Taking advantage of multiple frozen embryo transfers to
maximize pregnancies from one ovarian stimulation cycle
| Jun Tao, PhD, Randall H. Craig, MD, Rychllik Daniel, MD [E-mail: jtao@ftcivf.com] Fertility Treatment Center, Chandler, AZ |
| Introduction |
| Experiencing a successful pregnancy after an IVF procedure is the goal for all infertility patients. Ideally patients can achieve pregnancies from their first fresh ETs. Therefore, a transfer using the best quality embryos is always the first option in fresh cycles. In this way, patients seem to be provided the best opportunity to achieve pregnancies. |
| It is not unusual however, that many patients fail to achieve pregnancy after superior quality embryos are transferred in their fresh ETs. In addition to the possibilities of genetic abnormalities within embryos and poor embryo transfer technique, that may be responsible to implantation failure, suboptimal endometrium receptivity in fresh cycles can be another important reason for implantation failure since ovarian stimulation may have a negative influence on the potential of embryonic implantation (Nikas and Aghajanoval, 2002; Tropea et al., 2004; Devroey et al., 2004). |
| After undergoing ovarian stimulation, it is common for patients to produce fifteen or even more oocytes per cycle that consequently yield more embryos than need be used for the fresh transfer. Cryopreservation of these excess embryos has been popular for decades in infertility practice (Testart et al., 1986; Fugger et al., 1991). Compared to a repeat stimulation and fresh ET, frozen ET is both less expensive and less invasive. |
| Surprisingly, data reported by The Centers of Disease Control and Prevention (CDC, U.S. Department of Health and Human Services), indicates that the performance rate of national frozen ET cycles under age 41 from 2000 to 2002 are only 18.4% (ages <35), 19.7% (35-38), 20.6% (38-40), respectively (The numbers presented here are pooled data from three subgroups under age 41 and excludes cancelled cycles). This seems to suggest that around eighty percent of stimulated cycles do not produce enough excess embryos for subsequent frozen embryo transfer attempts. |
| Even in donor egg cycles, in which more oocytes are usually produced and embryo development is better, the ratio of frozen ETs to fresh ETs are still less than half. CDC data indicates 36.0% (<35 yrs), 39.2% (35-37 yrs), |
| and 41.4% (38-40 yrs), respectively, from 2000 to 2002. It may be worth while to investigate the reason for this low incidence of utilizing a program’s frozen-embryotransfer program. One possibility is that some patients stop pursuing frozen ET because of their successful pregnancies from fresh ETs. |
| However, national live birth rates per cycle under age 41 (non-donor after fresh ET) reported by CDC from 2000-2002 are 32.8% (<35 yrs), 35.2% (35-37 yrs) and 36.9% (38-40 yrs), respectively, which suggests that the majority of patients did not achieve successful pregnancies from their fresh ETs. If so, what is the reason of this low incidence of frozen ET? Is it that clinics or patients prefer to have another ovarian stimulation rather than use surplus frozen embryos from their previous stimulated cycles? |
| Our current retrospective study reports results in which patients who had fresh ETs in ovarian stimulation cycles returned for subsequent frozen ETs. The purpose is to: (1) compare endometrium receptivity between fresh ET and frozen ETs where embryos were obtained from the same ovarian stimulation cycle, (2) evaluate the significance of a frozen ET program, (3) study the possibility of maximizing the number of conceptions from a single oocyte retrieval cycle. |
| Materials and Methods |
| All patients under the age of 41 who had both fresh and at least one frozen embryo transfer from August 1999 to June 2005 were included in this study. Egg donor cycles and patients that had a fresh ET without a subsequent frozen ET were excluded from this study. Patients that had a frozen ET but did not have a fresh ET, due to ovarian hyperstimulation, were also excluded from this study. |
| Patient preparation for stimulated cycles as well as for frozen embryo transfer cycles has been described in detail (Tao et al, 2004). Fresh ET’s were performed on Day 4 and embryo development was evaluated daily. The criteria for Day 2 and Day 3 embryos was based on cell number, evenness of blastomeres and the percentage of fragmentation. Day 4 grading was based on the proportion of blastomeres that underwent compaction, |
| morphology of the multi-cellular mass, amount of fragmentation as well as the quality of the embryo on Day 2 and Day 3. |
| Embryos were graded from 1-3 with grade 3 indicating the best quality and grade one indicating the lowest quality (Tao et al., 2004). Grade 3 embryos have more than 75% of their blastomeres compacting and look sphere-shaped with a smooth profile. Grade 3 embryos had 4 and 8 cells on Day 2 and Day 3 respectively with relatively even-sized blastomere and less than 25% fragmentation. Grade 2 embryos had 50-75% of their blastomeres compacting or an irregular shaped profile with a larger percentage of blastomeres compacting. Grade 2 embryos had moderately uneven blastomeres on Day 2 and day3 with fragmentation between 25-50%. Grade 1 embryos had less than 50% of their blastomeres compacting or a higher percentage of compaction with a severely irregular shape. On day 2 and day 3 these embryos had a lower cell number, severely uneven blastomeres and multinuclei may have been observed. Grade 1 embryos usually had more than 50% fragmentation. |
| The fresh ET strategy was to transfer two Grade 3 compacted embryos on Day 4. The remaining embryos were frozen only if they underwent compaction or showed signs of possible compaction. The preference was to freeze grade 2 and grade 3 embryos although some grade 1 embryos were frozen. This was usually done when patients did not have any good embryos or sometimes due to patient request. The embryo freezing and thawing protocols utilized were previously described in detail (Tao et al, 2004). |
| The goal of our embryo thawing program is to have two grade 3 embryos available for transfer. If the embryos did not survive the thaw, or did not show any morphological recovery changes post thaw, more embryos were thawed. In some situations however, some patients did receive more than two grade 3 embryos at transfer. This occurred when patients had had repeated implantation failure, repeated miscarriages or even where some patients requested that more than 2 embryos be transferred. Patients who did not have any grade 3 embryos frozen had up to three, or very occasionally four, embryos of lesser quality transferred at ET. |
| Embryos were considered to have survived the thawing process if more than 50% of their cells remained intact. The morphological alterations were assessed for each embryo post-thaw and compared to their developmental status prior to freezing. Embryos that showed developmental progression were deemed suitable for transfer while those embryos that did not progress were |
| determined to be unsuitable for transfer. These unsuitable embryos were some times transferred at the patient’s request, otherwise they were either discarded the next day or refrozen, based on quality. |
| Frozen embryo transfers were performed 1-2 hours after the thawing process and mechanical assisted hatching was performed on all embryos that retained an intact zona pellucida after thawing. |
| Since embryo number does not reflect embryo quality, a parameter of total embryo score for each ET, was therefore utilized in this study to cover both embryo number and quality factors. Each embryo was given an individual score based upon the quality: for grade 1 embryo the score is the grade times 2 (1 x 2); for grade 2 embryo the grade times 2.5 (2 x 2.5), for grade 3 embryo the grade times 3 (3 x 3). The total score for each embryo transfer is the sum of each individual embryo score. |
| The ages of patients who had a fresh ET and subsequent series of frozen ETs are presented in Table |
| 1. The number of embryos thawed in frozen cycles, the number of embryos transferred and the total embryo scores in embryo transfer cycles are analyzed. In Table 1 ongoing/live birth rates are used as the parameter for statistical analysis between different embryo transfers. |
| The clinical pregnancy rates are used as a parameter for the implantation study in Table 2. Table 2 also shows accumulated clinical pregnancies after fresh ET and subsequent frozen ETs. The definition of accumulated clinical pregnancy in this study is the clinical pregnancy rate per oocyte retrieval, which means when three clinical pregnancies (for example, one fresh and two frozen ETs) are achieved from embryos yielded from one retrieval cycle, that retrieval cycle is counted as one clinical pregnancy cycle rather than three pregnancies. The number of patients who still have cryopreserved surplus embryos after each embryo transfer are also presented in Table 2. |
| Data are presented as means ±SDs or as percentages. Where appropriate, data were analyzed with unpaired Student’s t-tests or χ2– tests. Significance was accepted at P |
| < 0.05. |
| Results |
| The average age of patients who had fresh ETs and a subsequent series of frozen ETs are presented in Table 1. The average number of oocytes retrieved per cycle is 20 ± 8.9 and the average excess embryos cryopreserved after fresh ET is 6.5 ± 4.2 per patient. |
| Table 1 presents the number of embryos thawed in each frozen cycles. In this article, the first frozen ET is |
| Table 1: Characteristics and outcomes of fresh and frozen ETs. |
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| a Significant difference compared with Frozen ET (1) (P < 0.05) b Significant difference compared with Fresh ET (P < 0.01) |
| presented as frozen ET (1), and frozen ET (2) and frozen ET (3) stand for second and third frozen ET, respectively. The number of embryos thawed in frozen ET (2) and frozen ET (3) are significantly higher than frozen ET |
| (1) (P < 0.05) due to our policy to thaw the best quality embryos first and that left relatively inferior ones for subsequent cycles. |
| As shown in Table 1, the average number of embryos transferred in fresh ET (2.2 ± 0.5) is significantly lower than frozen ET (1) (2.4 ± 0.7, P < 0.01), frozen ET (2) |
| (2.7 ± 0.8, P < 0.01), and frozen ET (3) (3.2 ± 0.8, P < 0.05). In frozen ET (1), the average number of embryos transferred is significantly lower than that in frozen ET |
| (2) (P < 0.05). |
| c Significant difference compared with Fresh ET (P < 0.05) d Significant difference compared with Fresh ET (P < 0.001) |
| The average total embryo score per transfer in fresh ET is significantly higher than that in frozen ET (1) (P |
| < 0.001 or P=1.518E-9) and frozen ET (2) (P < 0.001) which indicates that embryo quality in fresh ET is significantly better than subsequent frozen ETs. However, even though fresh embryos had higher scores, the ongoing/live birth rates in frozen ET (1) and frozen ET (2) are significantly higher than that in fresh ET (P < 0.01). |
| Comparisons between positive hCG tests, clinical pregnancies, ongoing/live birth rates and implantation rates in fresh ETs with the series of frozen ETs are presented in Figure 1. Positive hCG tests, clinical pregnancies and ongoing/live birth rate in frozen ET |
| (1) and frozen ET (2) are all significantly higher than |
| Table 2. Clinical pregnancy rates after each ET and accumulated clinical pregnant rates after subsequent frozen ETs and the number of patients who have surplus frozen embryos after frozen ETs. |
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| those in fresh ET (P < 0.01). Implantation rates are also significantly higher in frozen ET (1) and frozen ET (2) than that in fresh ET (P < 0.05). Frozen ET (3), though having a small sample size, also has significantly higher clinical pregnancy and ongoing/live birth rates and implantation rates than in fresh ET (P < 0.05). |
| Table 2 compares fresh ET with subsequent cycles of frozen ETs using clinical pregnancy rates as an parameter. The data shows pregnancy rates after fresh and each frozen embryo transfer and accumulated times of conceptions in patients who experienced cycles of embryo transfers. After frozen ET (1), 100 clinical pregnancies were achieved that means that 120 out of 190 retrieval cycles achieved pregnancy after one fresh and one frozen ET. Twenty-eight of these 120 pregnant women experienced a second clinical pregnancy. |
| Of 42 patients who had a second frozen ET (frozen ET (2)), 25 clinical pregnancies were obtained. In these 42 patients, 33 experienced a clinical pregnancy from either fresh or frozen ETs, or both. Fourteen of these 42 patients were pregnant twice, 7 of which had one pregnancy from a fresh ET and one from one of the two frozen ETs. Another 7 had both pregnancies in frozen ETs but not in fresh ETs. In addition, four of these 42 women became pregnant three times, in all their fresh and frozen ETs. |
| Although the sample size of patients who had three frozen ETs (frozen ET (3)) is small, all 6 patients have been conceived as clinical pregnancies after frozen ET (3), three had one pregnancy and three had two pregnancies. Unfortunately, one patient who finally achieved a clinical pregnancy in her frozen ET (3) miscarried. |
| Based on the results in Table 2, patients who had one frozen embryo transfer (frozen ET (1)), obtained a clinical pregnancy rate that reached 67.0% compared with 26.8% after fresh ET. This rate increased to 78.6% after the second frozen embryo transfer (frozen ET (2)) was performed. Six patients had three frozen ETs, all experienced at least one clinical pregnancy. |
| After frozen ET (1), 59.8% patients still have reserved embryos remaining for future embryo transfer attempts. After frozen ET (2), twenty-five out of 42 (59.5%) patients had more embryos in cryopreservation. In 6 patients who had three frozen ETs, two of them have excess embryos remaining for future frozen cycles (Table 2). |
| Discussion |
| Fresh ETs are routinely performed following gonadotropin stimulated oocyte retrievals. It is quite common that patients fail to achieve pregnancy even though superior quality embryos are transferred in these |
| fresh cycles. Three main reasons can be given to explain this observation: (1) decreased endometrial receptivity, (2) inferior embryo viability, and (3) poor embryo transfer technique. |
| In the current study, patient success rates in the frozen cycles are significantly higher than that in fresh ETs even though those embryos were obtained from the same stimulated cycles and embryo transfer techniques were similar in both the fresh and frozen ETs. In addition, the same physician performed the embryo transfer procedure for all cycles of a given patient. The average embryo number transferred in frozen cycles, although significantly higher than in fresh cycles (P < 0.01), had on average a total embryo score significantly lower than in fresh cycles (P < 0.001). The high pregnancy rates obtained in frozen cycles are likely due to optimal endometrial receptivity which indicates that frozen-embryo-transfers can be as successful or better than fresh ETs for patients wishing to fulfill their dream of becoming pregnant. |
| Studies indicate that endometrial receptivity can be detrimentally affected by ovarian stimulation due to the supraphysiological concentrations of steroids used to produce large numbers of oocytes. Precocious luteal transformation, which is represented as premature expression of pinopodes and integrins (Bourgain and Devroey, 2003), can compromise embryonic implantation potential and waste superior quality embryos when transferred during fresh cycles. When patients only have a very few good quality embryos, which is common, it is a dilemma whether all good embryos should be transferred in the fresh ET or be frozen for a frozen- |
| Figure 1. Comparisons of positive hCG tests, clinical pregnancy rates, ongoing/live birth rates, and implantation rates between fresh ET and frozen ETs |
| a Significant difference compared with fresh ET (P < 0.01) b Significant difference compared with fresh ET (P < 0.05) |
| embryo-transfer cycle. |
| During frozen cycles serum estrogen and progesterone are administered to mimic the natural cycle. Therefore, better endometrial receptivity for embryo implantation can be expected. When surplus embryos, produced from fresh cycles, are frozen and thawed successfully, and those post-thaw embryos are determined to be viable, patients can achieve reasonably high pregnancy rates (El-Toukhy et al., 2003; Tao et al., 2004). |
| The advantages of frozen ET does not seem to be maximally utilized in routine infertility practice. Not only are the ratios of national frozen cycles to fresh cycles low, but the national pregnancy rates of frozen ETs is lower compared with fresh ET. Based on CDC reports, national live birth rates after frozen ETs under age 41 are 20.9%, 24.2%, and 25.6% from 2000 to 2002, respectively. |
| The following are possible explanations causing this lower frozen ET success: (1) the best quality embryos are chosen for fresh ET so that frozen cycle embryo quality is probably lower than those used in fresh transfers; (2) embryos are commonly frozen at the pronuclear or early cleavage stage which does not provide good embryo selection, (3) poor embryo survival rate or the high incidence of blastomere damage; (4) there is no readily and reliable method to determine post-thaw embryo viability when post-thaw embryo are transferred at cleavage stage as currently performed in most clinics. |
| We have reported the advantages of freezing embryos at the compact-morula stage (Tao et al., 2005). Embryos frozen at this stage provide a better selection than is the case for pronuclear and early cleavage stages and have great post-thaw survival rate. Most importantly, the compact-morula embryo demonstrates unique morphological alterations during thawing, and shortly after thawing, which enhances post-thaw embryo viability determination. Due to high post-thaw survival rates and the ability to determine embryo viability, only a limited number of embryos need be thawed and transferred to make embryo utilization very efficient. As shown in the results, not only do implantation rates in frozen ETs reach 33%, but after two to three frozen ETs, more than half of these patients still have embryos remaining in liquid nitrogen for future ET attempts. |
| Based on our experience, when embryos are frozen at the compact-morula stage and reexamined for viability after thawing thus the advantage of observing unique characteristics of post-thaw morphological alterations, embryos with relatively higher viability can be selected from inferior embryos. When patients have high number of inferior quality embryos, this strategy may even |
| provide patients multiple opportunities of embryo transfer with a reasonable pregnancy rate. |
| The current trend for embryo transfer is to transfer one to two embryos in order to avoid multiple gestations (Gardner et al., 2000; Papanikolaou et al., 2005). This concept works well when patients have superior quality embryos. But more often than not, patients have embryos of various quality, and many times only of inferior quality. In order to avoid significantly increasing the risk of multiple gestations but still provide patients with a reasonable chance to achieve pregnancy, the consideration of total embryo score, used in this article, may have some value as an additional reference in the decision of how many embryos should be transferred. Further study is needed to determine the proper total embryo score for embryo transfer that will give patients a good opportunity to get pregnant while lowering the risk of multiple gestation. |
| Conclusion |
| The great value of frozen ET is that patients, who have frozen surplus embryos in cryo-storage, can have multiple opportunities to achieve pregnancy. In patients with possible decreased endometrium receptivity after ovarian stimulation, successful cryopreservation and post-thaw viability determination, using our compact-morula freezing technique, can result in a better chance to achieve pregnancy. When patients have high numbers of frozen embryos that may be of low quality, using compact-morula stage freezing and post-thaw reselection can give reasonable pregnancy rates and maximize the efficient use of embryos obtained from a fresh retrieval. |
| Acknowledgements |
| We thank Wendy Lewis and Yingzhi Yang for their assistance in this manuscript preparation and statistics analysis. |
| References |
| Nikas G and Aghajanoval L. Endometrial pinopodes: some more understanding on human implantation? Reprod Biomed Online. 2002; 4 Suppl 3:18-23. |
| Tropea A., Miceli F., Minici F., Orlando M., Lamanna G., Gangale M., Catino S., Lanzone A., and Apa R. Endometrial evaluation in superovulation programs: relationship with successful outcome. Ann NY Acad Sci. 2004; 1034:211-8. |
| Devroey P., Bourgain C., Macklon NS., and Fauser BC. Reproductive biology and IVF: ovarian stimulation and endometrial receptivity. Trends Endocrinol Metab. 2004 Mar; 15:84-90. |
| Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, et al. High pregnancy rates after early human embryo freezing . Fertil Steril 1986;46:268-72. |
| Fugger EF, Bustillo M, Dorfmann AD, Schulman JD. Human preimplantation embryo cryopreservation: selected aspects. Hum Reprod 1991;6:131-5. |
| 2000 Assisted Reproductive Technology Success Rates, National 2003,79:1106-11. Summary and Fertility Clinic Reports, U.S. Department of Health and Tao J, Craig RH, Johnson M, Willams B, Lewis W, White J, Human Services, Centers for Disease Control and Prevention. Buehler N. Observed advantages of compact-morula embryo |
| 2001 Assisted Reproductive Technology Success Rates, National cryopreservation. The Clinical Embryologist, 2005, 8:3. Summary and Fertility Clinic Reports, U.S. Department of Health and Gardner DK., Lane M., Stevens J., Schlenker T. and Schoolcraft Human Services, Centers for Disease Control and Prevention. WB. Blastocyst score affects implantation and pregnancy outcome: |
| 2002 Assisted Reproductive Technology Success Rates, National towards a single blastocyst transfer. Fertil Steril. 2000; 73:1155-8. Summary and Fertility Clinic Reports, U.S. Department of Health and Papanikolaou EG., D’haeseleer E., Verheyen G., Van de Velde Human Services, Centers for Disease Control and Prevention. H., Camus M., Van Steirteghem A., Devroey P. and Tournaye H. |
| Tao J, Craig RH, Johnson M, Willams B, Lewis W, White J, Live birth rate is significantly higher after blastocyst transfer than Buehler N. Cryopreservation of human embryos at the morula stage after cleavage-stage embryo transfer when at least four embryos are and outcomes after transfer. Fertil Steril 2004; 82:108-18 available on day 3 of embryo culture. A randomized prospective |
| El-Toukhy T, Khalf Y, Al-Darazi K, Andritsos V, Taylor A, study. Hum Reprod. 2005; 20:3198-03. Braude P. Effect of blastomere loss on the outcome of frozen embryo replacement cycles. Fertil. Steril. |
| Corrigendum |
| The editor would like to bring your attention to a correction for the article from Dr. Jun Tao and |
| colleagues entitled: Taking advantage of multiple frozen embryo transfers to maximize pregnancies from |
| one ovarian stimulation cycle (Spring 9:1 2006). |
| Within the Introduction (paragraph #4), the percentages stated (18.4%, 19.7% and 20.6%), refer to the |
| ratio of FET cycles to Fresh Cycles in patients under age 41 during three years of calculations (2000, 2001, |
| 2002) respectively, and not by age group as was stated. Likewise, similar misinterpretations occurred in |
| paragraphs #5 and #6. The Clinical Embryologist regrets this editing error. |
| — Editor |