Integration of blastocyst transfer for all patients

FERTILITY AND STERILITY威 VOL. 77, NO. 4, APRIL 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

IN VITRO FERTILIZATION

Integration of blastocyst transfer for all patients Michael Wilson, Ph.D., Kathy Hartke, B.A., Michelle Kiehl, B.S., Jonetta Rodgers, M.S., Celeste Brabec, M.D., and Rodney Lyles, M.D. Reproductive Resource Center of Greater Kansas City, P.A., Overland Park, Kansas

Objective: To evaluate the efficacy of using day-5 embryo transfer (ET) for all patients. Design: Retrospective, non-randomized comparison of day-3 ET versus day-5 ET. Setting: Private practice. Patient(s): Women having in vitro fertilization and receiving either day-3 ET or day-5 ET. Intervention(s): Extended embryo culture through embryonic genome activation to select those embryos with higher implantation potential. Main Outcome Measure(s): The following parameters were compared for day-3 ET and day-5 ET: clinical and ongoing pregnancy rates, implantation rates, patients having cryopreservation, and liveborn rates. Result(s): Blastocyst embryo transfer (day-5 ET) increased the clinical and ongoing pregnancy rate for patients ⬍35 years of age and for the total program. The risk of high order multiple pregnancy was reduced by a decrease in the number of embryos transferred (2.0 vs. 2.7; day-5 ET vs. day-3 ET, respectively). However, the multiple pregnancy rate was not different due to an increase in the implantation rate (day-5 ET 43% vs. day-3 ET 27%). There was no difference in the percent of patients not having an ET (day-5 ET 2.8%; day-3 ET 1.3%). Conclusion(s): Blastocyst ET increased the clinical and ongoing pregnancy rate for patients ⬍35 years of age and for the total program, with a concomitant decrease in the number of embryos transferred. However, the decrease in the number of embryos transferred did not decrease the multiple rate, but did reduce the incidence of high order multiples. Most patients (97%) had an embryo transfer. Therefore, culturing embryos an additional 48 hours through embryonic genome activation allows for selection of the most viable embryos without compromising the patient’s opportunity to become pregnant. (Fertil Steril威 2002;77:693– 6. ©2002 by American Society for Reproductive Medicine.) Key Words: IVF, blastocyst, pregnancy, embryo culture

Received July 10, 2001; revised and accepted September 28, 2001. Reprint requests: Michael Wilson, Ph.D., Reproductive Resource Center of Greater Kansas City, P.A., 12200 West 106th Street, Suite 120, Overland Park, Kansas 66215 (FAX: 913-894-0841; E-mail: [email protected]). 0015-0282/02/$22.00 PII S0015-0282(01)03235-6

Reducing the number of embryos transferred to minimize high order multiple pregnancies without compromising the patient’s chance of achieving a pregnancy should be the goal of all assisted reproductive technology (ART) programs. To accomplish this goal, embryos that have the greatest potential for implantation must be selected for embryo transfer (ET). Advances in embryo culture media, specifically the development of sequential media, to promote embryonic growth through genome activation, blastocoele development, and embryonic expansion, have allowed for selection of those embryos with the greatest implantation potential (1). Several investigators (2– 6) have shown that embryo development in sequential media and blastocyst ET increases pregnancy and implan-

tation rates in selected patients. Marek et al. (7) demonstrated an increase in implantation rates for all patients when blastocyst transfer, rather than day-3 ET, was used. Moreover, Schoolcraft and Gardner (8) increased clinical pregnancy rates and implantation rates in their donor oocyte program using blastocyst transfer. Other investigators (9, 10) have shown that selection of embryos on day 3 is a poor predictor of blastocyst development. These data are a retrospective, nonrandomized analysis of outcomes following day-3 ET (419) or day-5 ET (570). Day-3 ET data were from two ovarian stimulation protocols (Fertinex and Gonal-F; Serono Laboratories, Randolph, MA) and embryo culture systems (human tubal fluid [HTF]; Irvine Scientific, Irvine, CA, ⫹ maternal plasma 693

[MP] and sequential media, IVF Science; Gothenburg, Sweden). When these data were compared, there was no difference (P⬎.05) in all parameters measured; therefore, data were combined. Day-5 ET data were from the same ovarian stimulation protocol (Gonal-F) and embryo culture system (sequential media) used for day-3 ET. One physician managed the stimulations, and performed the retrievals and embryo transfers for ⬎95% of the cycles analyzed.

MATERIAL AND METHODS Patients underwent controlled ovarian hyperstimulation using a GnRH analog (Lupron; TAP Pharmaceuticals, Waukegan, IL) and either a highly purified FSH (Fertinex) or a recombinant FSH (Gonal-F). Patients were monitored via ultrasonography and serum estradiol (E2) assay for follicular development. When at least two follicles with a mean diameter of 18 to 20 mm were present, patients were instructed to administer hCG. Follicular aspiration was scheduled 33 to 36 hours after hCG administration. Oocytes were retrieved in modified human tubal fluid (MHTF; Irvine Scientific), located and placed into either HTF or P1 (Irvine Scientific) ⫹ 0.5% human serum albumin (HSA) (Irvine Scientific) until insemination or sperm injection. Oocytes were inseminated in microdrops, either overnight in P1 ⫹ 0.5% HSA or for 2 hours in HTF ⫹ 0.5% HSA. Oocytes intended for intracytoplasmic sperm injection (ICSI) were placed into P1 plus hyaluronidase (80 IU/mL; Sigma, St. Louis, MO) 1 to 2 hours after retrieval to remove cumulus/corona cells. Oocytes were then placed into P1 ⫹ 0.5% HSA or G1.2 (IVF Science; Gothenburg, Sweden) microdrops under oil until injection. Following injection, oocytes were moved into P1 ⫹ 0.5% HSA or G1.2. All oocytes were evaluated for fertilization at 16 to 19 hours after insemination/ICSI. Normal, two pronuclear (2PN) zygotes were cultured in approximately 30 ␮L drops (HTF ⫹ 15% MP [day-3 ET embryos]; S1 or G1.2 [day-3 ET and day-5 ET embryos]) under oil, in groups of two or more. Embryos were evaluated and moved into new drops of media daily.

Day-3 Embryo Transfer From January 1997 to December 1998, embryos for day-3 ET were selected the morning of day 3 and moved into the transfer dish in HTF ⫹ 15% MP or G2.2 (IVF Science). Remaining embryos were transferred into new drops of media (HTF ⫹ 15% MP; S2 or G2.2) and were cultured an additional 48 hours. Embryos that developed to blastocyst stage were cryopreserved on day 5 or day 6 using the protocol described by Menezo et al. (11).

Day-5 Embryo Transfer From June 1998 to December 2000, for extended culture, embryos were moved into approximately 30 ␮L drops of S2/G2.2 (IVF Science) under oil on day 3, then were evaluated and moved into new media daily. Embryos for ET were 694

Wilson et al.

Blastocyst transfer for all patients

selected the morning of day 5 and moved into the transfer dish in S2/G2.2. Supernumerary blastocysts on day 5 were cryopreserved (11). Remaining embryos were cultured an additional 24 hours, and those that developed to blastocyst stage were cryopreserved on day 6. All embryos were transferred with a 23-cm Wallace (Edwards-Wallace; Cooper Surgical, Shelton, CT) catheter. Institutional review board approval was not obtained, as this study was a nonrandomized retrospective analysis of data that was not patient specific. Data were analyzed by chi-square and group comparisons.

RESULTS Extending embryo culture to day 5 before ET increased (P⬍.005) the clinical and ongoing pregnancy rates for patients ⬍35 years of age (Table 1). The clinical and ongoing pregnancy rates were not different (P⬎.05) for patients ⬎35 years of age or for donor oocyte recipients. However, when all data were combined, the clinical and ongoing pregnancy rates were greater (P⬍.005) for day-5 ET compared to day-3 ET. There was no difference (P⬎.05) in the multiple pregnancy rate (see Table 1). Selecting embryos for ET on day 5 decreased (P⬍.005) the miscarriage rate for patients 38 to 40 years of age, but there was no difference in the miscarriage rate (P⬎.05) for patients in other age groups. When cycles were analyzed for several parameters (Table 2), there was no difference (P⬎.05) in age, number of oocytes retrieved, fertilization rate, and number of 2PN embryos available for culture. Cancellation of ET was not different (P⬎.05) between day-5 ET and day-3 ET. The increased pregnancy rate for day-5 ET was due in part to an increased (P⬍.001) implantation rate, although fewer (P⬍.001) embryos were transferred on day 5. Additionally, more (P⬍.001) day-5 ET patients had supernumerary embryos cryopreserved (see Table 2), with no difference (P⬎.05) in the average number of embryos cryopreserved per patient (day-3 ET 3.6; day-5 ET 3.3). When data from conventional in vitro fertilization (IVF) and ICSI were compared, the clinical and ongoing pregnancy rates were greater (P⬍.05) for day-5 ET for both IVF (day-3 ET 50%/45%; day-5 ET 61%/55%) and ICSI (day-3 ET 44%/37%; day-5 ET 55%/51%), respectively. The number of oocytes retrieved and the fertilization rate were not different (P ⬎.05) between day-5 ET and day-3 ET for either IVF or ICSI. In 250 of 989 retrievals (day-3 ET 140; day-5 ET 110), three or less embryos (2PN) were available for culture. There was no difference (P⬎.05) in the clinical pregnancy rate (36%, 35%), ongoing pregnancy rate (31%, 30%), multiple pregnancy rate (24%, 19%), or miscarriage rate (14%, 14%) for day-3 ET and day-5 ET, respectively. Greater than 97% of these patients had an ET with a mean of 2.2 (day-3 ET) and 1.9 (day-5) embryos transferred. However, this group (⬍3 embryos) of patients had clinical and ongoing pregVol. 77, No. 4, April 2002

TABLE 1 Comparison of pregnancy, multiple, and miscarriage rates of day-3 ET and day-5 ET for different age groups.

⬍35

Patient age Day of ET No. of recipients Mean no. of embryos transfered Clinical pregnancy rate (%) Ongoing pregnancy rate (%) Implantation rate Multiple rate (%) Miscarriage rate (%)

3 192 2.7 53 48 30 38 8

35–37 5

291 2.0a 66b 62b 50b 43 7

3 89 2.6 45 42 25 30 8

⬎40

38–40 5

118 2.1a 52 46 34c 23 11

Donor oocyte recipient

3

5

3

5

3

5

64 2.9 38 23 16 21 38

73 2.2a 37 32 21 15 15b

13 3.3 46 38 21 50 17

12 2.2a 25 25 11 0 0

61 2.4 48 43 35 48 10

76 1.9 57 49 43 30 14

Total 3 419 2.7 48 42 27 36 12

5 570 2.0a 57b 52b 43b 35 9

Note: Chi-square and group comparisons. P⬍.001 compared to day-3 ET. b P⬍.005 compared to day-3 ET. c P⬍.05 compared to day-3 ET. a

Wilson. Blastocyst transfer for all patients. Fertil Steril 2002.

nancy rates that were lower (P⬍.05) than for day-3 ET and day-5 ET patients with ⬎3 embryos available for culture (day-3 ET 57%/50%; day-5 ET 65%/59%). Analysis of the 989 retrieval cycles revealed that in 60 (6%) cycles, three or fewer oocytes were retrieved. Eightythree percent of these patients had an embryo transfer with a 33% ongoing pregnancy rate. When these cycles were analyzed by day of transfer, there was no difference (P⬎.05) in clinical pregnancy rates, ongoing pregnancy rates, multiple rates, or miscarriage rates (day-3 ET 41%, 38%, 43%, 7%; day-5 ET 30%, 27%, 25%, 13%). Birth data for day-3 ET and day-5 ET were compared. There was no difference (P⬎.05) in the boy/girl ratio between day-3 ET and day-5 ET for IVF, ICSI, donor recipient,

or for the total program. Also, there was no difference (P⬎.05) between day-3 ET and day-5 ET for singleton (day-3 ET 119/64%; day-5 ET 194/66%), twin (day-3 ET 54/29%; day-5 ET 87/30%), or triplet (day-3 ET 12/6%; day-5 ET 12/4%) births. However, 11 of 12 triplet births from day-5 ET were due to monozygotic twinning. The other triplet birth was a 41-year-old to whom three embryos were transferred. There were no quadruplet pregnancies in day-3 ET or day-5 ET.

DISCUSSION In the preliminary study, day-5 ET was initially used for patients who had failed implantation with quality embryos

TABLE 2 Comparison of day-3 ET and day-5 ET for several parameters (nondonor cycles). Day-3 ET Embryo transfer No. of retrievals Mean patient age Mean no. of oocytes/retrieval Fertilization rate Mean no. of 2PN embryos Mean no. of embryos transferred Clinical pregnancy rate Implantation rate Ongoing pregnancy rate Patients with cryopreserved embryos No embryo transfer

Day-5 ET

n

%

n

%

P value

358.00 33.90 9.40 — 6.11 2.60 172.00 — 150.00 72.00 4.00

—

494.00 33.60 10.90 — 6.65 2.00 284.00 — 260.00 190.00 16.00

—

NS NS NS NS NS .001 .005 .001 .005 .001 NS

— 68 — — 48 27 42 21 1.3

— 68.0 — — 57.0 43.0 53.0 38.0 2.8

Note: Chi-square and group comparisons. NS ⫽ not significant (P⬎.05). Wilson. Blastocyst transfer for all patients. Fertil Steril 2002.

FERTILITY & STERILITY威

695

and day-3 ET. During this preliminary study, the pregnancy rate for day-5 ET was greater than for day-3 ET (data not shown). Data analyzed for this study have shown that the integration of blastocyst (day-5 ET) culture for all patients not only increased the clinical and ongoing pregnancy rate, but virtually eliminated the incidence of high-order multiple pregnancies due to a decrease in the number of embryos transferred. Additionally, extending embryo culture allows for the selection of the most viable embryos, as evidenced by an increase in the implantation rate. Scholtes and Zeilmaker (12) demonstrated that, by selecting cavitating embryos on day 5, pregnancy and implantation rates were greater than for day-3 ET for all age groups. Additionally, there was a reduction in the miscarriage rate for patients 38 to 40 years of age. Data from this study agree with that of Scholtes and Zeilmaker (12); pregnancy and implantation rates were greater for patients ⬍35 years of age and for the total program, and the miscarriage rate for patients 38 to 40 years of age was reduced.

percent of patients with an ET as compared to patients with ⬎3 oocytes at retrieval. However, there was no difference between day-3 ET and day-5 ET in the percentage of patients having an ET, when ⱕ3 oocytes were retrieved. Therefore, these data suggest that extending embryo culture to day-5 prior to embryo selection for ET does not further compromise a patient’s cycle with ⱕ3 oocytes or ⱕ3 embryos. However, as would be expected, the clinical and ongoing pregnancy rates for these patients (ⱕ3 oocytes or ⱕ3 embryos) were less than for patients who had sufficient embryos for selection. With almost 300 babies born from day-5 ET, there was no difference in the boy/girl ratio when compared to day-3 ET.

In these analyses, there was no difference between day-3 ET and day-5 ET in patient age, the number of pronuclear embryos available for culture, or percentage of patients who did not have an ET. This differs from the results of Marek et al. (7), who found an increase in the number of canceled ETs with day 5 ET. However, in agreement with Marek et al. (7), more day-5 ET patients had supernumerary embryos cryopreserved. When day-3 ET and day-5 ET data were compared by procedure (IVF/ICSI), clinical and ongoing pregnancy rates were greater for day-5 ET, with fewer embryos transferred on day 5.

1. Gardner DK, Lane M. Culture and selection of viable blastocyst: a feasible proposition for human IVF? Hum Reprod Update 1997;3:367– 82. 2. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocyst increases implantation rates and reduced the need for multiple embryo transfers. Fertil Steril 1998;69:84 – 8. 3. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434 – 40. 4. Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N, Wood C. Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod 1998;13:169 –77. 5. Behr B, Pool TB, Milki AA, Moore D, Gebhardt J, Dasig D. Preliminary clinical experience with human blastocyst development in vitro without co-culture. Hum Reprod 1999;14:454 –7. 6. Gardner DK, Lane M, Schoolcraft WB. Culture and transfer of viable blastocyst: a feasible proposition for human IVF. Hum Reprod 2000; 15:9 –23. 7. Marek D, Langley M, Gardner DK, Confer N, Doody KM, Doody KJ. Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program. Fertil Steril 1999;72:1035– 40. 8. Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril 2000;74: 482– 6. 9. Rijindas PM, Jason CAM. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 1998;13:2869 –73. 10. Graham J, Han T, Porter R, Levy M, Stillman R, Tucker MJ. Day 3 morphology is a poor predictor of blastocyst quality in extended culture. Fertil Steril 2000;74:495–7. 11. Menezo Y, Nicollet B, Herbaut N, Andre D. Freezing cocultured human blastocyst. Fertil Steril 1992;58:977– 80. 12. Scholtes M, Zeilmaker G. 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. 13. Scott L, Alvero R, Leondires M, Miller B. The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod 2000;15:2394 – 403. 14. Coskun S, Hollanders J, Al-Hassan S, Al-Sufyan H, Al-Mayman H, Jaroudi K. Day 5 versus day 3 embryo transfer: a controlled randomized trial. Hum Reprod 2000;15:1947–52.

Scott et al. (13) and Coskun et al. (14) have reported that they were unable to show a difference in pregnancy rates between day-3 ET and day-5 ET when day-5 ET was limited to those patients with a predetermined number of embryos. Our data has shown that the clinical and ongoing pregnancy rates were greater with day-5 ET for patients ⬍35 years of age and for the total program when no patient selection occurred. There was no difference in the clinical and ongoing pregnancy rates between day-5 ET and day-3 ET for other age groups, although the number of embryos transferred on day 5 was fewer. Additionally, when ⱕ3 oocytes or ⱕ3 embryos were available, there was no difference in the clinical or ongoing pregnancy rates between day-5ET and day-3ET. When ⱕ3 embryos were available for culture, the percent of patients who had an ET was not different from patients who had ⬎3 embryos available for culture. For those patients with ⱕ3 oocytes, there was a decrease in the

696

Wilson et al.

Blastocyst transfer for all patients

Overall, these data suggest that day-5 ET improves several important parameters for a successful ART program without compromising the patient’s opportunity for an embryo transfer or pregnancy. References

Vol. 77, No. 4, April 2002