Blastocyst culture and transfer increases the efficiency of oocyte donation

FERTILITY AND STERILITY VOL. 74, NO. 3, SEPTEMBER 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Blastocyst culture and transfer increases the efficiency of oocyte donation William B. Schoolcraft, M.D., and David K. Gardner, D. Phil. Colorado Center for Reproductive Medicine, Englewood, Colorado Objective: To determine the impact of blastocyst transfer on an oocyte donation program. Design: Retrospective review of embryo transfer in an IVF clinic. Setting: Private assisted reproductive technology unit. Patient(s): Two hundred and twenty nine patients undergoing oocyte donation. Intervention(s): Culture of pronucleate embryos to either day 3 or day 5 followed by embryo transfer. Main Outcome Measure(s): Implantation rates, pregnancy rates, and multiple gestations were analyzed. Result(s): Implantation rates and pregnancy rates were significantly increased by moving to extended embryo culture and transfer on day 5. After day 3 transfers, implantation and pregnancy rates were 47.1% and 75%, respectively. In contrast, on day 5 these rates were increased to 65.8% and 87.6%. Concomitantly, there were significantly fewer embryos transferred on day 5 (2.1) compared to day 3 (3.2). Conclusion(s): Blastocyst transfer is a highly effective treatment for patients who receive donor oocytes, allowing excellent pregnancy rates while significantly reducing the incidence of high-order multiple gestations. (Fertil Steril 2000;74:482 6. 2000 by American Society for Reproductive Medicine.) Key Words: Embryo culture, implantation, IVF, sequential media, viability Received December 3, 1999; revised and accepted March 14, 2000. Reprint requests: William B. Schoolcraft, M.D., Colorado Center for Reproductive Medicine, 799 East Hampden Ave., Suite 300, Englewood, Colorado 80110 (FAX: 303-788-4438; E-mail: bschoolcraft @colocrm.com). 0015-0282/00/$20.00 PII S0015-0282(00)00685-0 Since the first report of successful oocyte donation in 1984 by Lutjen et al. (1), this technique has become an increasingly important part of assisted reproductive technologies. Although initial indications for oocyte donation included premature ovarian failure and the avoidance of genetically transmissible disease, oocyte donation is most commonly used today in cases of advanced reproductive age, impaired ovarian reserve, and repetitive IVF failure because of oocyte defects. The aging of the reproductive population has led to a dramatic increase in the use of oocyte donation. Although pregnancy rates with oocyte donation are significantly higher than with standard IVF, according to the 1995 ASRM-SART registry (2) (ongoing pregnancy rates of 36% versus 22.5%, respectively), multiple gestations represent a significant complication of this technology. The 1995 SART report revealed a 41% incidence of multiple gestation, with 5% of these being high-order multiple gestations (triplets or greater). Because patients undergoing selective embryo reduction were excluded from this analysis, the true incidence of high-order multiple gestation may well exceed 10%. Recently, blastocyst culture and transfer in the human with use of sequential culture systems has been shown to be an effective means of limiting the number of embryos transferred and, therefore, the incidence of high-order multiple gestation, while maintaining excellent pregnancy rates (3 6). As the quality of embryos is ultimately dependent on the quality of the gametes from which the embryo is derived (7), it is plausible that embryo development to the blastocyst stage will be higher in oocyte donors, a hypothesis recently confirmed by Patton et al. (8), who found blastocyst implantation rates in donor recipients (52%) to be higher than blastocyst implantation rates in IVF patients under 40 years of age (31%). We therefore present our recent experience with oocyte donation at the Colorado Center for Reproductive Medicine and compare the outcome of recipient cycles having cleavage stage embryo transfer on day 3 to blastocyst transfer on day 5. 482

MATERIALS AND METHODS A retrospective review of all patients undergoing oocyte donation between January 1998 and August 1999 at the Colorado Center for Reproductive Medicine was performed. Cycle outcome was analyzed comparing day 3 (n 116) versus day 5 (n 113) embryo transfer. IRB approval was obtained. All patients were offered blastocyst transfer and counseled that in the majority of cases only two blastocysts would be transferred. Exceptions to the two-blastocyst rule were made when only low scoring blastocysts were available for transfer. Forty nine percent of patients elected to have blastocyst transfer. Oocyte donors consisted of paid anonymous volunteers or unpaid volunteers known to the recipient. The screening of oocyte donors included: basal FSH and estradiol levels to assess ovarian reserve, screening ultrasound including the assessment of resting follicle counts, sexually transmitted disease screening, complete physical examination, psychological evaluation, and genetic screening by an external consultant geneticist that included family pedigree and karyotyping. Oocyte donors initially underwent GnRH-a down-regulation at a dose of 0.5 mg per day SC, either starting in the midluteal phase of the prior menstrual cycle, or while receiving oral contraceptive pills. After successful down-regulation (E 2 35 pg/ml, no follicle 1.0 cm), hmg with recombinant FSH or pure recombinant FSH was initiated. The starting dose for FSH ranged from 2 to 4 ampules per day depending on basal FSH, resting follicle count, and age and body weight. HCG was administered when at least two follicles reached a mean diameter of 1.8 cm, and oocyte retrieval was scheduled for 36 hours later. Semen preparation was carried out with a 50%-70%-95% discontinuous PureSperm (Nidacon, Gothenburg) gradient. The pellet was washed in Ham s F-10 medium and stored in the incubator until insemination. One hundred thousand sperm per ml were added to each oocyte. All inseminations occurred in Ham s F-10 medium fetal calf serum. Assessment of fertilization took place 15 to 18 hours after insemination. Cumulus and corona cells were removed by dissection with 27-gauge disposable needles in an organ culture dish. All gamete and embryo manipulations occurred in a pediatric isolette designed to control humidity, temperature, and ph fluctuations. For day three embryo transfers, the culture system consisted of Ham s F-10 medium with 15% fetal cord serum or medium G1.2 (4). There was no significant difference in the performance of either medium with respect to implantation rates for the day 3 transfers. Embryos not transferred on day 3 were cultured to the blastocyst stage and cryopreserved in glycerol. For day 5 embryo transfers, embryos were cultured in G1.2 and G2.2 sequential media. The details of these culture methods have been previously discussed (4). All TABLE 1 Characteristics of oocyte donors. Variable Day 3 Day 5 P value No. of donors 116 113 Mean ( SEM) age of donors in years 28.8 0.44 27.8 0.41 NS Age range 21 41 21 37 Mean ( SEM) FSH level 6.3 0.16 6.6 0.20 NS Mean ( SEM) no. of oocytes 19.9 0.9 22.8 0.8.05 patients who underwent blastocyst transfer had embryos replaced on day 5. Blastocysts that were not transferred were cryopreserved. Oocyte donation recipients were initially down-regulated with GnRH-a followed by escalating doses of transdermal E 2 until an endometrial thickness of 8 mm and a triple layer pattern were achieved. E 2 levels were maintained between 3 pg/ml and 900 pg/ml. Leuprolide acetate was discontinued, and progesterone (50 mg in oil IM) was commenced on the day prior to the donor s oocyte retrieval. Embryo transfers were performed using a Wallace catheter under ultrasound guidance. Luteal support for all patients consisted of progesterone in oil, which was begun the night before the retrieval of oocytes from the donor. The initial dose was 50 mg or 1 ml daily the first two days, increasing to 100 mg or 2 ml daily from the third day of luteal support onward (9). Recipients concurrently received transdermal estrogen at a dose of 0.4 mg daily. Blastocysts were therefore transferred on the seventh day of progesterone administration. Clinical pregnancy was defined by the sonographic appearance of a fetal heart beat in the uterus six weeks after oocyte retrieval. Statistical Analysis The chi square or Student s unpaired t-test were used as appropriate. RESULTS The characteristics of the oocyte donors are presented in Table 1. There was no difference between the age and FSH level of the donors used for either day 3 or day 5 recipients. However, more oocytes were recovered from donors designated for the day 5 transfers. Table 2 shows the outcome of embryo transfer of donated oocytes after culture to day 3 compared to transfer on day 5. Patients who underwent embryo transfer on day 3 received significantly more embryos (3.2) than patients who underwent blastocyst transfer on day 5 (2.1). Subsequently, embryo freezing was performed for more patients on day 5 than on day 3, although the mean number of embryos frozen on each day was the same. Fifty eight percent of pronucleate FERTILITY & STERILITY 483

TABLE 2 Effect of day of transfer on implantation and pregnancy outcome in oocyte donors. Variable Day 3 Day 5 P value No. of patients 116 113 Mean ( SEM) age of recipient (y) 39.9 0.43 41.3 0.41 NS Age range (y) 28 48 27 50 Patients with ICSI (%) 26.7 32.9 NS Mean ( SEM) no. of pronuclear embryos (mean SEM) 11.9 0.62 14.2 0.53.01 Blastocyst development (%) 58.2 Mean ( SEM) no. of embryos transferred (mean SEM) 3.2 0.05 2.1 0.04.01 Patients with embryo freezing (%) 60.3 83.6.01 Mean ( SEM) no. of frozen embryos 5.2 0.59 5.6 0.43 NS Implantation rate (fetal sac) (%) 47.1 65.8.01 Implantation rate (fetal heart) (%) 41.6 65.0.01 Clinical pregnancy rate per retrieval (%) 75.0 87.6.05 No. of deliveries and ongoing pregnancies/no. of retrievals (%) 68.1 79.5 NS Twins (%) 25.0 41.6.05 Triplets (%) 15.5 2.6*.001 Multiples (%) 40.5 44.2 NS * Includes two incidences of monozygotic twinning. embryos undergoing extended culture reached the blastocyst stage and 84% of patients received blastocysts scoring 3AA or higher (see figure 1). Implantation rates when expressed as either fetal sac or fetal heart per embryo transferred were significantly increased by the transfer of blastocysts on day 5 (65.8% and 65%, respectively) compared to day 3 (47.1% and 41.6%, respectively; P 0.01). Even though more embryos were transferred on day 3, day 5 embryo transfer resulted in a significant increase in the clinical pregnancy rate (P.05). There was no overall significant difference in the number of multiple gestations conceived when embryos were transferred at the cleavage stage on day 3 or at the blastocyst stage on day 5. However, there was a significant reduction in highorder multiple gestations when blastocysts were transferred. Maternal age of the recipient did not have a significant effect on implantation or pregnancy rates (Table 3). When the effect of gonadotropin stimulation on the oocyte donors was examined, the presence of LH had no effect on any parameter measured. DISCUSSION When oocytes from young fertile volunteers are used and the recipient uterus is artificially prepared to maximize uterine receptivity, oocyte donation may represent an ideal model to assess the maximal efficiency of human conception. Indeed, the high pregnancy and implantation rates described in this series suggest that close to a 90% chance of pregnancy per embryo transfer is a reality with just two blastocysts transferred, culminating in a delivery/ongoing pregnancy rate of close to 80%. That blastocysts have implantation rates of 65% in recipients with an average age of 41.3 strongly suggests that it is the embryo, and not the uterus, which is the major determinant as to whether implantation is achieved. This observation is certainly consistent with the findings of Navot et al. (10), who demonstrated that the age-related decline in fertility resulted from a decline in oocyte potential rather than a decreased capacity of the uterus to receive a blastocyst. These data therefore allow for a reappraisal of the relationship between the embryo, uterus, and transfer efficiency parameters in the determination of ultimate IVF success. The efficacy of culture systems for human embryos are routinely compared to those used for other mammals, such as the mouse and cow. However, such comparisons are biased as the majority of laboratory and domestic animals are of known pedigree. Furthermore, the majority of reports on other mammals are from the embryos of fertile parents. In contrast, the human population represents the most genetically diverse group of mammals, and by default individuals who attend an infertility program are reproductively compromised. Therefore, the ability to assess embryo development in oocyte donors represents a more accurate reflection of the development potential of the human embryo in vitro. Data in this analysis showed that close to 60% of pronucleate embryos were able to form blastocysts. This is in comparison to just below 50% blastocyst development reported for general IVF patients attending the same clinic and using the same culture system (4), indicating that a more viable cohort of oocytes were obtained from the donors. Furthermore, the quality of the blastocysts as assessed using a three-part scoring system (5) was higher in the oocyte 484 Schoolcraft and Gardner Blastocyst transfer improves oocyte donation Vol. 74, No. 3, September 2000

FIGURE 1 Photomicrograph of human blastocysts developed from donated oocytes on the morning of day 5. Four blastocysts can be seen to be hatching from the zona pellucida. donors than the general IVF population. This could reflect the quality of oocytes, as the quality of an embryo is ultimately dependent upon the quality of the gametes from which it is derived (7). It has been demonstrated that blastocyst quality directly affects implantation rate and pregnancy outcome (11). It is proposed, therefore, that blastocyst development from donated oocytes is a valuable quality assurance parameter in the IVF laboratory, with which the efficacy of a given culture system can be evaluated more TABLE 3 Effect of maternal age of recipient when receiving blastocysts. 35 35 39 39 No. of recipients 8 27 78 No. of blastocysts received 2.0 0.00 2.1 0.07 2.1 0.05 Implantation rate (fetal sac) (%) 68.8 66.1 65.4 Implantation rate (fetal heart) (%) 68.8 64.3 63.6 Clinical pregnancy rate (%) 87.5 88.9 88.5 meaningfully. However, when interpreting blastocyst data from oocyte donors one has to acknowledge that embryo development could still be compromised because of unknown paternal effects on the embryo. Interestingly, there was no notable effect of recipient age on either implantation or pregnancy rates when blastocysts were transferred on day 5. This observation is in contrast to the findings of Yaron et al. (12), who observed a significant decrease in pregnancy rates with increasing recipient age, with 36.8% of patients under 30 becoming pregnant, compared to only 17.8% of recipients over 40. Similarly, Borini et al. (13), who used shared oocytes from a single donor, observed that both oocyte and uterine age impacted on implantation rates. These data suggest that the older uterus provides a less suitable environment that a younger one. Importantly, both Yaron et al. (12) and Borini et al. (13) transferred embryos on day 2 or 3, and not day 5. The fact that the pregnancy rate for patients over 40 was only 17.8% after the transfer of cleavage stage embryos (12), compared to 88.5% after the transfer of blastocysts, indicates that the blastocyst is more suited to a uterine environment, especially one that may be compromised in older patients. FERTILITY & STERILITY 485

TABLE 4 Effect of stimulation protocol on transfer outcome for patients receiving blastocysts. Pure FSH Presence of LH No. of patients 50 63 Mean age of recipient ( SEM) 41.4 0.6 41.2 0.6 Mean number of oocytes ( SEM) 23.9 1.4 21.9 0.9 Mean number of fertilized oocytes ( SEM) 15.1 0.9 13.4 0.6 Blastocyst formation (%) 59.2 57.4 Mean number of embryos transferred ( SEM) 2.1 0.04 2.1 0.06 Implantation rate (% fetal sac) 61.2 69.5 NS Implantation rate (% fetal heart) 59.2 69.5 NS Pregnancy rate (%) 84.0 90.5 NS It has been reported that the stimulation protocol can have a significant impact on implantation and pregnancy rates after blastocyst transfer in non-oocyte donor patients (11). The presence of LH in the patient stimulation regime was associated with significantly higher implantation and pregnancy rates. However, in this study, where the recipients had not undergone hyperstimulation, the presence of LH in the stimulation protocol of the oocyte donors did not result in a significant increase in either parameter. It is therefore plausible that LH that is present during the follicular phase is acting on the endometrium or ovarian function and not on the oocyte per se. This therefore warrants further investigation in a prospective randomized trial. The ability to successfully cryopreserve such blastocysts is significant because this results in high survival and pregnancy rates (14). With the introduction of improved cryopreservation techniques, such as container-less vitrification (15), success rates should further improve, making the transition to single blastocyst transfers more acceptable. In conclusion, the data reported here suggest that blastocyst transfer is a highly successful treatment in oocyte donation. The high implantation rates obtained for all age groups strongly supports the move to single blastocyst transfers, thereby overcoming the problems of multiple gestations. Acknowledgments: The authors gratefully acknowledge the staff of our clinical and research laboratories at the Colorado Center for Reproductive Medicine: John Stevens, B.S., Terry Schlenker, M.A., Michelle Lane, Ph.D., Kathy Hooper, B.S., Mike Baird, B.A., Susie Guadagnoli, B.S., Mary Rawlins, B.S. and Lyla Wagley, M.Sc., Mary Jo Steiert, R.N., and Cheri Schoolcraft, R.N. References 1. Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P. The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature 1984;307:174 5. 2. Assisted reproductive technology in the United States and Canada: 1995 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 1998;69:389 98. 3. Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update 1997;3: 367 82. 4. 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. 5. Gardner DK, Schoolcraft WB. In-vitro culture of human blastocysts. in Towards Reproductive Certainty: Infertility and Genetics Beyond 1999. eds. Jansen, R. and Mortimer, D. Parthenon Press, Carnforth, pp 378 88. 6. Milki AA, Fisch JD, Behr B. Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer. Fertil Steril 1999; 72:225 8. 7. Gardner DK, Schoolcraft WB. Human embryo viability: what determines developmental potential, and can it be assessed? J Assist Reprod Gen 1998;15:455 8. 8. Patton PE, Sadler-Fredd K, Burry KA, Gorrill MJ, Johnson A, Larson JM, Wolf DP. Development and integration of an extended embryo culture program. Fertil Steril 1999;72:418 22. 9. Meldrum DR. Female reproductive aging: ovarian and uterine factors. Fertil Steril 1993;59:1 5. 10. Navot D, Drews MR, Bergh PA, Guzman I, Karstaedt A, Scott RT Garrisi GJ, Hofmann GE. Age-related decline in female fertility is not due to diminished capacity of the uterus to sustain embryo implantation. Fertil Steril 1994;61:97 101. 11. Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999;72;604 9. 12. Yaron Y, Ochshorn Y, Amit A, Kogosowski A, Yovel I, Lessing JB. Oocyte donation in Israel: a study of 1001 initiated treatment cycles Hum Reprod 1998;13:1819 24. 13. Borini A, Bianchi L, Violini F, Maccolini A, Cattoli M Flamigni C. Oocyte donation program: pregnancy and implantation rates in women of different ages sharing oocytes from single donor. Fertil Steril 1996; 65:94 7. 14. Marek D, Langley M, Confer N, Cram L, Underwood L, Doody KM, Doody KJ. Frozen embryos transfer (FET) implantation and ongoing pregnancy rates resulting from day 3 multi-cell embryos compares with day 3 multi-cell embryos cultured to day 5 and day 5 blastocyst embryos. Proc Am Soc Rep Med 1999; O-008. 15. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999;72:1073 8. 486 Schoolcraft and Gardner Blastocyst transfer improves oocyte donation Vol. 74, No. 3, September 2000