However, further studies are required to substantiate our hypothesis. Second, we made changes to the method of loading embryos into the straws. In the standard method, during the filling of the straws with freezing solutions, embryos are drawn into the middle column along with the freezing solution. Sometimes the embryos might not stay together, and some might move toward the upper meniscus of the second column, where seeding is performed. It is possible that during seeding on the meniscus, some embryos might be exposed to a sudden change in temperature, resulting in formation of ice crystals in embryos that are only partially dehydrated. This might result in lysis of the blastomeres on warming. In the modified method described here, embryos were prevented from moving to the upper meniscus by filling the freezing straws with the freezing solution first and then loading the embryos with an “L”-shaped pulled Pasteur pipette. This ensures that embryos remain together close to the lower meniscus of the freezing column and do not move within the column, even if the straw is turned upside-down. However, caution must be exercised not to introduce air bubbles and to avoid breakage of the “L”-shaped pipette.
Third, we made changes to the warming rates. The optimal warming rate of embryos depends on the cooling procedure. Some freezing programs terminate the cooling of embryos at 30°C, whereas others continue to 150°C and then plunge into LN2. In both instances, straws have been warmed by exposure to room temperature for 30 seconds and then immersion in a water bath at 30°C for 30 seconds. However, each method of cooling achieves a different degree of dehydration, with different requirements of warming rate to ensure minimal damage to blastomeres. If a small amount of intracellular water is present in the embryos when the cooling is stopped at 30°C and the straw is plunged into LN2, small ice crystals can form. Under these circumstances, rapid warming rates are essential (room temperature for 30 seconds, followed by a water bath at 30°C for 30 seconds). A slow warming rate would allow the small ice crystals time to grow into larger, harmful crystals (20, 21). If embryos are cooled slowly to 150°C, they will be nearly completely dehydrated when they are plunged into LN2. These embryos might be damaged by rapid warming because of extremely rapid changes in osmolarity in the dehydrated embryo. Slow warming, which allows more time for cells to rehydrate, is best for severely dehydrated embryos. Thus, in the present study embryos were cooled to 150°C before being plunged into LN2 and warmed by leaving the straws at room temperature for 2 minutes. This gave the embryos more time to rehydrate and minimized damage to the blastomeres (20, 21). Fourth, in routine practice, cryoprotectant is removed from the embryos in three to four steps of progressively lower concentrations of cryoprotectant. This reduces swelling and subsequent lyses of blastomeres. An alternative and more rapid method for removing cryoprotectant from embryos is to use a high concentration of a non penetrating molecule, such as sucrose. The high extra cellular concentration of sucrose counterbalances the high concentration of propanediol in the embryo because it reduces the differences in the osmolarity between the inside and the outside of the cell (22). Embryos containing propanediol shrink when placed in a concentrated sucrose solution (0.5 mol/L), indicating that both propanediol and water are leaving the cell; because of this it was difficult to assess the survival of embryos immediately. The use of 0.5 mol/L and 0.2 mol/L sucrose in two steps each of 5 minutes in the modified thawing solutions achieved this aim here and is simpler than the step-wise reduction of cryoprotectant concentration in the standard method. The second criterion that is used to assess the efficiency of an embryo-freezing program is the ability of embryos to grow further in vitro and particularly in vivo. Ideally it is better to freeze embryos on day 2 and compare the development of these embryos after thawing to the rate of development of fresh embryos on day 3. However, the practice in this laboratory is to choose the best embryos on day 3 for transfer and freeze the supernumerary embryos. Indirect evidence of further development of frozen – thawed embryos comes from the development of these embryos in vivo. Of the 56 patients who underwent frozen ET, 23 achieved clinical pregnancy, with an implantation rate of 18.1% and a clinical pregnancy rate of 41%. This further confirms the efficacy of the modified cryopreservation method. The implantation and pregnancy rates reported here are greater than those obtained with our standard method and those reported elsewhere (11, 14–18). Thus, modifications to various steps in the cryopreservation protocol improve the post-thaw survival, with concomitant increase in implantation rates, and this could be due to higher numbers of embryos with 100% blastomere survival. Consequently, we observed a 13% twinning rate among patients in the modified method group as compared with none in the standard method group. The three patients who had two eight-cell embryos each transferred had all their blastomeres intact. It is difficult to exactly pinpoint whether the improvement in post-thaw survival was due to the improved freezing or thawing protocol. However, the improvement could be attributed mainly to changes made to both the freezing and thawing protocols, particularly the loading of embryos into the straws and the warming rates at room temperature. It has been reported that the highest implantation and pregnancy rates come from embryos with all their blastomeres intact (14). Thus, it is important to achieve 100% survival of blastomeres in the maximum number of embryos, which in turn results in higher implantation and pregnancy rates. Obviously, results reported here confirm this. In conclusion, this is the first report of high post-thaw embryo survival after modification of the standard method. The proper study design would have been a prospectively randomized study, with half of the cycles or half of the embryos per cycle frozen with the standard and half with the modified protocol. However, the differences in post-thaw survival were such that a prospective randomized study was unethical.”