In vitro development and pregnancy outcomes for human embryos cultured in either a single medium or in a sequential media system

Transcription

1 In vitro development and pregnancy outcomes for human embryos cultured in either a single medium or in a sequential media system Soledad Sepulveda, Ph.D., Javier Garcia, Ph.D., Elard Arriaga, Lic., Julio Diaz, M.D., Luis Noriega-Portella, M.D., and Luis Noriega-Hoces, M.D. Grupo PRANOR, Instituto de Ginecologıa y Reproduccion, Lima, Peru Objective: To compare a single medium, with medium renewal on day 3, with a sequential media system for development of human embryos to the blastocyst stage and subsequent pregnancy outcome. Design: Prospectively randomized study using donor oocytes. Setting: Private infertility clinic. Patient(s): Oocytes were collected from 47 donors (mean SD age ¼ years) from March to October 2006 and used for 79 patients ( years). The patients were prospectively randomized to have their embryos cultured in a single medium or in a sequential media system. Intervention(s): The donor oocytes were fertilized with fresh patient partner sperm and the resultant zygotes cultured in either a single medium or a sequential media system from day 1 until the blastocyst stage. After endometrial preparation the embryos were transferred to the patients on day 5 or day 6. Main Outcome Measure(s): In vitro embryo developmental characteristics and pregnancy and implantation rates. Result(s): In vitro development rates on days 3, 4, and 5 and implantation rates were significantly greater for embryos cultured in the single medium than for those cultured in the sequential media system. Conclusion(s): A single medium was as good as or better than a sequential media system for human embryo culture from the zygote to blastocyst stage. The idea that a sequential media system is required for optimal development of the human embryo to the blastocyst stage is highly questionable. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Embryo culture, sequential media, single medium On the basis of the differences in composition of oviductal and uterine secretions, as well as changes in embryo metabolism during early development, it has been suggested that a sequential media system is required for optimal development of the human embryo to the blastocyst stage (1). In such sequential media culture systems, the medium used for culture from days 1 to 3 of development differs in composition and/or concentration of the components from the medium used for subsequent culture from day 3 to the blastocyst stage (2, 3). As noted by Biggers et al. (4) the idea that sequential media are required for optimal embryo development has an intuitive appeal, but it is not supported by direct experimental evidence. Two studies have compared sequential media systems with a single culture medium for development of the human embryo to the blastocyst stage. Using sibling zygotes, the development after culture from day 1 to day 5 in KSOM aa, without medium renewal on day 3, was not different from that using Received October 16, 2007; revised February 26, 2008; accepted February 27, 2008; published online April 25, Embryo culture medium was supplied free of charge by IVFonline, Guelph, Ontario, Canada. Presented in abstract form at the Taller General RED Latinoamericana de Reproduccion Asistida, Mar del Plata, Argentina, April 27 29, Reprint requests: Soledad Sepulveda, Ph.D., Instituto de Ginecologia y Reproduccion, Manuel Olguin 1045, Surco, Lima, Peru (FAX: ; soledads@fertilidadperu.com). sequential culture in P-1 from day 1 to day 3 followed by culture in CCM from day 3 to day 5 (5). In a prospective, randomized study (6), three culture treatments were compared: culture in Rotterdam medium from day 1 to 5, culture in Rotterdam medium from day 1 to day 5 with a change to fresh medium on day 3, and culture in the sequential media of G1 from day 1 to day 3 followed by culture in G2 from day 3 to day 5. There were no differences in blastulation, implantation, or pregnancy rates among the three culture treatments. In both those studies (5, 6), the embryos were derived from oocytes collected from IVF patients, without patient selection. However, the success rates for IVF decrease dramatically with increased patient age (7). Consequently, the statistical analysis of the results of those studies may have been affected by within-group variability in oocyte quality associated with patient age. The objective of the study described here was to compare in vitro development and pregnancy rates for embryos cultured in a single medium, with medium renewal on day 3, or in sequential media. To minimize within-group variability in oocyte quality due to age, all the oocytes were obtained from young anonymous donors. The oocytes were donated to patients who had been prospectively randomized for culture of their embryos in either a sequential culture system or a single culture medium /09/$36.00 Fertility and Sterility â Vol. 91, No. 5, May doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 MATERIALS AND METHODS In this study, only standard assisted reproductive technology (ART) laboratory procedures were used. The comparison made was of culture of the embryos between two commercially available media, which are widely used in human ART. No other experimental treatments were used. Consequently, institutional review board approval was not required. Stimulation and Oocyte Collection Oocytes were obtained from 47 oocyte donors (age range ¼ years; mean SD ¼ years). Follicle-stimulating hormone (Puregon; Organon, Quimica Suiza, Peru) was used for follicular stimulation after down-regulation, in daily doses of IU. Dosage was administered once daily and was based on initial follicular dynamics on day 2 or 3 (day 0 ¼ day of menses). Follicular response was measured by transvaginal ultrasound as well as by measurement of estrogen activity using hormone assay. Follicular stimulation was continued until the oocyte donor had at least two follicles of R18 mm; final oocyte maturation was induced with hcg (SC injection of 10,000 IU Pregnyl; Organon). Oocyte pickup was performed 35 to 36 hours after hcg administration. Oocytes were recovered in N-2-hydroxyethylpiperazine-N 0-2-ethanesulfonic acid (HEPES)-buffered human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA) supplemented with 10% vol/vol synthetic serum substitute (SSS; Irvine Scientific). After retrieval, cumulus oocyte complexes were trimmed of excess cumulus cells and cultured in 100-mL drops of HTF þ 10% SSS under oil in 60-mm plates (Nalge Nunc International, Rochester, NY). A total of 913 oocytes were collected from the 47 donors ( oocytes per donor). The oocytes from each donor were divided among 1 to 4 patients before fertilization by conventional insemination or intracytoplasmic sperm injection (ICSI). Sperm Collection Semen was collected by masturbation from the patients partners. Motile spermatozoa were separated from the seminal plasma by centrifugation through 1.0 ml 95% and 45% Isolate gradients (Irvine Scientific) for 15 minutes at 300 g. The sperm were then washed with sperm-washing medium (Irvine Scientific). For oligospermic samples the sperm were washed and resuspended in varying amounts of sperm-washing medium depending on initial concentration and motility and then placed into 15-mL drops of HEPESbuffered HTF þ 10% SSS for ICSI. Fertilization For conventional insemination, approximately 60,000 spermatozoa were placed in 100-mL drops of HTF plus 10% SSS containing one to five oocytes. For ICSI, injected oocytes were placed in 100-mL drops of HTF plus 10% SSS. After insemination or ICSI on day 0 (Table 1), all oocytes were cultured for hours, at 37.1 C in an atmosphere of 5.6% CO 2 in air. Embryo Recipients A total of 80 patients, 40 per group, were randomly assigned to have their embryos cultured in either Global medium (IVFonline, Guelph, ON, Canada) or in Irvine Early Cleavage Medium (ECM)/Multiblast (Irvine Scientific), as described below. For clinical reasons, ET was performed on day 3 for one recipient in the ECM/Multiblast treatment group. The in vitro embryo development and pregnancy data for that patient were not included in the statistical analysis. As shown in Table 1, patient age did not differ between the culture treatment groups. To prepare the endometrium for transfer, each recipient was given daily oral doses of estradiol valerate from 2 to 6 mg, in ascending order. Administration began on day 2 or 3 of the cycle. Recipients needed to have a minimal endometrial thickness of 8 mm before receiving P. Recipients received 600 mg per day of micronized P (Geslutin; Tecnofarma, Lima, Peru) beginning the day of oocyte aspiration until the day of the pregnancy test. If the pregnancy test was positive, P supplementation was continued for an additional 2 months. Embryo Culture The embryo culture media were prepared and used according to the manufacturers instructions. The CO 2 concentration in the incubator was 5.6%, resulting in a ph of approximately 7.30 in all media. The morning after fertilization (day 1), zygotes with two pronuclei were randomly divided to be cultured individually, under oil, in 15-mL droplets of either ECM (n ¼ 322) or Global medium (n ¼ 287) from day 1 to day 3. Both media were supplemented with 10% vol/vol SSS. On day 3, the embryos that had been cultured in ECM þ 10% SSS from day 1 to 3 were moved to 15-mL droplets of Multiblast þ 10% SSS. The embryos that had been cultured in Global þ 10% SSS from day 1 to 3 were moved to fresh 15-mL droplets of Global þ 10% SSS. TABLE 1 Patient characteristics. Parameter Global ECM/ Multiblast No. of patients Patient age (y) Range Mean SD Standard IVF (% of patients) ICSI (% of patients) P 1766 Sepulveda et al. Single vs. sequential culture media Vol. 91, No. 5, May 2009

3 Evaluation of Embryo Development On day 2 the embryos were evaluated for cell number, fragmentation, and multinucleation; on day 3 for cell number, compaction, fragmentation, and multinucleation; on day 4 for development to morula; and on day 5 for development to blastocyst and expansion. Embryos that were full blastocysts or further in development were assigned inner cell mass (ICM) and trophectoderm scores. The ICM score was evaluated as follows: 3 ¼ compact area, many cells present; 2 ¼ cells are loosely grouped; or 1 ¼ very few cells present. The trophectoderm was scored as follows: 3 ¼ many cells forming a tight epithelial network of cells; 2 ¼ few cells forming a loose network of cells; or 1 ¼ very few or large cells. Embryo transfer was performed with a Frydman Ultrasoft catheter (Laboratoire CCD, Paris, France) that had been previously washed with culture medium. The catheter was completely filled with the embryos in the last 10 ml of the catheter. All transfers were performed as described by Mansour (8). Pregnancy Determinations Approximately 12 days after transfer, pregnancy was determined by quantitative b-subunit hcg. Transvaginal ultrasonography was used to detect gestational sacs and fetal heartbeats at approximately 21 and 28 days after transfer, respectively. Embryo Transfer For one patient in the ECM/Multiblast treatment group, all embryos were frozen on day 5, and there was, therefore, no transfer. Embryos were transferred to the remaining patients on day 5 (Global, n ¼ 36 patients; ECM/Multiblast, n ¼ 30 patients) or on day 6 (Global, n ¼ 4 patients; ECM/Multiblast, n ¼ 8 patients). Each patient in the Global group received two embryos. In the ECM/Multiblast group, one patient received one embryo, one received three embryos, and all others received two embryos. The mean number of embryos transferred did not differ (P¼1.000) between the Global ( ) and ECM/Multiblast ( ) groups. Embryos that were not transferred were either frozen or discarded. Statistical Analysis Proportions were compared by c 2 analysis or Fisher s exact test, where appropriate. All other measures were compared by Kruskal-Wallis tests. RESULTS The proportions of zygotes that reached given developmental stages during culture from day 1 to day 5 are shown in Table 2. On day 2 the cleavage rate was not different between embryos cultured in Global and ECM (98.3% vs. 97.5%; P¼.585), but there was a tendency toward a larger proportion of the zygotes to have developed to exactly four cells in Global than in ECM (45.3% vs. 37.9%; P¼.064). On day TABLE 2 Numbers and morphology of embryos at the indicated stages. Parameter Global ECM/Multiblast P No. of zygotes cultured Day 2 No. of embryos Cleaved (% of zygotes) No. of cells (mean SD) Exactly 4 cells (% of zygotes) %20% fragmentation (% of embryos) Multinucleated (% of embryos) Day 3 No. of embryos No. of cells (mean S.D) R6 cells (% of zygotes) Compacted (% of zygotes) <.001 %20% fragmentation (% of embryos) Multinucleated (% of embryos) Day 4 Morula (% of zygotes) <.001 Day 5 Blastocyst (% of zygotes) Full to hatching blastocyst (% of zygotes) Fertility and Sterility â 1767

4 TABLE 3 Developmental stages of transferred embryos and ICM and trophectoderm grades for transferred full to hatching blastocysts. Parameter Global ECM/Multiblast P No. of embryos transferred Cleaved or morulae (% of embryos transferred) Early or mid-blastocysts (% of embryos transferred) Full to hatching blastocysts (% of embryos transferred) Inner cell mass grade (mean SD) Trophectoderm grade (mean SD) Note: There was no overall difference in the proportions of transferred embryos at the given stages between the two culture treatments (P¼.448). 3, the proportion of zygotes that had developed to six or more cells was significantly greater for Global than for ECM (74.9% vs. 66.8%; P¼.026), as was the compaction rate (22.6% vs. 10.2%; P<.001). On day 4, a significantly greater proportion of the zygotes had developed to the morula stage in Global than in ECM/Multiblast (64.5% vs. 50.6%; P<.001). On day 5, a significantly greater proportion of the zygotes had developed to the blastocyst stage in Global than in ECM/Multiblast (42.9% vs. 31.1%; P¼.002) and to the full to hatching blastocyst stage (24.7% vs. 13.7%; P<.001). Measurements of embryo morphology on days 2 and 3 are shown in Table 2. On day 2 the mean cell number was significantly greater for embryos cultured in Global than for those in ECM ( vs ; P¼.022) and the rate of multinucleation was significantly less (2.5% vs. 6.4%; P¼.020). The proportion of embryos having 20% or less fragmentation was not different between the media (94.3% vs. 92.0%; P¼.266). On day 3 there were no significant differences between Global and ECM for mean cell number ( vs ; P¼.101), rate of multinucleation (1.1% vs. 0.6%; P¼.568), or proportion of embryos having 20% or less fragmentation (92.9% vs. 90.4%; P¼.275). As shown in Table 3, there was no difference in the proportions of developmental stages of transferred embryos between the media treatment groups. There were also no differences in ICM and trophectoderm scores for transferred full to hatching blastocysts between the two media treatment groups (Table 3). Pregnancy and implantation rates are shown in Table 4. The biochemical (positive for hcg), clinical (positive for fetal sacs), and ongoing (positive for fetal heartbeats) pregnancy rates tended to be greater after culture in Global medium than in ECM/Multiblast, but the differences were not statically significant. However, the implantation rates for embryos cultured in Global were significantly greater than those for embryos cultured in ECM/Multiblast, as measured by the proportion of transferred embryos that developed into fetal sacs (57.5% vs. 36.8%; P¼.008) and by fetal heartbeats (53.8% vs. 35.5%; P¼.020). DISCUSSION In this study, the in vitro development and specific morphologic attributes of embryos cultured in a single medium (Global) were as good, and in many instances better, than TABLE 4 Pregnancy and implantation rates. Parameter Global ECM/Multiblast P No. of transfers Positive for hcg (% of transfers) Positive for fetal sacs (% of transfers) Positive for fetal heartbeats (% of transfers) No. of embryos transferred Fetal sacs (% of embryos transferred) Fetal heartbeats (% of embryos transferred) Sepulveda et al. Single vs. sequential culture media Vol. 91, No. 5, May 2009

5 for embryos cultured in a sequential media system (ECM/ Multiblast). For example, a larger percentage of zygotes cultured in Global were classified as compacted embryos on day 3. Compaction has been positively associated with higher implantation rates after embryo replacement on day 3 (9). The proportions of zygotes that developed to the blastocyst stage and to the full to hatching blastocyst stage were also significantly greater in Global medium than in ECM/Multiblast. Blastocyst development is regulated by the expression of specific gene families that encode for cell polarity, cell junctional, cytoskeletal, ion transporter, and water channel gene products (10), and the degree of expansion of the human blastocyst is positively associated with pregnancy and implantation rates (11, 12). Together, these observations suggest that culture in Global medium is more favorable for transcriptional activity and functional potential of the embryo compared with culture in ECM/Multiblast. The best (usually two) embryos were selected for transfer, and the morphologic scores of transferred embryos did not differ between the culture groups. However, the implantation rates for embryos cultured in Global were significantly greater than for embryos cultured in ECM/Multiblast. This would suggest that, compared with culture in Global, culture in ECM/Multiblast had a deleterious effect on the potential viability of the embryos that was not apparent by morphologic assessment. The better in vitro development and subsequent implantation rates for embryos cultured in Global compared with those cultured in ECM/Multiblast may be related to two significant differences between the culture systems. First, Global is derived from KSOM aa, an elaboration of simplex optimization medium (SOM). The concentrations of the 11 components of SOM were determined in a manner functionally analogous to an 11-dimensional multifactorial design (13, 14), ensuring the optimal balance among those basic components for in vitro embryo development. In contrast, the concentrations of the basic components of sequential media were based on their measured concentrations in oviduct and uterine fluids (1), which may not be optimal for in vitro development. Second, the Global used in this study contained all 20 amino acids (including alanyl-glutamine as a source of glutamine), whereas ECM contains only alanyl-glutamine. It has been shown that there is a measurable turnover of 17 amino acids by human embryos from day 2 to day 3, and that amino acid turnover is related to subsequent development to the blastocyst stage (15). The same group subsequently showed that the turnovers of Ser, Gly, Arg, and Leu by cleavage-stage human embryos from day 1 to day 2 were related to pregnancy after transfer on day 2 (16). It has also been suggested that methionine is particularly important for embryo development because it is involved in the initiation of all protein synthesis through Met-tRNA methylation of DNA and histones through S-adenosyl methionine, and in the control of apoptosis (17). The provision of all 20 amino acids in Global throughout culture from day 1 onward may provide a significant advantage for embryo development, compared with culture in ECM/Multiblast, in which all the amino acids are only present after day 3. Our results agree with previous studies that showed no advantage of sequential media systems over a single medium for culture of the human embryo from the zygote to the blastocyst stage (5, 6) and, in particular, that Global medium is as good or better than the sequential media for development of the human embryo (18, 19). Another important aspect of this study is that the oocytes were derived from carefully screened oocyte donors. This would greatly reduce the within-group variability often associated with the use of oocytes from infertile patients. Therefore, this would allow us to truly evaluate the effects of media composition on embryonic development, pregnancy and implantation rates. In conclusion, the results of this study show that in vitro development to the blastocyst stage and subsequent implantation rates were better for human embryos cultured in a single medium, Global, than for those cultured in a sequential media system, ECM/Multiblast. Taken together with the results of previous studies (5, 6, 18, 19), the suggestion that a sequential media system is required for optimal development of the human embryo to the blastocyst stage (1) is highly questionable. REFERENCES 1. Gardner DK, Lane M. Development of viable mammalian embryos in vitro: evolution of sequential media. In: Cibelli JB, Lanza RP, Campbell KHS, West MD, eds. Principles of cloning. Amsterdam: Academic Press, 2002: Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update 1997;3: Gardner DK, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod 1998;3: Biggers JD, McGinnis LK, Lawitts JA. One-step versus two-step culture of mouse preimplantation embryos: is there a difference? Hum Reprod 2005;20: Biggers JD, Racowsky C. The development of fertilized human ova to the blastocyst stage in KSOM(AA) medium: is a two-step protocol necessary? Reprod Biomed Online 2002;5: Macklon NS, Pieters MH, Hassan MA, Jeucken PH, Eijkemans MJ, Fauser BC. A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development. Hum Reprod 2002;17: U.S. Department of Health and Human Services assisted reproductive technology success rates: national summary and fertility clinic reports. Atlanta, GA: Centers for Disease Control and Prevention, Mansour R. Minimizing embryo expulsion after embryo transfer: a randomized controlled study. Hum Reprod 2005;20: Skiadas CC, Jackson KV, Racowsky C. Early compaction on day 3 may be associated with increased implantation potential. Fertil Steril 2006;86: Watson AJ, Natale DR, Barcroft LC. Molecular regulation of blastocyst formation. Anim Reprod Sci 2004;82 83: Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73: Fertility and Sterility â 1769

6 12. Nilsson S, Waldenstrom U, Engstrom AB, Hellberg D. Promising results with 306 single blastocyst transfers. Fertil Steril 2005;83: Lawitts JA, Biggers JD. Optimization of mouse embryo culture media using simplex methods. J Reprod Fert 1991;91: Lawitts JA, Biggers JD. Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media. Molec Reprod Devel 1992;31: Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002;17: Brison DR, Houghton FD, Falconer D, Roberts SA, Hawkhead J, Humpherson PG, et al. Identification of viable embryos in IVF by noninvasive measurement of amino acid turnover. Hum Reprod 2004;19: Menezo Y. Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment. Reprod BioMed Online 2006;12: Zech N, Stecher A, Zech H, Uher P, Vanderzwalmen P. Prospective analysis of embryo development to day 5 and transfer outcomes in sequential medium (G1.3-G2.3) vs a one step protocol (Global medium) [abstract]. Human Reprod 2006;21(Suppl 1):i Angus S, Grunert GM, Dunn RC, Valdes CT, Schenk LM, Mangal RK. No advantage of using the sequential GIII media versus the single media Global [abstract]. Fert Steril 2006;86(Suppl 2):S Sepulveda et al. Single vs. sequential culture media Vol. 91, No. 5, May 2009