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Comparison of in vitro maturation media of immature oocytes: the effectiveness of blastocyst culture media
Comparison of in vitro maturation media of immature oocytes: the effectiveness of blastocyst culture media Miran Kim, M.D., Ph.D.,a Sun Jung Hong, M.Sc.,a Jae Hoon Lee, M.Sc.,a,b Churl K. Min, Ph.D.,b Kyung Joo Hwang, M.D., Ph.D.,a and Rae Woong Park, M.D., Ph.D.c a
Department of Obstetrics and Gynecology, Ajou University School of Medicine, b Department of Biological Science, College of Natural Science, Ajou University, and c Department of Biomedical Informatics, Ajou University School of Medicine, Suwon, South Korea
Objective: To compare three different in vitro maturation (IVM) media for immature oocytes. Design: Experimental study. Setting: In vitro fertilization laboratory. Animal(s): BDF1 female and male mice. Intervention(s): Retrieval and maturation of cumulus-enclosed germinal vesicle–stage oocytes according to one of three protocols: group A, conventional IVM medium; group B, blastocyst culture medium; and group C, tissue culture medium (TCM) 199. Main Outcome Measure(s): Maturation, fertilization, and developmental rates of immature oocytes. Result(s): A total of 653 immature oocytes were cultured in vitro and then analyzed. No difference was found in maturation rates and fertilization rates in comparing groups A and B. However, the IVM rates were statistically significantly increased in groups A and B compared with group C. No difference was found in fertilization rates between media, but the developmental competency to blastocyst stage was statistically significantly higher in group B compared with group C. Conclusion(s): The developmental competency of immature oocytes did not differ between conventional IVM medium and blastocyst culture medium, but TCM-199 was found to be unsuitable. Evidence from mice as test subjects suggests that both conventional IVM medium and blastocyst culture medium are suitable for IVM, and that blastocyst culture medium may be a good choice for conventional IVM of immature oocytes. (Fertil Steril 2011;95:554–7. 2011 by American Society for Reproductive Medicine.) Key Words: Blastocyst culture media, conventional IVM culture media, immature oocytes, in vitro maturation, tissue culture media 199 (TCM-199)
In vitro maturation (IVM) of immature oocytes obtained from small antral follicles is a promising technique for infertility treatment or fertility preservation. Despite more than 1,000 healthy infants having been delivered through the IVM process as of 2008 (1, 2), only a few hospitals practice the technique. Many trials have attempted to improve oocyte maturation and embryo developmental rates. One study demonstrated an increased beneficial effect using retinoids for goat embryonic development (3). The maturation media for both humans and animals are usually supplemented with various protein sources such as fetal bovine serum, fetal cord serum, or maternal serum. It is believed that these serums contain albumin and other unknown constituents that are helpful for immature oocyte maturation. Although different protein sources have been used, all of the studies have given comparable results (4). A variety of culture media are available, and the choice of the base medium for IVM is considered particularly difficult. Currently, the most commonly used culture medium for IVM is conventional Received March 12, 2010; revised September 21, 2010; accepted October 13, 2010; published online November 20, 2010. M.K. has nothing to disclose. S.J.H. has nothing to disclose. J.H.L. has nothing to disclose. C.K.M. has nothing to disclose. K.J.H. has nothing to disclose. R.W.P. has nothing to disclose. Supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007-611-E00011). Reprint requests: Kyung Joo Hwang, M.D., Ph.D., Department of Obstetrics and Gynecology, Ajou University Hospital, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-721, South Korea (E-mail: kjhwang@ajou. ac.kr).
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IVM medium. Commercialized IVM media such as MediCult (Origio A/S, Jyllinge, Denmark) and Sage (CooperSurgical, Trumbull, CT) have demonstrated reasonable fertilization, implantation, and clinical pregnancy rates (5, 6). During IVM, it is important that the culture medium provide the oocytes with energy metabolites of the glycolytic pathway, such as pyruvate and adenosine triphosphate (ATP) (7). On this basis, the pyruvate supplementation found in both blastocyst culture medium and tissue culture medium 199 (TCM-199) is essential. All three media appear to be valuable for use in in vitro culture of immature oocytes. Our study compared the maturation, fertilization, and developmental competency of immature mice oocytes after in vitro culture with the three culture media.
MATERIALS AND METHODS The experimental protocols and animal handling procedures were reviewed and approved by the Animal Ethics Committee of Ajou University. Oocytes were obtained from 6- to 8-week-old BDF1 female mice (Orient Bio, Sungnam, South Korea) raised in the research colonies of the investigators at the animal laboratory. Germinal vesicle (GV)–stage oocytes for IVM were isolated from large antral follicles 48 hours after the mice were given an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Sigma Chemical, St. Louis, MO). The mice were killed 48 hours later by cervical dislocation.
In Vitro Maturation, Fertilization, and Development Oocyte–cumulus complexes with a tight, unexpanded cumulus oophorus and an oocyte at the GV-stage were selected for IVM. The IVM, fertilization, and
Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.10.035
TABLE 1 Developmental competency of immature oocytes among different in vitro maturation media. Group A B C
Total CEO
Matured oocytes (%)
Cleaved oocytes (%)
To the blastocyst stage (%)
P value
230 223 200
121 (52.6)a 115 (51.6)a 78 (39.0)
61 (50.4) 68 (59.1) 37 (47.4)
3 (4.9) 10 (14.7)b 1 (2.7)
.005 .010
Note: CEO ¼ cumulus-enclosed oocyte; A ¼ conventional in vitro maturation (IVM) medium; B ¼ blastocyst culture medium; C ¼ tissue culture medium (TCM) 199. a P< .05 vs. in vitro culture with TCM-199 (chi-square test). b P< .05 vs. in vitro culture with TCM-199 (P¼ .049, Fisher’s exact test, one-sided). Kim. Blastocyst culture media for IVM. Fertil Steril 2011.
development were carried out as follows. The oocytes were washed with human tubal fluid (HTF)–N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES; Irvine Scientific, Santa Ana, CA) to remove blood and debris. Cumulus-enclosed GV-stage oocytes were retrieved and matured according to the protocol: group A ¼ conventional IVM medium (Sage; CooperSurgical); group B ¼ blastocyst culture medium (BMI, Suwon, South Korea); and group C ¼ TCM-199 (Sigma). Immature oocytes were randomly assigned to culture plates containing 1 mL of each culture media. All media were supplemented with 10% synthetic substitutive serum (SSS; Irvine Scientific, Santa Ana, CA), and human menopausal gonadotropin (0.075 IU/mL, IVF-M; LG, Seoul, South Korea). All media were also supplemented with pyruvate (22 mg/mL), streptomycin (0.06 mg/mL), and penicillin (0.06 mg/mL). Dishes were then incubated in a humidified atmosphere of 5% CO2 at 37 C. After 24 hours of culture, all the cumulus–oocyte complexes surrounding the oocytes were removed with hyaluronidase solution (H 4272 type IVS; Sigma) at a dilution of 80 IU/mL for a maximum of 30 seconds and then washed with human tubal fluid (HTF) supplemented with 10% SSS. Mature oocytes were determined by the presence of a first polar body. Epididymal spermatozoa were retrieved from the cauda epididymis of 6- to 8-week-old BDF1 male mice (Orient Bio), and the sperm suspensions were preincubated for 1.5 hours in capacitation medium (CooperSurgical/Sage). The matured oocytes were then inseminated by sperm at a final dilution of 2 106/mL and incubated at 37 C in humidified 5% CO2 in air. Inseminated oocytes were washed away from sperm by gentle pipetting 6 hours later, then placed in one of the three different media. After insemination, the oocytes were cultured singularly in 25 mL HTF supplemented with 10% SSS and evaluated 16 to 18 hours later. Fertilization was considered normal when two pronuclei were identified. After fertilization, we assessed embryo developmental results for 5 days. We analyzed the oocyte maturation, fertilization, and developmental rates of the blastocyst stage.
Statistical Analysis Data collected on oocyte maturation and fertilization rates were analyzed by Pearson chi-square analysis. P<.05 was considered statistically significant.
RESULTS A total of 505 immature oocytes (group A: 230; group B: 223; and group C: 200) were cultured in vitro and then analyzed. The maturation rate (121 out of 230, 52.6%; 115 out of 223,51.6%; and 78 out of 200, 39.0%, respectively) per retrieved immature oocyte was statistically higher in groups A and B than in group C. The fertilization rate (61 out of 121, 50.4%; 68 out of 115, 59.1%; and 37 out of 78, 47.4%, respectively) per matured oocyte was not different among the three culture media. The developmental rate to the blastocyst stage (3 out of 61, 4.9%; 10 out of 68, 14.7%; and 1 out of 37, 2.7%, respectively) was statistically significantly higher in group B than in group C (Table 1). Our study demonstrated that blastocyst culture medium has a comparable or even superior effect on Fertility and Sterility
maturation and developmental competency to blastocyst stage of immature oocytes in in vitro culture conditions.
DISCUSSION Our study demonstrated that blastocyst culture medium and conventional IVM medium had a superior effect in the mouse model for immature oocyte maturation, fertilization, and subsequent embryo development compared with TCM-199. Since the first report of the spontaneous gonadotropin-independent meiotic maturation of mammalian oocytes in vitro (8), developmental successes with animal models have presented clinicians with the possibility of using oocytes matured in vitro (9). By virtue of these efforts, IVM of human oocytes became possible. Up to now, only conventional IVM medium has been widely used. However, as the small trial numbers of IVM and funding restrictions indicate, more feasible and economic IVM culture media are needed. We compared each formulation among different media (Table 2). According this analysis, taurine and calcium lactate were the only components that were present in blastocyst culture medium but not in the two other media. Taurine, which is a b-amino acid, acts as an antioxidant in vivo in the oviductal fluid. Large amounts of taurine and hypotaurine are present in gametes and in the embryo environment in all the species reported to date. The beneficial effect of supplementing culture medium with taurine on in vitro embryo development has been reported in several species, including the mouse (10), cow (11, 12), and rabbit (13). It has been demonstrated that enriching the culture medium with taurine and melatonin can improve in vitro oocyte meiotic maturation and embryo development (14). Increased oxidative stress appears to play a major role in impairing in vitro mammalian embryo development, and several defense mechanisms are present in mammalian embryo development. In vivo, hypotaurine and taurine are synthesized and secreted by oviductal epithelial cells. Hypotaurine can neutralize hydroxyl radicals and prevent lipid peroxidation. The byproduct of hypotaurine, after free radical scavenging, is taurine. Taurine has indirect antioxidant effects. One study proposed that somatic cells might secrete embryotropic factors and may lower the damage caused by oxygen (15). We hypothesized that blastocyst culture media containing taurine would have a superior effect on immature oocyte in vitro maturation and development to blastocyst stage through the role of the free radical scavenging effect. Nuclear changes during oocyte maturation and fertilization are coordinated with organelle movements to ensure normal embryo development. Cytoplasmic changes occur, and mitochondria undergo a complex stage-specific intracellular translocation that seems to be a critical step for their subsequent development
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TABLE 2 Formulations of different in vitro maturation media.
Formulation Basic media componenta Nonessential amino acidsb Essential amino acidsc Vitaminsd Magnesium sulfate heptahydrate Magnesium chloride Sodium phosphate Calcium chloride Calcium lactate Taurine Cysteamine L-cysteine Sodium lactate Plasma protein fraction
IVM media washing
IVM media maturation
IVM media maintenance
Blastocyst culture medium
x x
x x
x x
x x
x x
x
x x x x x
x x
x x
x x x
TCM-199 x x x x
x x x x x
x
x
x x x x
x
x
x
a
Sodium chloride, potassium chloride, sodium bicarbonate, D-glucose, sodium pyruvate, phenol red, gentamicin. Alanyl-glutamine, asparagines, aspartic acid, glycine, proline, serine. Arginine, L-cystine dihydrochloride, histidine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, tyrosine, valine. d D-calcium pantothenate, choline chloride, folic acid, I-inositol, nicotinamide, pyridoxine HCl, riboflavin, thiamine. b c
Kim. Blastocyst culture media for IVM. Fertil Steril 2011.
(16, 17). During oocyte maturation and fertilization, dramatic mitochondrial distribution changes occur with the aim of bringing mitochondria to the region of the cell where a higher energy source, such as ATP (18) or calcium release, is required (19). We previously reported on the role of extracellular ATP as an inducer of apoptotic cell death in human granulosa cells and that treatment with human chorionic gonadotropin (hCG) eliminated both ATP-induced mitochondrial depolarization and apoptosis (20). In the previous study, the receptor type that binds ATP, thus mediating ATP-induced apoptosis, was determined by using the calcium imaging and patch-clamp techniques. One study evaluated mitochondrial organization in prepubertal goat oocytes cultured with calcium lactate during IVM and fertilization (21). The accumulation of mitochondria in the perinuclear area indicated that pronuclear opposition and syngamy may require high concentrations of ATP or free calcium. Abnormal and asymmetric mitochondrial distribution around pronuclei could directly affect mitochondrial distribution to the daughter cells, thereby producing blastomeres with an incomplete mitochondrial number that have low-energy production (17). Although the precise mechanism is unknown, blastocyst culture media containing calcium lactate may positively affect mitochondrial function for obtaining sufficient ATP production. Some investigators demonstrated that there is no difference between TCM-100 medium and conventional IVM medium (6, 22). However, our results indicate that blastocyst culture medium is
superior to TCM-199. We cannot precisely explain this phenomenon, but we were able to duplicate these results. Further studies on human immature oocytes are underway, and we hope to obtain human data prospectively. Supplementation or replacement of standard culture medium appears to be necessary to optimize the maturation of immature oocytes. Epidermal growth factor, gonadotropins, and essential and nonessential amino acids were used to optimize the maturation of porcine oocytes (23, 24). For human oocyte maturation, human menopausal gonadotropin, pregnant mare serum gonadotropin, follicle-stimulating hormone (FSH) and luteinizing hormone, pyruvic acid, estradiol (E2), recombinant FSH, insulin, transferrin, and selenium have been added to the IVM media (25–27). Undefined protein sources, such as fetal bovine serum, human maternal serum, synthetic human serum, and human follicular and peritoneal fluid have also been used to supplement maturation media (28–30). Taken together, no one recipe for maturation medium has suited all researchers, and further studies are needed to address the mechanism of oocyte maturation in vitro. Most animal testing leads to human trials. The studies to optimize culture conditions for oocytes and embryo development using animal models are limited by the availability of resources and the fact that the exact mechanism of immature oocyte maturation itself is unknown. Nonetheless, clinically this result means blastocyst culture medium may be a good alternative to the IVM culture medium.
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Blastocyst culture media for IVM
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14. Manjunatha B, Devaraj M, Gupta P, Ravindra J, Nandi S. Effect of taurine and melatonin in the culture medium on buffalo in vitro embryo development. Reprod Domest Anim 2009;44:12–6. 15. Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995;1:91–148. 16. Bavister BD, Squirrell JM. Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod 2000;15(Suppl 2):189–98. 17. Van Blerkom J, Davis P, Mathwig V, Alexander S. Domains of high-polarized and low polarized mitochondria may occur in mouse and human oocytes and early embryos. Hum Reprod 2002;17:393–406. 18. Van Blerkom J, Runner MN. Mitochondrial reorganization during resumption of arrested meiosis in the mouse oocyte. Am J Anat 1984;171:335–55. 19. Sousa M, Barros A, Silva J, Tesarik J. Developmental changes in calcium content of ultrastructurally distinct subcellular compartments of preimplantation human embryos. Mol Hum Reprod 1997;3:83–90. 20. Park DW, Cho T, Kim MR, Kim YA, Min CK, Hwang KJ. ATP-induced apoptosis of human granulosa luteal cells cultured in vitro. Fertil Steril 2003;80:993–1002. 21. Velilla E, RodrIguez-Gonzalez E, Vidal F, Izquierdo D, Paramio M-T. Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization. Mol Reprod Dev 2006;73:617–26. 22. Filali M, Hesters L, Fanchin R, Tachdjian G, Frydman R, Frydman N. Retrospective comparison of two media for in vitro maturation of oocytes. Reprod Biomed Online 2008;16:250–6. 23. Bevers MM, Izadyar F. Role of growth hormone and growth hormone receptor in oocyte maturation. Mol Cell Endocrinol 2002;197:173–8.
24. Hong JY, Yong HY, Lee BC, Hwang WS, Lim JM, Lee ES. Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media. Theriogenology 2004;62:1473–82. 25. Zhang X, Zerafa A, Wong J, Armstrong DT, Khamsi F. Human menopausal gonadotropin during in vitro maturation of human oocytes retrieved from small follicles enhances in vitro fertilization and cleavage rates. Fertil Steril 1993;59:850–3. 26. Cha KY, Chung HM, Lee DR, Kwon H, Chung MK, Park LS, et al. Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization–embryo transfer. Fertil Steril 2005;83:1461–5. 27. Isachenko E, Rahimi G, Isachenko V, Nawroth F. Invitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation. Reprod Biomed Online 2004;8: 553–7. 28. Chian R-C, Buckett WM, Jalil AKA, Son W-Y, Sylvestre C, Rao D, et al. Natural-cycle in vitro fertilization combined with in vitro maturation of immature oocytes is a potential approach in infertility treatment. Fertil Steril 2004;82:1675–8. 29. Mikkelsen AL. Strategies in human in-vitro maturation and their clinical outcome. Reprod Biomed Online 2005;10:593–9. 30. Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55: 109–13.
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Department of Obstetrics and Gynecology, Ajou University School of Medicine, b Department of Biological Science, College of Natural Science, Ajou University, and c Department of Biomedical Informatics, Ajou University School of Medicine, Suwon, South Korea
Objective: To compare three different in vitro maturation (IVM) media for immature oocytes. Design: Experimental study. Setting: In vitro fertilization laboratory. Animal(s): BDF1 female and male mice. Intervention(s): Retrieval and maturation of cumulus-enclosed germinal vesicle–stage oocytes according to one of three protocols: group A, conventional IVM medium; group B, blastocyst culture medium; and group C, tissue culture medium (TCM) 199. Main Outcome Measure(s): Maturation, fertilization, and developmental rates of immature oocytes. Result(s): A total of 653 immature oocytes were cultured in vitro and then analyzed. No difference was found in maturation rates and fertilization rates in comparing groups A and B. However, the IVM rates were statistically significantly increased in groups A and B compared with group C. No difference was found in fertilization rates between media, but the developmental competency to blastocyst stage was statistically significantly higher in group B compared with group C. Conclusion(s): The developmental competency of immature oocytes did not differ between conventional IVM medium and blastocyst culture medium, but TCM-199 was found to be unsuitable. Evidence from mice as test subjects suggests that both conventional IVM medium and blastocyst culture medium are suitable for IVM, and that blastocyst culture medium may be a good choice for conventional IVM of immature oocytes. (Fertil Steril 2011;95:554–7. 2011 by American Society for Reproductive Medicine.) Key Words: Blastocyst culture media, conventional IVM culture media, immature oocytes, in vitro maturation, tissue culture media 199 (TCM-199)
In vitro maturation (IVM) of immature oocytes obtained from small antral follicles is a promising technique for infertility treatment or fertility preservation. Despite more than 1,000 healthy infants having been delivered through the IVM process as of 2008 (1, 2), only a few hospitals practice the technique. Many trials have attempted to improve oocyte maturation and embryo developmental rates. One study demonstrated an increased beneficial effect using retinoids for goat embryonic development (3). The maturation media for both humans and animals are usually supplemented with various protein sources such as fetal bovine serum, fetal cord serum, or maternal serum. It is believed that these serums contain albumin and other unknown constituents that are helpful for immature oocyte maturation. Although different protein sources have been used, all of the studies have given comparable results (4). A variety of culture media are available, and the choice of the base medium for IVM is considered particularly difficult. Currently, the most commonly used culture medium for IVM is conventional Received March 12, 2010; revised September 21, 2010; accepted October 13, 2010; published online November 20, 2010. M.K. has nothing to disclose. S.J.H. has nothing to disclose. J.H.L. has nothing to disclose. C.K.M. has nothing to disclose. K.J.H. has nothing to disclose. R.W.P. has nothing to disclose. Supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007-611-E00011). Reprint requests: Kyung Joo Hwang, M.D., Ph.D., Department of Obstetrics and Gynecology, Ajou University Hospital, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-721, South Korea (E-mail: kjhwang@ajou. ac.kr).
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IVM medium. Commercialized IVM media such as MediCult (Origio A/S, Jyllinge, Denmark) and Sage (CooperSurgical, Trumbull, CT) have demonstrated reasonable fertilization, implantation, and clinical pregnancy rates (5, 6). During IVM, it is important that the culture medium provide the oocytes with energy metabolites of the glycolytic pathway, such as pyruvate and adenosine triphosphate (ATP) (7). On this basis, the pyruvate supplementation found in both blastocyst culture medium and tissue culture medium 199 (TCM-199) is essential. All three media appear to be valuable for use in in vitro culture of immature oocytes. Our study compared the maturation, fertilization, and developmental competency of immature mice oocytes after in vitro culture with the three culture media.
MATERIALS AND METHODS The experimental protocols and animal handling procedures were reviewed and approved by the Animal Ethics Committee of Ajou University. Oocytes were obtained from 6- to 8-week-old BDF1 female mice (Orient Bio, Sungnam, South Korea) raised in the research colonies of the investigators at the animal laboratory. Germinal vesicle (GV)–stage oocytes for IVM were isolated from large antral follicles 48 hours after the mice were given an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Sigma Chemical, St. Louis, MO). The mice were killed 48 hours later by cervical dislocation.
In Vitro Maturation, Fertilization, and Development Oocyte–cumulus complexes with a tight, unexpanded cumulus oophorus and an oocyte at the GV-stage were selected for IVM. The IVM, fertilization, and
Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.10.035
TABLE 1 Developmental competency of immature oocytes among different in vitro maturation media. Group A B C
Total CEO
Matured oocytes (%)
Cleaved oocytes (%)
To the blastocyst stage (%)
P value
230 223 200
121 (52.6)a 115 (51.6)a 78 (39.0)
61 (50.4) 68 (59.1) 37 (47.4)
3 (4.9) 10 (14.7)b 1 (2.7)
.005 .010
Note: CEO ¼ cumulus-enclosed oocyte; A ¼ conventional in vitro maturation (IVM) medium; B ¼ blastocyst culture medium; C ¼ tissue culture medium (TCM) 199. a P< .05 vs. in vitro culture with TCM-199 (chi-square test). b P< .05 vs. in vitro culture with TCM-199 (P¼ .049, Fisher’s exact test, one-sided). Kim. Blastocyst culture media for IVM. Fertil Steril 2011.
development were carried out as follows. The oocytes were washed with human tubal fluid (HTF)–N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES; Irvine Scientific, Santa Ana, CA) to remove blood and debris. Cumulus-enclosed GV-stage oocytes were retrieved and matured according to the protocol: group A ¼ conventional IVM medium (Sage; CooperSurgical); group B ¼ blastocyst culture medium (BMI, Suwon, South Korea); and group C ¼ TCM-199 (Sigma). Immature oocytes were randomly assigned to culture plates containing 1 mL of each culture media. All media were supplemented with 10% synthetic substitutive serum (SSS; Irvine Scientific, Santa Ana, CA), and human menopausal gonadotropin (0.075 IU/mL, IVF-M; LG, Seoul, South Korea). All media were also supplemented with pyruvate (22 mg/mL), streptomycin (0.06 mg/mL), and penicillin (0.06 mg/mL). Dishes were then incubated in a humidified atmosphere of 5% CO2 at 37 C. After 24 hours of culture, all the cumulus–oocyte complexes surrounding the oocytes were removed with hyaluronidase solution (H 4272 type IVS; Sigma) at a dilution of 80 IU/mL for a maximum of 30 seconds and then washed with human tubal fluid (HTF) supplemented with 10% SSS. Mature oocytes were determined by the presence of a first polar body. Epididymal spermatozoa were retrieved from the cauda epididymis of 6- to 8-week-old BDF1 male mice (Orient Bio), and the sperm suspensions were preincubated for 1.5 hours in capacitation medium (CooperSurgical/Sage). The matured oocytes were then inseminated by sperm at a final dilution of 2 106/mL and incubated at 37 C in humidified 5% CO2 in air. Inseminated oocytes were washed away from sperm by gentle pipetting 6 hours later, then placed in one of the three different media. After insemination, the oocytes were cultured singularly in 25 mL HTF supplemented with 10% SSS and evaluated 16 to 18 hours later. Fertilization was considered normal when two pronuclei were identified. After fertilization, we assessed embryo developmental results for 5 days. We analyzed the oocyte maturation, fertilization, and developmental rates of the blastocyst stage.
Statistical Analysis Data collected on oocyte maturation and fertilization rates were analyzed by Pearson chi-square analysis. P<.05 was considered statistically significant.
RESULTS A total of 505 immature oocytes (group A: 230; group B: 223; and group C: 200) were cultured in vitro and then analyzed. The maturation rate (121 out of 230, 52.6%; 115 out of 223,51.6%; and 78 out of 200, 39.0%, respectively) per retrieved immature oocyte was statistically higher in groups A and B than in group C. The fertilization rate (61 out of 121, 50.4%; 68 out of 115, 59.1%; and 37 out of 78, 47.4%, respectively) per matured oocyte was not different among the three culture media. The developmental rate to the blastocyst stage (3 out of 61, 4.9%; 10 out of 68, 14.7%; and 1 out of 37, 2.7%, respectively) was statistically significantly higher in group B than in group C (Table 1). Our study demonstrated that blastocyst culture medium has a comparable or even superior effect on Fertility and Sterility
maturation and developmental competency to blastocyst stage of immature oocytes in in vitro culture conditions.
DISCUSSION Our study demonstrated that blastocyst culture medium and conventional IVM medium had a superior effect in the mouse model for immature oocyte maturation, fertilization, and subsequent embryo development compared with TCM-199. Since the first report of the spontaneous gonadotropin-independent meiotic maturation of mammalian oocytes in vitro (8), developmental successes with animal models have presented clinicians with the possibility of using oocytes matured in vitro (9). By virtue of these efforts, IVM of human oocytes became possible. Up to now, only conventional IVM medium has been widely used. However, as the small trial numbers of IVM and funding restrictions indicate, more feasible and economic IVM culture media are needed. We compared each formulation among different media (Table 2). According this analysis, taurine and calcium lactate were the only components that were present in blastocyst culture medium but not in the two other media. Taurine, which is a b-amino acid, acts as an antioxidant in vivo in the oviductal fluid. Large amounts of taurine and hypotaurine are present in gametes and in the embryo environment in all the species reported to date. The beneficial effect of supplementing culture medium with taurine on in vitro embryo development has been reported in several species, including the mouse (10), cow (11, 12), and rabbit (13). It has been demonstrated that enriching the culture medium with taurine and melatonin can improve in vitro oocyte meiotic maturation and embryo development (14). Increased oxidative stress appears to play a major role in impairing in vitro mammalian embryo development, and several defense mechanisms are present in mammalian embryo development. In vivo, hypotaurine and taurine are synthesized and secreted by oviductal epithelial cells. Hypotaurine can neutralize hydroxyl radicals and prevent lipid peroxidation. The byproduct of hypotaurine, after free radical scavenging, is taurine. Taurine has indirect antioxidant effects. One study proposed that somatic cells might secrete embryotropic factors and may lower the damage caused by oxygen (15). We hypothesized that blastocyst culture media containing taurine would have a superior effect on immature oocyte in vitro maturation and development to blastocyst stage through the role of the free radical scavenging effect. Nuclear changes during oocyte maturation and fertilization are coordinated with organelle movements to ensure normal embryo development. Cytoplasmic changes occur, and mitochondria undergo a complex stage-specific intracellular translocation that seems to be a critical step for their subsequent development
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TABLE 2 Formulations of different in vitro maturation media.
Formulation Basic media componenta Nonessential amino acidsb Essential amino acidsc Vitaminsd Magnesium sulfate heptahydrate Magnesium chloride Sodium phosphate Calcium chloride Calcium lactate Taurine Cysteamine L-cysteine Sodium lactate Plasma protein fraction
IVM media washing
IVM media maturation
IVM media maintenance
Blastocyst culture medium
x x
x x
x x
x x
x x
x
x x x x x
x x
x x
x x x
TCM-199 x x x x
x x x x x
x
x
x x x x
x
x
x
a
Sodium chloride, potassium chloride, sodium bicarbonate, D-glucose, sodium pyruvate, phenol red, gentamicin. Alanyl-glutamine, asparagines, aspartic acid, glycine, proline, serine. Arginine, L-cystine dihydrochloride, histidine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, tyrosine, valine. d D-calcium pantothenate, choline chloride, folic acid, I-inositol, nicotinamide, pyridoxine HCl, riboflavin, thiamine. b c
Kim. Blastocyst culture media for IVM. Fertil Steril 2011.
(16, 17). During oocyte maturation and fertilization, dramatic mitochondrial distribution changes occur with the aim of bringing mitochondria to the region of the cell where a higher energy source, such as ATP (18) or calcium release, is required (19). We previously reported on the role of extracellular ATP as an inducer of apoptotic cell death in human granulosa cells and that treatment with human chorionic gonadotropin (hCG) eliminated both ATP-induced mitochondrial depolarization and apoptosis (20). In the previous study, the receptor type that binds ATP, thus mediating ATP-induced apoptosis, was determined by using the calcium imaging and patch-clamp techniques. One study evaluated mitochondrial organization in prepubertal goat oocytes cultured with calcium lactate during IVM and fertilization (21). The accumulation of mitochondria in the perinuclear area indicated that pronuclear opposition and syngamy may require high concentrations of ATP or free calcium. Abnormal and asymmetric mitochondrial distribution around pronuclei could directly affect mitochondrial distribution to the daughter cells, thereby producing blastomeres with an incomplete mitochondrial number that have low-energy production (17). Although the precise mechanism is unknown, blastocyst culture media containing calcium lactate may positively affect mitochondrial function for obtaining sufficient ATP production. Some investigators demonstrated that there is no difference between TCM-100 medium and conventional IVM medium (6, 22). However, our results indicate that blastocyst culture medium is
superior to TCM-199. We cannot precisely explain this phenomenon, but we were able to duplicate these results. Further studies on human immature oocytes are underway, and we hope to obtain human data prospectively. Supplementation or replacement of standard culture medium appears to be necessary to optimize the maturation of immature oocytes. Epidermal growth factor, gonadotropins, and essential and nonessential amino acids were used to optimize the maturation of porcine oocytes (23, 24). For human oocyte maturation, human menopausal gonadotropin, pregnant mare serum gonadotropin, follicle-stimulating hormone (FSH) and luteinizing hormone, pyruvic acid, estradiol (E2), recombinant FSH, insulin, transferrin, and selenium have been added to the IVM media (25–27). Undefined protein sources, such as fetal bovine serum, human maternal serum, synthetic human serum, and human follicular and peritoneal fluid have also been used to supplement maturation media (28–30). Taken together, no one recipe for maturation medium has suited all researchers, and further studies are needed to address the mechanism of oocyte maturation in vitro. Most animal testing leads to human trials. The studies to optimize culture conditions for oocytes and embryo development using animal models are limited by the availability of resources and the fact that the exact mechanism of immature oocyte maturation itself is unknown. Nonetheless, clinically this result means blastocyst culture medium may be a good alternative to the IVM culture medium.
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