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Blastocyst transfer in day-5 embryo transfer depends primarily on the number of oocytes retrieved and not on age
FERTILITY AND STERILITY@ VOL.. 69, NO. I, JANUARY 1998 Copyright 01998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Blastocyst transfer in day-5 embryo transfer depends primarily on the number of oocytes retrieved and not on age _
Michael C. W. Scholtes, M.D., and Gerard H. Zeilmaker, Ph.D. Department
of Endocrinology
and Reproduction,
Erasmus University Rotterdam,
Rotterdam,
the Netherlands
Objective: To analyze the effects of patient age and treatment cycle number on the occurrence of blastocyst transfer and subsequent implantation. Design: Prospective study. Setting: Department of endocrinology and reproduction. Patient(s): All 1,099 women had day-5 transfers after IVF or intracytoplasmic sperm injection treatment. Intervention(s): All patients were checked for embryo development in vitro in consecutive day-5 transfer cycles. Two blastocysts or three lesser-developed embryos were transferred. Main Outcome Measure(s): Blastocyst formation rate or clinical pregnancy/implantation rate. Result(s): Of 929 patients in the first cycle, 545 (59%) had at least one blastocyst available for ET. Among 151 patients with a blastocyst in cycle 1, 77 developed one or more blastocysts in cycle 2 (51%). Fifty of 143 patients without a blastocyst in cycle 1 had at least one blastocyst in cycle 2 (35%). After subdivision of all day-5 ETs according to the first four cycles, the following implantation rates per embryo were found for ET with one or more blastocysts: cycle 1 (n = 545), 23%; cycle 2 (n = 264) 23%; cycle 3 (n = llO), 14%; and cycle 4 (n = 27), 12%, and with noncavitating embryos, respectively: (n = 384) 6%, (n = 193) 6%, (n = 94) 2%, and (n = 35) 3%. The negative correlation of the age of the woman on blastulation depended primarily on the number of oocytes retrieved. Conclusion(s): The blastocyst implantation rate decreased after cycle 2. Biologic ovarian age, rather than chronoloaic age, determines the frequency of blastocyst transfer or pregnancy rate. (FertilStez’a’ 1998;69:78-83. 01998 by American Society for Reproductive Medicine.) Key Words: Blastulation, blastocyst, consecutive cycles, embryo development, embryo transfer, implantation rate, female age Received
May 6, 1997.
Reprint requests: Gerard H. Zeilmaker, Ph.D., Department of Endocrinology and Reproduction, Erasmus University, Postbox 1738, 3000 DR Rotterdam, the Netherlands (FAX: 31-l O4366832). 00150282/98/$19.00 PII s0015-0292(97)00450-0
78
In a previous study, it was demonstrated that after prolonged culture of embryos for 5 days, blastocysts formed, with a high implantation rate (1). Transfer of only two blastocysts led to a high pregnancy rate (PR) without the risk of triplets. Because the intention of IVF treatment is to provide a chance for subfertile couples to deliver a mature infant, after a low-risk single-
ton or gemellary pregnancy with minimal perinatal complications, prolonged embryo culture offers the best opportunity. In the present study, we analyzed successive IVF-ET cycles to determine whether blastocyst formation is more likely to occur under certain circumstances. The effect of patient age in relation to blastocyst transfer also was studied.
of all day-5 ETs according to cycle number, ET with at least one blastocyst or without cavitating embryos, and the implantationrate per embryo.
Subdivision
Implantation
Implantation rate per embryo No. of ETs with blastocysts
Cycle no.
23* 23* 14 12
545 264 110 21
1 2 3 4
Note. NS = not significant. * P = 0.02 (vs. implantation t P = 0.08 (vs. implantation
(%)
384 193 94 35
rate per embryo (%)
P value
6t 6t
0.001 0.001 0.002 0.08 (NS)
2 3
rate in cycles 3 and 4). rate in cycles 3 and 4).
Finally, successive day-5 transfers were analyzed to demonstrate the possible effect of the number of treatment cycles on blastocyst formation and pregnancy induction.
MATERIALS AND METHODS This study included all couples undergoing ET after 5 days of embryo culture from an ongoing prospective randomized, IRB-approved study of ET after 3-5 days conducted between January 1994 and December 1995 (1). Two different groups were identified. Group 1 comprised 1,099 patients having day-5 ET in one or more subsequent IVF-ET or intracytoplasmic sperm injection (ICSI)-ET cycles. Group 2 included couples having day-5 ET (n = 294) exclusively in all subsequent IVF-ET cycles. The effect of patient age on the frequency of blastocyst transfer was studied by age groups of 3 years from 22 to 47 years of age. Ovarian stimulation consisted of a long-acting GnRH agonist (GnRH-a), 200 pg intranasally administered twice daily starting in the late luteal phase. After ultrasonic evidence of ovarian suppression, hMG or FSH was given at a dose of 150 IU/d IM or SC. Higher or lower initial doses were used if necessary, as dictated by any previous response to ovarian stimulation. Intramuscular hCG was administered at a dose of 10,000 IU when the mean diameter of two or more dominant follicles was 18 mm. The GnRH-a and hMG were discontinued when hCG was given. Transvaginal oocyte retrieval was performed 35 hours after administration of hCG. Luteal support generally consisted of 600 mg of progesterone administered vaginally, in combination with 1,500 IU of hCG on days 2, 5, and 8 after ovum pickup, when no signs of increased risk for ovarian hyperstimulation syndrome were present. To analyze the effect of the number of oocytes on the frequency of blastocyst transfer, we compared patients with up to four and five or more oocytes by ovum pickup. After fertilization, the embryos were cultured in a standard medium without coculture, under mineral oil with strict COZ control. Rapid CO, recovery occurred. Details of this FERTILITY
No. of ETs without blastocysts
& STERILITY
@
element of the study can be found as referenced (1) or at the intemet address: http://huizen.dds.nV-ghz/starlab.html). Embryo development on day 5 varied from 8 to 32 cell stages, morula, morula-blastula stage (differentiating morula stages without a visible blastocele), and cavitating embryos: from early blastocysts with or without a clear inner cell mass (ICM) to expanding blastocysts with or without a normal amount of ICM. On day 3 after OPU, a maximum of three embryos usually was transferred. However, after 5 days of embryo culture, ET was reduced to two embryos whenever two blastocysts with a clear ICM were available. In other cases, the couples were consulted individually regarding the number of embryos to be transferred, with the maximum number being three. Pregnancies were recorded by serum hCG assay (>25 IV/L) 217 days after ovum pickup, and multiple pregnancies were confirmed by multiple positive heartbeats recorded by ultrasonography 31 days after ovum pickup. Statistics were calculated by 2 analysis and by CochranMantel-Haenszel statistics and logistic regression.
RESULTS In the first IVF-ET cycle, consisting of 929 patients having day-5 ET, 545 (59%) had at least one blastocyst available for ET and 384 did not achieve blastulation. Among 151 patients with a blastocyst in cycle 1, 77 developed one or more blastocysts in cycle 2 (51%). Interestingly, 50 of 143 (35%) patients without a blastocyst in cycle 1 had at least one blastocyst in cycle 2 (P = 0.006). In total, 946 ETs were carried out with one or more blastocysts, leading to 321 clinical pregnancies (34%). Among 586 day-5 ETs without blastocysts, only 47 women became pregnant (8%) (P = 0.001). Eight of these pregnancies resulted from 228 transfers in which one or more eight-cell embryos were the most advanced stages available (3.5%). The other preg79
Percentage of ETs with one or more blastocyst(s) according to the number of oocytes retrieved.
5-10
IO-15
15-20
No. of oocytes retreived
nancies in this patient category came from transfers of morulae or differentiating morulae (10.9%). Subdivision according to the cycle number of all day-5 ETs (Table 1) showed comparable implantation rates per embryo in the first (n = 929) and second (n = 457) IVF-ET cycles after transfer with at least one blastocyst (23% and 23%), and for lesser stages of embryonic development of 6% and 6%. In contrast, in cycles 3 (n = 204) and 4 (n = 62), a decrease in implantation rates was found for blastocysts (P = 0.02), with 14% and 12%, respectively, and for lesser stages (P = 0.08), with 2% and 3%, respectively. It appears that on day 5, the implantation chance for a blastocyst transfer was three times higher than for noncavitating embryos (relative risk, 3.025). Taking into account the cycle number of IVF treatment, the age of the woman had a strong correlation with the capacity for blastulation (P = 0.0001). The chance of blastocyst transfer decreased by 14% per age group of + 3 years. In addition, by regression analysis, the chance of blastocyst transfer decreased by 13% per cycle number. The apparent effect of female age on the chance of blastocyst transfer seemed to be mediated by the number of oocytes retrieved. The relation between the number of oocytes and the percentage of blastocyst transfers is demonstrated in Figure 1 (P = 0.0001, odds ratio 1.395). When only patients with more than four oocytes were considered, 80
Scholtes and Zeilmaker
the chance of blastocyst transfer decreased per age group by 9.9% (P = O.Ol), but was significantly better (P = 0.0001, odds ratio 1.901) in comparison with patients with fewer than five oocytes (Fig. 2). Finally, the increasing proportion of patients with fewer than five oocytes (P = 0.001) per age group upon ovum pickup further explained the decreasing percentage of patients achieving blastocyst transfer (Fig. 3).
DISCUSSION Because the replacement of two blastocysts on day 5 leads to a high pregnancy rate (PR) (1) and prevents triplets, the chance to attain a blastocyst transfer was investigated in consecutive IVF-ET cycles, in relation to patient age and ovarian response on gonadotropins. Furthermore, successive day-5 IVF-ET cycles were analyzed according to PRs and implantation rates per embryo. The implantation rate per embryo reflects the outcome of the combination of embryonal (e.g., genomic) and extraembryonal factors. Important factors determining the implantation rate are age (2), quality of the embryos, quality and duration of culture (1,3), previous IVF-ET cycles and pregnancies (4), uterine anomalies (5,6), and general health problems (7). The decision of how many embryos to transfer (8) could be based on the presence of ovarian hyperstimulation syndrome, repetitive poor responses to ovarian hyperstimulation, impaired accessability of sperm (micro-
Consecutive day-5 blastocyst transfer
Vol. 69, No. 1, January 1998
Percentage of transfers with one or more blastocyst(s): the influence of the number of oocytes retrieved according to age. + = patients with one to four oocytes; ??= patients with more than four oocytes.
22-25
25-28
28-31
31-34
34-37
Age group
epididymal sperm aspiration, testicular sperm aspiration, cryosperm), quality of cryopreservation programs, and costeffectiveness. In 1990, Muggleton-Harris et al. (9) stated that the culture of human preimplantation embryos from the one-cell to the morula/blastocyst stage of development was not satisfactory; therefore, an improvement in the standard conditions used to culture the embryo was needed. The infrequent occurrence of blastocyst formation in vitro can have several possible causes. During a prolonged period, culture conditions can play an important role, especially when the incubators are opened between pronucleus inspection and ET for other purposes, thus leading to prolonged disturbance of temperature and CO, tension. In the present study and in the previous one (l), a rapid CO, recovery system was used, leading to complete recovery of CO, tension within 2 minutes after door opening. Using a system with one standard medium and strict control of culture conditions may have solved this culture problem. Although other groups are using coculture systems more or less successfully (lo), we refrained from coculture for reasons of unknown medical risks. The capacity of the zygotes to develop into blastocysts furthermore may be influenced by the embryonic genome (and gene activation) (11). FERTILITY & STERILITY@
Menezo et al. (12,13) showed that embryos with developmental arrest exhibited a high rate of aneuploidy and mosaicism. Bavister (14) reviewed the subject of coculture extensively and was critical about the use of coculture. The fact that transfer of up to two blastocysts is possible in 59% of all couples in the first cycle is of paramount importance. It is clear from these observations that prolonged embryo culture is the best method to prevent the occurrence of triplets, even when in the first treatment cycle no blastocysts were obtained. By transferring no more than two blastocysts, one can prevent triplets, exept, of course, in rare cases of monozygous twinning. The percentage of pronucleate embryos reaching the blastocyst stage seems to be less meaningful. It may be influenced primarily by individual variation in the actual number of pronucleate embryos, and possibly by paternal factors or polycystic ovary syndrome (15, 16). Our study indicates that it is not possible to select a category of patients by the capacity of blastulation in IVF culture. Absence of development of cavitating embryos in the first cycle seems to lead to a slightly lower chance of blastocyst formation in the subsequent cycle. During prolonged embryo culture, the effects of activation of the embryonic genome after the four- to eight-cell stage become visible. This may lead to natural selection according to the 81
Number of oocytes retrieved versus age: the percentage of ovum pickup with fewer than five oocytes.
0
22-25
25-28
28-31
31-34
34-37
37-40
40-43
43-47
Age group
potency for further development, as seems to be confirmed by the high implantation rate of blastocysts. We hypothesize that the capacity of an embryo to develop into the blastocyst stage depends primarily on the embryonic genome, as on day 5, considerable variability occurred between embryos growing in the same culture drop. It is still unclear whether lower implantation rates of morphologically optimal blastocysts in subsequent cycles after the second IVF-ET cycle are caused by embryonal factors or by extraembryonal factors such as disturbances in implantation. Theoretically, more definite answers could be obtained by egg donation programs in which embryos originating from older egg donors would be implanted in younger recipients and vice versa. Obviously, such an experiment would be unethical. Although data reported by others are partially contradictory as to lower PRs and implantation rates in subsequent cycles, larger studies confirm our findings (17-19). Because the occurrence of blastocyst formation in successive treatment cycles varies widely, it seems that we can rule out a parental factor. It is more likely that the embryonic genome plays an important role in determining the chance of ET with blastocysts. Even in the older patient, the influence of age is mediated by the number of oocytes obtained. The chance of blastocyst transfer depends on the number of oocytes, especially after 82
Scholtes and Zeilmaker
37 years of age. Roest et al. (20) demonstrated that the lower number of oocytes in the older patient was the principal factor for a lower PR. Prolonged embryo culture results in a smaller number of embryos available for cryopreservation, as embryos with developmental arrest will not be cryopreserved. Unfortunately, blastocyst/ICM viability seems to be impaired after thawing (21,22). Failure to achieve blastocyst formation is a bad prognostic factor for conception because of the low (2-6%) implantation rate of noncavitating day-5 embryos. Although the embryo’s potential to develop may be limited by the embryonal genome, careful attention nevertheless should be given to optimizing culture conditions. References 1. Scholtes MCW, Zeilmaker GH. A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil Steril 1996;65:1245-8. 2. Hull MGR, Fleming CF, Hughes AO, McDermott A. The age-related decline in female fecundity: a qualitative controlled study of implanting capacity and survival of individual embryos after in vitro fertilization. Fertil Steril 1996;65:783-90. 3. van OS HC, Alberda ATh, Janssen-Caspers HAB, Leerentveld RA, Scholtes MCW, Zeilmaker GH. The influence of the interval between in vitro fertilization and embryo transfer and some other variables on treatment outcome. Fertil Steril 1989;51:360-2. 4. Molloy D, Doody ML, Breen T. Second time around: a study of patients seeking second assisted reproduction pregnancies. Fertil Steril 1995; 64:546-51. 5. Nickerson CW. Infertility and the uterine contour. Am J Obstet Gynecol 1977;129:268-73.
Consecutive day-5 blastocyst transfer
Vol. 69, No.
1,
January 1998
6. Sorensen SS. Minor mullerian anomalies and oligomenorrhoea in infertile women: a new syndrome. Am J Obstet Gynecol 1981;140:636. 7. Ashkenazi J, Farhi J, Dicker D, Feldberg D, Shalev J, Ben-Rafael Z. Acute pelvic inflammatory disease after oocyte retrieval: adverse effects on the results of implantation. Fertil Steril 1994;61:526-8. 8. Roest J, van Heusden AM, Verhoeff A, Mous HVM, Zeilmaker GH. A triplet pregnancy after in vitro fertilization is a procedure-related complication that should be prevented by replacement of two embryos only. Fertil Steril 1997;67:290-5. 9. Muggleton-Harris AL, Findley I, Whittingham DG. Improvement of the culture conditions for the development of human preimplantation embryos. Hum Reprod 1990;5:217-20. 10. Bongso A, Ng SC, Fong CY, Ratnam S. Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil Steril 1991;56:179-91. 11. Braude F, Bolton V. Moore S. Human eene exuression first occurs between the 4- and 8-cell stages of pr%mpla&tion development. Nature 1988;332:459-61. 12. Menezo YJ, Nicollet B, Dumont M, Hazout A, Janny L. Factors affecting human blastocvst formation in vitro and freezing at the blastoc$st stage. Acta Eur Fertil 1993;24:207-13. 13. Menezo YJ, Ben-Khalifa M. Cytogenetic and cryobiology of human cocultured embryos: a 3-year experience. J Assist Reprod Genet 1995; 12:35-40. 14. Bavister B. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1:91-148.
FERTILITY
& STERILITY@
15. Janny L, Menezo YJ. Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol Reprod Dev 1994;38:36-42. 16. Deaton JL, Dempsey RA, Miller KA. Serum from women with polycystic ovary syndrome inhibits fertilization and embryonic development in the murine in vitro fertilization model. Fertil Steril 1996,65: 1224-8. 17. Tan SL, Royston P, Campbell S, Jacobs HS, Betts J, Mason B, et al. Cumulative conception and livebirth rates after in-vitro fertilization. Lancet 1992;339:1390-4. 18. Fertilisation in vitro National (FIVNAT). Evolution of prognostic criteria of in vitro fertilization according to the rank of attempts. Contracept Fertil Sex 1994;22:282-6. 19. Alsalili M, Yupze A, Tummon 1, Parker J, Martin J, Daniel S, et al. Cumulative pregnancy rates and pregnancy outcome after in-vitro fertilization: >50OClcvcles at one centre. Hum Reorod 1995:10:470-4. 20. Roest J, van Heusden AM, Mous H, Zeilmaker’GH, Verhoeff A. The ovarian response as a predictor for successful in vitro fertilization treatment after the age of 40 years. Fertil Steril 1997$X:969-73. 21. Takagi M, Sakonju L, Otoi T, Hamana K, Suzuki T. Postthaw viability of the inner cell mass of in vitro-matured/in vitro-fertilized bovine embryos frozen in various cryoprotectants. Cryobiology 1994;31:398405. 22. Kaufman RA, Menezo Y, Hazout A, Nicollet B, Dumont M, Servy EJ. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril 1995;64:1125-9.
83
Blastocyst transfer in day-5 embryo transfer depends primarily on the number of oocytes retrieved and not on age _
Michael C. W. Scholtes, M.D., and Gerard H. Zeilmaker, Ph.D. Department
of Endocrinology
and Reproduction,
Erasmus University Rotterdam,
Rotterdam,
the Netherlands
Objective: To analyze the effects of patient age and treatment cycle number on the occurrence of blastocyst transfer and subsequent implantation. Design: Prospective study. Setting: Department of endocrinology and reproduction. Patient(s): All 1,099 women had day-5 transfers after IVF or intracytoplasmic sperm injection treatment. Intervention(s): All patients were checked for embryo development in vitro in consecutive day-5 transfer cycles. Two blastocysts or three lesser-developed embryos were transferred. Main Outcome Measure(s): Blastocyst formation rate or clinical pregnancy/implantation rate. Result(s): Of 929 patients in the first cycle, 545 (59%) had at least one blastocyst available for ET. Among 151 patients with a blastocyst in cycle 1, 77 developed one or more blastocysts in cycle 2 (51%). Fifty of 143 patients without a blastocyst in cycle 1 had at least one blastocyst in cycle 2 (35%). After subdivision of all day-5 ETs according to the first four cycles, the following implantation rates per embryo were found for ET with one or more blastocysts: cycle 1 (n = 545), 23%; cycle 2 (n = 264) 23%; cycle 3 (n = llO), 14%; and cycle 4 (n = 27), 12%, and with noncavitating embryos, respectively: (n = 384) 6%, (n = 193) 6%, (n = 94) 2%, and (n = 35) 3%. The negative correlation of the age of the woman on blastulation depended primarily on the number of oocytes retrieved. Conclusion(s): The blastocyst implantation rate decreased after cycle 2. Biologic ovarian age, rather than chronoloaic age, determines the frequency of blastocyst transfer or pregnancy rate. (FertilStez’a’ 1998;69:78-83. 01998 by American Society for Reproductive Medicine.) Key Words: Blastulation, blastocyst, consecutive cycles, embryo development, embryo transfer, implantation rate, female age Received
May 6, 1997.
Reprint requests: Gerard H. Zeilmaker, Ph.D., Department of Endocrinology and Reproduction, Erasmus University, Postbox 1738, 3000 DR Rotterdam, the Netherlands (FAX: 31-l O4366832). 00150282/98/$19.00 PII s0015-0292(97)00450-0
78
In a previous study, it was demonstrated that after prolonged culture of embryos for 5 days, blastocysts formed, with a high implantation rate (1). Transfer of only two blastocysts led to a high pregnancy rate (PR) without the risk of triplets. Because the intention of IVF treatment is to provide a chance for subfertile couples to deliver a mature infant, after a low-risk single-
ton or gemellary pregnancy with minimal perinatal complications, prolonged embryo culture offers the best opportunity. In the present study, we analyzed successive IVF-ET cycles to determine whether blastocyst formation is more likely to occur under certain circumstances. The effect of patient age in relation to blastocyst transfer also was studied.
of all day-5 ETs according to cycle number, ET with at least one blastocyst or without cavitating embryos, and the implantationrate per embryo.
Subdivision
Implantation
Implantation rate per embryo No. of ETs with blastocysts
Cycle no.
23* 23* 14 12
545 264 110 21
1 2 3 4
Note. NS = not significant. * P = 0.02 (vs. implantation t P = 0.08 (vs. implantation
(%)
384 193 94 35
rate per embryo (%)
P value
6t 6t
0.001 0.001 0.002 0.08 (NS)
2 3
rate in cycles 3 and 4). rate in cycles 3 and 4).
Finally, successive day-5 transfers were analyzed to demonstrate the possible effect of the number of treatment cycles on blastocyst formation and pregnancy induction.
MATERIALS AND METHODS This study included all couples undergoing ET after 5 days of embryo culture from an ongoing prospective randomized, IRB-approved study of ET after 3-5 days conducted between January 1994 and December 1995 (1). Two different groups were identified. Group 1 comprised 1,099 patients having day-5 ET in one or more subsequent IVF-ET or intracytoplasmic sperm injection (ICSI)-ET cycles. Group 2 included couples having day-5 ET (n = 294) exclusively in all subsequent IVF-ET cycles. The effect of patient age on the frequency of blastocyst transfer was studied by age groups of 3 years from 22 to 47 years of age. Ovarian stimulation consisted of a long-acting GnRH agonist (GnRH-a), 200 pg intranasally administered twice daily starting in the late luteal phase. After ultrasonic evidence of ovarian suppression, hMG or FSH was given at a dose of 150 IU/d IM or SC. Higher or lower initial doses were used if necessary, as dictated by any previous response to ovarian stimulation. Intramuscular hCG was administered at a dose of 10,000 IU when the mean diameter of two or more dominant follicles was 18 mm. The GnRH-a and hMG were discontinued when hCG was given. Transvaginal oocyte retrieval was performed 35 hours after administration of hCG. Luteal support generally consisted of 600 mg of progesterone administered vaginally, in combination with 1,500 IU of hCG on days 2, 5, and 8 after ovum pickup, when no signs of increased risk for ovarian hyperstimulation syndrome were present. To analyze the effect of the number of oocytes on the frequency of blastocyst transfer, we compared patients with up to four and five or more oocytes by ovum pickup. After fertilization, the embryos were cultured in a standard medium without coculture, under mineral oil with strict COZ control. Rapid CO, recovery occurred. Details of this FERTILITY
No. of ETs without blastocysts
& STERILITY
@
element of the study can be found as referenced (1) or at the intemet address: http://huizen.dds.nV-ghz/starlab.html). Embryo development on day 5 varied from 8 to 32 cell stages, morula, morula-blastula stage (differentiating morula stages without a visible blastocele), and cavitating embryos: from early blastocysts with or without a clear inner cell mass (ICM) to expanding blastocysts with or without a normal amount of ICM. On day 3 after OPU, a maximum of three embryos usually was transferred. However, after 5 days of embryo culture, ET was reduced to two embryos whenever two blastocysts with a clear ICM were available. In other cases, the couples were consulted individually regarding the number of embryos to be transferred, with the maximum number being three. Pregnancies were recorded by serum hCG assay (>25 IV/L) 217 days after ovum pickup, and multiple pregnancies were confirmed by multiple positive heartbeats recorded by ultrasonography 31 days after ovum pickup. Statistics were calculated by 2 analysis and by CochranMantel-Haenszel statistics and logistic regression.
RESULTS In the first IVF-ET cycle, consisting of 929 patients having day-5 ET, 545 (59%) had at least one blastocyst available for ET and 384 did not achieve blastulation. Among 151 patients with a blastocyst in cycle 1, 77 developed one or more blastocysts in cycle 2 (51%). Interestingly, 50 of 143 (35%) patients without a blastocyst in cycle 1 had at least one blastocyst in cycle 2 (P = 0.006). In total, 946 ETs were carried out with one or more blastocysts, leading to 321 clinical pregnancies (34%). Among 586 day-5 ETs without blastocysts, only 47 women became pregnant (8%) (P = 0.001). Eight of these pregnancies resulted from 228 transfers in which one or more eight-cell embryos were the most advanced stages available (3.5%). The other preg79
Percentage of ETs with one or more blastocyst(s) according to the number of oocytes retrieved.
5-10
IO-15
15-20
No. of oocytes retreived
nancies in this patient category came from transfers of morulae or differentiating morulae (10.9%). Subdivision according to the cycle number of all day-5 ETs (Table 1) showed comparable implantation rates per embryo in the first (n = 929) and second (n = 457) IVF-ET cycles after transfer with at least one blastocyst (23% and 23%), and for lesser stages of embryonic development of 6% and 6%. In contrast, in cycles 3 (n = 204) and 4 (n = 62), a decrease in implantation rates was found for blastocysts (P = 0.02), with 14% and 12%, respectively, and for lesser stages (P = 0.08), with 2% and 3%, respectively. It appears that on day 5, the implantation chance for a blastocyst transfer was three times higher than for noncavitating embryos (relative risk, 3.025). Taking into account the cycle number of IVF treatment, the age of the woman had a strong correlation with the capacity for blastulation (P = 0.0001). The chance of blastocyst transfer decreased by 14% per age group of + 3 years. In addition, by regression analysis, the chance of blastocyst transfer decreased by 13% per cycle number. The apparent effect of female age on the chance of blastocyst transfer seemed to be mediated by the number of oocytes retrieved. The relation between the number of oocytes and the percentage of blastocyst transfers is demonstrated in Figure 1 (P = 0.0001, odds ratio 1.395). When only patients with more than four oocytes were considered, 80
Scholtes and Zeilmaker
the chance of blastocyst transfer decreased per age group by 9.9% (P = O.Ol), but was significantly better (P = 0.0001, odds ratio 1.901) in comparison with patients with fewer than five oocytes (Fig. 2). Finally, the increasing proportion of patients with fewer than five oocytes (P = 0.001) per age group upon ovum pickup further explained the decreasing percentage of patients achieving blastocyst transfer (Fig. 3).
DISCUSSION Because the replacement of two blastocysts on day 5 leads to a high pregnancy rate (PR) (1) and prevents triplets, the chance to attain a blastocyst transfer was investigated in consecutive IVF-ET cycles, in relation to patient age and ovarian response on gonadotropins. Furthermore, successive day-5 IVF-ET cycles were analyzed according to PRs and implantation rates per embryo. The implantation rate per embryo reflects the outcome of the combination of embryonal (e.g., genomic) and extraembryonal factors. Important factors determining the implantation rate are age (2), quality of the embryos, quality and duration of culture (1,3), previous IVF-ET cycles and pregnancies (4), uterine anomalies (5,6), and general health problems (7). The decision of how many embryos to transfer (8) could be based on the presence of ovarian hyperstimulation syndrome, repetitive poor responses to ovarian hyperstimulation, impaired accessability of sperm (micro-
Consecutive day-5 blastocyst transfer
Vol. 69, No. 1, January 1998
Percentage of transfers with one or more blastocyst(s): the influence of the number of oocytes retrieved according to age. + = patients with one to four oocytes; ??= patients with more than four oocytes.
22-25
25-28
28-31
31-34
34-37
Age group
epididymal sperm aspiration, testicular sperm aspiration, cryosperm), quality of cryopreservation programs, and costeffectiveness. In 1990, Muggleton-Harris et al. (9) stated that the culture of human preimplantation embryos from the one-cell to the morula/blastocyst stage of development was not satisfactory; therefore, an improvement in the standard conditions used to culture the embryo was needed. The infrequent occurrence of blastocyst formation in vitro can have several possible causes. During a prolonged period, culture conditions can play an important role, especially when the incubators are opened between pronucleus inspection and ET for other purposes, thus leading to prolonged disturbance of temperature and CO, tension. In the present study and in the previous one (l), a rapid CO, recovery system was used, leading to complete recovery of CO, tension within 2 minutes after door opening. Using a system with one standard medium and strict control of culture conditions may have solved this culture problem. Although other groups are using coculture systems more or less successfully (lo), we refrained from coculture for reasons of unknown medical risks. The capacity of the zygotes to develop into blastocysts furthermore may be influenced by the embryonic genome (and gene activation) (11). FERTILITY & STERILITY@
Menezo et al. (12,13) showed that embryos with developmental arrest exhibited a high rate of aneuploidy and mosaicism. Bavister (14) reviewed the subject of coculture extensively and was critical about the use of coculture. The fact that transfer of up to two blastocysts is possible in 59% of all couples in the first cycle is of paramount importance. It is clear from these observations that prolonged embryo culture is the best method to prevent the occurrence of triplets, even when in the first treatment cycle no blastocysts were obtained. By transferring no more than two blastocysts, one can prevent triplets, exept, of course, in rare cases of monozygous twinning. The percentage of pronucleate embryos reaching the blastocyst stage seems to be less meaningful. It may be influenced primarily by individual variation in the actual number of pronucleate embryos, and possibly by paternal factors or polycystic ovary syndrome (15, 16). Our study indicates that it is not possible to select a category of patients by the capacity of blastulation in IVF culture. Absence of development of cavitating embryos in the first cycle seems to lead to a slightly lower chance of blastocyst formation in the subsequent cycle. During prolonged embryo culture, the effects of activation of the embryonic genome after the four- to eight-cell stage become visible. This may lead to natural selection according to the 81
Number of oocytes retrieved versus age: the percentage of ovum pickup with fewer than five oocytes.
0
22-25
25-28
28-31
31-34
34-37
37-40
40-43
43-47
Age group
potency for further development, as seems to be confirmed by the high implantation rate of blastocysts. We hypothesize that the capacity of an embryo to develop into the blastocyst stage depends primarily on the embryonic genome, as on day 5, considerable variability occurred between embryos growing in the same culture drop. It is still unclear whether lower implantation rates of morphologically optimal blastocysts in subsequent cycles after the second IVF-ET cycle are caused by embryonal factors or by extraembryonal factors such as disturbances in implantation. Theoretically, more definite answers could be obtained by egg donation programs in which embryos originating from older egg donors would be implanted in younger recipients and vice versa. Obviously, such an experiment would be unethical. Although data reported by others are partially contradictory as to lower PRs and implantation rates in subsequent cycles, larger studies confirm our findings (17-19). Because the occurrence of blastocyst formation in successive treatment cycles varies widely, it seems that we can rule out a parental factor. It is more likely that the embryonic genome plays an important role in determining the chance of ET with blastocysts. Even in the older patient, the influence of age is mediated by the number of oocytes obtained. The chance of blastocyst transfer depends on the number of oocytes, especially after 82
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37 years of age. Roest et al. (20) demonstrated that the lower number of oocytes in the older patient was the principal factor for a lower PR. Prolonged embryo culture results in a smaller number of embryos available for cryopreservation, as embryos with developmental arrest will not be cryopreserved. Unfortunately, blastocyst/ICM viability seems to be impaired after thawing (21,22). Failure to achieve blastocyst formation is a bad prognostic factor for conception because of the low (2-6%) implantation rate of noncavitating day-5 embryos. Although the embryo’s potential to develop may be limited by the embryonal genome, careful attention nevertheless should be given to optimizing culture conditions. References 1. Scholtes MCW, Zeilmaker GH. A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil Steril 1996;65:1245-8. 2. Hull MGR, Fleming CF, Hughes AO, McDermott A. The age-related decline in female fecundity: a qualitative controlled study of implanting capacity and survival of individual embryos after in vitro fertilization. Fertil Steril 1996;65:783-90. 3. van OS HC, Alberda ATh, Janssen-Caspers HAB, Leerentveld RA, Scholtes MCW, Zeilmaker GH. The influence of the interval between in vitro fertilization and embryo transfer and some other variables on treatment outcome. Fertil Steril 1989;51:360-2. 4. Molloy D, Doody ML, Breen T. Second time around: a study of patients seeking second assisted reproduction pregnancies. Fertil Steril 1995; 64:546-51. 5. Nickerson CW. Infertility and the uterine contour. Am J Obstet Gynecol 1977;129:268-73.
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6. Sorensen SS. Minor mullerian anomalies and oligomenorrhoea in infertile women: a new syndrome. Am J Obstet Gynecol 1981;140:636. 7. Ashkenazi J, Farhi J, Dicker D, Feldberg D, Shalev J, Ben-Rafael Z. Acute pelvic inflammatory disease after oocyte retrieval: adverse effects on the results of implantation. Fertil Steril 1994;61:526-8. 8. Roest J, van Heusden AM, Verhoeff A, Mous HVM, Zeilmaker GH. A triplet pregnancy after in vitro fertilization is a procedure-related complication that should be prevented by replacement of two embryos only. Fertil Steril 1997;67:290-5. 9. Muggleton-Harris AL, Findley I, Whittingham DG. Improvement of the culture conditions for the development of human preimplantation embryos. Hum Reprod 1990;5:217-20. 10. Bongso A, Ng SC, Fong CY, Ratnam S. Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil Steril 1991;56:179-91. 11. Braude F, Bolton V. Moore S. Human eene exuression first occurs between the 4- and 8-cell stages of pr%mpla&tion development. Nature 1988;332:459-61. 12. Menezo YJ, Nicollet B, Dumont M, Hazout A, Janny L. Factors affecting human blastocvst formation in vitro and freezing at the blastoc$st stage. Acta Eur Fertil 1993;24:207-13. 13. Menezo YJ, Ben-Khalifa M. Cytogenetic and cryobiology of human cocultured embryos: a 3-year experience. J Assist Reprod Genet 1995; 12:35-40. 14. Bavister B. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1:91-148.
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15. Janny L, Menezo YJ. Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol Reprod Dev 1994;38:36-42. 16. Deaton JL, Dempsey RA, Miller KA. Serum from women with polycystic ovary syndrome inhibits fertilization and embryonic development in the murine in vitro fertilization model. Fertil Steril 1996,65: 1224-8. 17. Tan SL, Royston P, Campbell S, Jacobs HS, Betts J, Mason B, et al. Cumulative conception and livebirth rates after in-vitro fertilization. Lancet 1992;339:1390-4. 18. Fertilisation in vitro National (FIVNAT). Evolution of prognostic criteria of in vitro fertilization according to the rank of attempts. Contracept Fertil Sex 1994;22:282-6. 19. Alsalili M, Yupze A, Tummon 1, Parker J, Martin J, Daniel S, et al. Cumulative pregnancy rates and pregnancy outcome after in-vitro fertilization: >50OClcvcles at one centre. Hum Reorod 1995:10:470-4. 20. Roest J, van Heusden AM, Mous H, Zeilmaker’GH, Verhoeff A. The ovarian response as a predictor for successful in vitro fertilization treatment after the age of 40 years. Fertil Steril 1997$X:969-73. 21. Takagi M, Sakonju L, Otoi T, Hamana K, Suzuki T. Postthaw viability of the inner cell mass of in vitro-matured/in vitro-fertilized bovine embryos frozen in various cryoprotectants. Cryobiology 1994;31:398405. 22. Kaufman RA, Menezo Y, Hazout A, Nicollet B, Dumont M, Servy EJ. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril 1995;64:1125-9.
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