- Email: [email protected]
Birth after 12 hours of oocyte refrigeration
CASE REPORT Birth after 12 hours of oocyte refrigeration Onder Coban, B.S.,a Oguzhan Hacifazlioglu, B.S.,a H. Nadir Ciray, M.D., Ph.D.,b Ulun Ulug, M.D.,b H. Ibrahim Tekin, M.D.,a and Mustafa Bahceci, M.D.b a
Cyprus IVF Center, Famagusta, Cyprus; and b Bahceci Women’s Health Care Center, Istanbul, Turkey
Objective: To assess cycle outcome after oocyte refrigeration. Design: Case report. Setting: Private IVF center. Patient(s): One couple in a donor oocyte program. Intervention(s): Intracytoplasmic sperm injection and blastocyst culture after refrigeration of oocytes for 12 hours. Main Outcome Measure(s): Birth. Result(s): Fourteen two-pronuclei zygotes from 17 metaphase II refrigerated oocytes resulted in transfer of two blastocysts at day 5 and cryopreservation of six excess embryos at day 6. The patient delivered one healthy male baby after 38 weeks’ gestation. Conclusion(s): The successful outcome of oocyte refrigeration indicates that this protocol could be useful in circumstances in which a delay in obtaining spermatozoa arises. (Fertil Steril 2010;94:2769.e5–e7. 2010 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, oocyte and gamete storage, refrigeration
Manipulations associated with assisted reproduction treatment are a sequence of time-dependent events. For example, insemination requires the simultaneous presence of both gametes at the embryology laboratory. Oocyte retrieval is usually scheduled at 35–36 hours after hCG injection, enabling maximum number of mature (metaphase II) oocytes to be collected from the patient. Oocytes displaying a polar body are subjected to insemination or microinjection within 3 hours after retrieval (1). Under some circumstances there may be a risk that a fresh semen sample cannot be available during the period that insemination should be performed. The risk can usually be predicted and circumvented by storage of the sperm in advance. However, if the risk has not been predicted, the laboratory has to make a decision either to prolong the insemination period or to preserve the oocytes under favorable conditions, preferably by long-term gamete storage because short-term storage is not practiced in human assisted reproductive technology. Prolonging the insemination period for extended periods after retrieval is not a situation where an embryologist will jump into because consequences, such as increase in zona hardening, parthenogenetic activation, impaired embryo development (2), and changes in alignment of chromosomes (3), have been reported in mouse oocytes. Oocyte postmaturity can cause fertilization abnormalities that have been shown in vitro maturation studies (4). To circumvent such problems, the metabolism of the oocyte could be Received January 6, 2010; revised April 12, 2010; accepted April 28, 2010; published online June 9, 2010. O.C. has nothing to disclose. O.H. has nothing to disclose. H.N.C. has nothing to disclose. U.U. has nothing to disclose. H.I.T. has nothing to disclose. M.B. has nothing to disclose. Reprint requests: H. Nadir Ciray, M.D., Ph.D., Bahceci Women’s Health Care Center, Abdi Ipekci cd., 44/17, Nisantasi, Istanbul, Turkey (TEL: þ90-212-230-0809; FAX: þ90-212-230-3990; E-mail: nadirc@ superonline.com).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.04.070
slowed by refrigeration of the gamete until insemination, as diminished metabolic activity of zygotes after exposure to 4 C up to 36 hours and subsequent high blastocyst yield has been shown in animal studies (5). To our knowledge, successful refrigeration of human oocytes has never been reported. In routine laboratory practice, long term storage (cryopreservation) of oocytes has limited application because of low outcome measures; therefore, it is still recommended as an experimental procedure by the Practice Committee opinion of the American Society of Reproductive Medicine (6). Introduction of novel techniques and protocols enabled oocyte cryopreservation to be a more widespread practice during recent years. Up to 97% survival rates have been reported after vitrification (7). Survival rates and the outcome of cycles after cryopreservation of oocytes have been reported to be higher after vitrification compared with slow freezing (8) and similar to those obtained from using of fresh oocytes (9). However, vitrification technique demands a training period and obtaining acceptable survival rates after vitrification of oocytes requires skilled and experienced embryologists before the procedure will be offered to patients as a routine practice. This article reports birth after microinjection of human oocytes exposed to short-term storage (i.e., refrigeration) for 12 hours.
CASE REPORT A 58-year-old patient and her 60-year-old husband were referred to the oocyte donation program. The couple had a history of intracytoplasmic sperm injection (ICSI) from a donation program previously at another IVF clinic, after which their first child was born. The husband had a vas ligation operation; therefore, samples of sperm were obtained through epididymal aspiration during the previous and current trials. A suitable donor was prepared according to the flexible multiple GnRH antagonist protocol. Gonadotrophins (Puregon; Schering-
Fertility and Sterility Vol. 94, No. 7, December 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
2769.e5
Plough, Istanbul, Turkey), at doses of 150–300 IU, were started on day 2. When the leading follicle reached 14 mm in diameter, 0.25 mg of ganirelix (Orgalutran; Schering-Plough) was administered daily until the day of hCG injection. The starting regimen was fixed for the first 4 days, and thereafter the gonadotrophin dose was adjusted according to the individual ovarian response. Human menopausal gonadotrophin (Menogon, Ferring, Malm€o, Sweden), at a dose of 75 IU per day, was also administered starting the same day as the antagonist. When at least two follicles reached 19 mm in diameter, hCG (Pregnyl; Scherin-Plough) 10,000 IU IM was administered. Oocytes were retrieved 35 hours after hCG injection. The endometrial preparation of the recipient was performed with estradiol as a transdermal therapeutic system; 2 mg (Estraderm TTS; Novartis, Istanbul, Turkey) was initiated on cycle day 2 and the dose was gradually increased every 2 days. Progesterone in oil 50 mg was added by cycle day 15, and embryos were transferred on cycle day 18. The couple was traveling abroad to where the clinic was located. The estimated arrival time was a few hours before the oocyte retrieval. However, the donor has administered hCG injection earlier than the scheduled time; therefore, the oocytes had to be retrieved approximately 12 hours earlier than the recipients’ arrival. At the time of treatment, the procedures regarding oocyte vitrification were not fully active in the clinic, because the initial experiments resulted in poor survival rates. The embryologists (O.C. and O.H.) considered refrigeration of the oocytes until insemination, because this could have slowed down their metabolism and prolong cell viability (5). A total of 20 oocytes were stripped from their cumulus cells 2 hours after retrieval; 17 displayed polar body extrusion and were distributed into a bicarbonate-buffer solution supplemented with synthetic serum substitute (Quinn’s Advantage Fertilization Medium; SAGE In-Vitro Fertilization, Trumbull, CT) in a four-well dish (0.8 mL/well) covered with mineral oil. The dish was subsequently placed into a conventional refrigerator (in air) of which the actual temperature recordings taken by a digital thermometer during the day of the event are given in Figure 1. Informed consent was obtained from the couple before the procedure. The husband’s operation was approximately 11 hours
FIGURE 1 The actual temperature values of the refrigerator recorded by a digital thermometer at the time of short-term storage of the oocyte. (Annual calibrations of this thermometer were performed against a standardized thermometer.) The left column represents the temperature values in degrees Celsius, and the horizontal line is the time course during the storage of the gametes (12 hours).
Coban. Birth after oocyte refrigeration. Fertil Steril 2010.
2769.e6
Coban et al.
Birth after oocyte refrigeration
after oocyte retrieval, and motile sperm were obtained. The sample was prepared by density gradient centrifugation and subsequent washing in a bicarbonate-buffer solution (SAGE, Trumbull, CT). The oocytes were removed from the refrigerator immediately before ICSI. The cytoplasm of the oocytes looked slightly blurry, although this condition did not affect the microinjection procedure. After ICSI, oocytes were taken into the Quinn’s Advantage Cleavage Medium supplemented with synthetic serum substitute (SAGE) and were incubated under 5.5% CO2 þ 5% O2. Embryo culture has been performed in microdrops (30 mL) under oil. At 16 hours, 14 oocytes displayed two pronuclei, after which culture media was replaced. Embryos were cultured for a total of 6 days in microdrops under oil. At day 3, the culture media (supplemented with synthetic serum substitute) has been replaced (Quinn’s Advantage Blastocyst Medium, SAGE, Trumbull, CT). At day 5, two grade 4BB blastocysts (10) were transferred to the patient, whose urinary bladder was full, under ultrasound guidance using a Wallace catheter. A total of six excess blastocysts were frozen. After 12 days, the result of pregnancy test was positive, and a gestational sac with a heartbeat was detected by ultrasound examination 1 month after transfer. The patient delivered a healthy boy (2,800 g) at 38 weeks’ gestation.
DISCUSSION In this case report, delaying ICSI up to 12 hours did not harm the outcome when the oocytes were refrigerated. Microinjection after a minimum of one hour of in vitro matured human oocytes up to 8 hours after extrusion of the first polar body resulted in high fertilization rates and good embryo quality (11). Prolonged incubation of metaphase II oocytes to a maximum of 8 hours did not compromise fertilization and pregnancy rates (12–15). The longest period of delay between retrieval and in vitro insemination without interfering outcome has been reported as 16 hours (16). These data contradict the potentially deleterious effect of aging to yield poor outcomes. Refrigeration of oocytes seems, at a minimum, not to harm embryo development and, subsequently, the outcome. Methods used to maintain embryos in a metabolically inhibited but viable state include cooling at refrigerated temperatures (short-term storage) (5). In several species, including rabbits (17), cows (18, 19), sheep (20) and mice (21–29), successful refrigeration of preimplantation embryos at 0–7 C has been reported. Although short-term storage of unfertilized oocytes is not a common practice, there have been many studies on the refrigeration of fertilized oocytes in several mouse strains (5, 22, 26). Initial attempts resulted in poor embryo development and low blastocyst yield (22, 26). A higher survival rate and better subsequent embryo development have been reported when oocytes have been refrigerated in bicarbonate buffer compared with bicarbonate-free media (21). Therefore, the use of the physiologic bicarbonate buffer solution could have contributed to the outcome by corresponding to the significantly reduced or inhibited metabolic demands of human oocytes during storage (30). Recently, blastocyst development up to 50% has been reported (5), and the difference between the results has been attributed to selection of mouse strains and/or use of improved media (5). Molecular assessment of fertilized mouse oocytes exposed to 4 C for up to 36 hours revealed temporary suppression of several events related to cell cycle progression and metabolism of the zygote (5). These events included DNA replication, cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene, and elevated mRNA Vol. 94, No. 7, December 2010
expression of zygotic gene activation-related genes (5). The disassembly of the meiotic spindle of mouse oocytes was reversible after cooling and rewarming (31). These findings indicate that metabolism of the zygote, which has a higher metabolic activity than an unfertilized oocyte, could be temporarily diminished by refrigeration. It is likely that successful outcome obtained from the present study on human oocytes resulted from prolonged survival of the cells because of lower metabolic activity, as has been observed in the previously mentioned studies.
Delivery of an infant after transfer of two blastocysts obtained from insemination of human oocytes refrigerated for 12 hours may offer an alternative for long-term storage of female gametes or prolonging the insemination period when the availability of the sperm is delayed. Although refrigeration of gametes is a simple procedure, future studies to assess survival and fertilization rates, as well as embryo development when compared with prolonged incubation and/or to cryopreservation of oocytes, are needed.
REFERENCES 1. Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:1014–9. 2. Fukuda A, Roudebush WE, Thatcher SS. Influences of in vitro oocyte aging on microfertilization in the mouse with reference to zona hardening. J Assist Reprod Genet 1992;9:378–83. 3. Saito H, Koike K, Saito T, Nohara M, Kawagoe S, Hiroi M. Aging changes in the alignment of chromosomes after human chorionic gonadotropin stimulation may be a possible cause of decreased fertility in mice. Horm Res 1993;39(Suppl 1):28–31. 4. Goud P, Goud A, Van Oostveldt P, Van der Elst J, Dhont M. Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity. Fertil Steril 1999;72:245–52. 5. Sakurai T, Kimura M, Sato M. Temporary developmental arrest after storage of fertilized mouse oocytes at 4 degrees C: effects on embryonic development, maternal mRNA processing and cell cycle. Mol Hum Reprod 2005;11:325–33. 6. Practice Committee opinion of the American Society of Reproductive Medicine. Essential elements of informed consent for elective oocyte cryopreservation: a Practice Committee opinion. Fertil Steril 2008;90:S134–5. 7. Cobo A, Bellver J, Domingo J, Perez S, Crespo J, Pellicer A, et al. New options in assisted reproduction technology: the Cryotop method of oocyte vitrification. Reprod Biomed Online 2008;17:68–72. 8. Fadini R, Brambillasca F, Renzini MM, Merola M, Comi R, De Ponti E, et al. Human oocyte cryopreservation: comparison between slow and ultrarapid methods. Reprod Biomed Online 2009;19:171–80. 9. Rienzi L, Romano S, Albricci L, Maggiulli R, Capalbo A, Baroni E, et al. Embryo development of
Fertility and Sterility
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fresh ‘versus’ vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum Reprod 2010;25:66–73. Gardner DK, Lane M. Towards a single embryo transfer. Reprod Biomed Online 2003;6:470–81. Hyun CS, Cha JH, Son WY, Yoon SH, Kim KA, Lim JH. Optimal ICSI timing after the first polar body extrusion in in vitro matured human oocytes. Hum Reprod 2007;22:1991–5. Andrews MM, Fishel SB, Rowe PH, Berry JA, Lisi F, Rinaldi L. Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa. Reprod Biomed Online 2001;2:89–97. Jacobs M, Stolwijk AM, Wetzels AM. The effect of insemination/injection time on the results of IVF and ICSI. Hum Reprod 2001;16:1708–13. Van de Velde H, De Vos A, Joris H, Nagy ZP, Van Steirteghem AC. Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:3160–4. Yanagida K, Yazawa H, Katayose H, Suzuki K, Hoshi K, Sato A. Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection. Hum Reprod 1998;13:2223–6. Harrison KL, Wilson LM, Breen TM, Pope AK, Cummins JM, Hennessey JF. Fertilization of human oocytes in relation to varying delay before insemination. Fertil Steril 1988;50:294–7. Chang MC. The effects of low temperature on fertilized rabbit ova in vitro, and the normal development of ova kept at low temperature for several days. J Gen Physiol 1948;31:385–410. Bondurant RH, Anderson CB, Boland MP, Cupps PT, Hughes MA. Preliminary studies on bovine embryo survival following short-term storage at 4 degrees C. Theriogenology 1982;17:223–30. Lindner GM, Anderson GB, BonDurant RH, Cupps PT. Survival of bovine embryos stored at 4 degrees C. Theriogenology 1983;20:311–9.
20. Harper MJ, Rowson LE. Attempted Storage of Sheep Ova at 7 Degrees Centigrade. J Reprod Fertil 1963;6: 183–91. 21. Herr CM, Wright R Jr. Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media. Theriogenology 1988;30:159–68. 22. Herr CM, Wright RW Jr. Cold storage of mouse embryos of different stages of development. Theriogenology 1988;29:765–70. 23. Kasai M. Nonfreezing technique for short-term storage of mouse embryos. J In Vitro Fert Embryo Transf 1986;3:10–4. 24. Kasai M, Niwa K, Iritani A. Protective effect of sucrose on the survival of mouse and rat embryos stored at 0 degree C. J Reprod Fertil 1983;68: 377–80. 25. Miyoshi I, Ishikawa K, Kasai M, Kasai N. Useful short-range transport of mouse embryos by means of a nonfreezing technique. Lab Anim Sci 1992;42: 198–201. 26. Nakamura K, Tsunoda Y. Viability of nuclei of twocell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos. Cryobiology 1992;29:493–9. 27. Sherman JK, Lin TP. Temperature shock and coldstorage of unfertilized mouse eggs. Fertil Steril 1959;10:384–96. 28. Tsuchiya H, Ogonuki N, Kuwana T, Sankai T, Kanayama K. Short-term preservation of mouse oocytes at 5 degrees C. Exp Anim 2001;50:441–3. 29. Wiggins PM, Rowlandson J, Ferguson AB. Preservation of murine embryos in a state of dormancy at 4 degrees C. Am J Physiol 1999;276: C291–9. 30. Quinn P, Warnes GM, Kerin JF, Kirby C. Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fertil Steril 1984;41:202–9. 31. Pickering SJ, Johnson MH. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1987;2:207–16.
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Cyprus IVF Center, Famagusta, Cyprus; and b Bahceci Women’s Health Care Center, Istanbul, Turkey
Objective: To assess cycle outcome after oocyte refrigeration. Design: Case report. Setting: Private IVF center. Patient(s): One couple in a donor oocyte program. Intervention(s): Intracytoplasmic sperm injection and blastocyst culture after refrigeration of oocytes for 12 hours. Main Outcome Measure(s): Birth. Result(s): Fourteen two-pronuclei zygotes from 17 metaphase II refrigerated oocytes resulted in transfer of two blastocysts at day 5 and cryopreservation of six excess embryos at day 6. The patient delivered one healthy male baby after 38 weeks’ gestation. Conclusion(s): The successful outcome of oocyte refrigeration indicates that this protocol could be useful in circumstances in which a delay in obtaining spermatozoa arises. (Fertil Steril 2010;94:2769.e5–e7. 2010 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, oocyte and gamete storage, refrigeration
Manipulations associated with assisted reproduction treatment are a sequence of time-dependent events. For example, insemination requires the simultaneous presence of both gametes at the embryology laboratory. Oocyte retrieval is usually scheduled at 35–36 hours after hCG injection, enabling maximum number of mature (metaphase II) oocytes to be collected from the patient. Oocytes displaying a polar body are subjected to insemination or microinjection within 3 hours after retrieval (1). Under some circumstances there may be a risk that a fresh semen sample cannot be available during the period that insemination should be performed. The risk can usually be predicted and circumvented by storage of the sperm in advance. However, if the risk has not been predicted, the laboratory has to make a decision either to prolong the insemination period or to preserve the oocytes under favorable conditions, preferably by long-term gamete storage because short-term storage is not practiced in human assisted reproductive technology. Prolonging the insemination period for extended periods after retrieval is not a situation where an embryologist will jump into because consequences, such as increase in zona hardening, parthenogenetic activation, impaired embryo development (2), and changes in alignment of chromosomes (3), have been reported in mouse oocytes. Oocyte postmaturity can cause fertilization abnormalities that have been shown in vitro maturation studies (4). To circumvent such problems, the metabolism of the oocyte could be Received January 6, 2010; revised April 12, 2010; accepted April 28, 2010; published online June 9, 2010. O.C. has nothing to disclose. O.H. has nothing to disclose. H.N.C. has nothing to disclose. U.U. has nothing to disclose. H.I.T. has nothing to disclose. M.B. has nothing to disclose. Reprint requests: H. Nadir Ciray, M.D., Ph.D., Bahceci Women’s Health Care Center, Abdi Ipekci cd., 44/17, Nisantasi, Istanbul, Turkey (TEL: þ90-212-230-0809; FAX: þ90-212-230-3990; E-mail: nadirc@ superonline.com).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.04.070
slowed by refrigeration of the gamete until insemination, as diminished metabolic activity of zygotes after exposure to 4 C up to 36 hours and subsequent high blastocyst yield has been shown in animal studies (5). To our knowledge, successful refrigeration of human oocytes has never been reported. In routine laboratory practice, long term storage (cryopreservation) of oocytes has limited application because of low outcome measures; therefore, it is still recommended as an experimental procedure by the Practice Committee opinion of the American Society of Reproductive Medicine (6). Introduction of novel techniques and protocols enabled oocyte cryopreservation to be a more widespread practice during recent years. Up to 97% survival rates have been reported after vitrification (7). Survival rates and the outcome of cycles after cryopreservation of oocytes have been reported to be higher after vitrification compared with slow freezing (8) and similar to those obtained from using of fresh oocytes (9). However, vitrification technique demands a training period and obtaining acceptable survival rates after vitrification of oocytes requires skilled and experienced embryologists before the procedure will be offered to patients as a routine practice. This article reports birth after microinjection of human oocytes exposed to short-term storage (i.e., refrigeration) for 12 hours.
CASE REPORT A 58-year-old patient and her 60-year-old husband were referred to the oocyte donation program. The couple had a history of intracytoplasmic sperm injection (ICSI) from a donation program previously at another IVF clinic, after which their first child was born. The husband had a vas ligation operation; therefore, samples of sperm were obtained through epididymal aspiration during the previous and current trials. A suitable donor was prepared according to the flexible multiple GnRH antagonist protocol. Gonadotrophins (Puregon; Schering-
Fertility and Sterility Vol. 94, No. 7, December 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
2769.e5
Plough, Istanbul, Turkey), at doses of 150–300 IU, were started on day 2. When the leading follicle reached 14 mm in diameter, 0.25 mg of ganirelix (Orgalutran; Schering-Plough) was administered daily until the day of hCG injection. The starting regimen was fixed for the first 4 days, and thereafter the gonadotrophin dose was adjusted according to the individual ovarian response. Human menopausal gonadotrophin (Menogon, Ferring, Malm€o, Sweden), at a dose of 75 IU per day, was also administered starting the same day as the antagonist. When at least two follicles reached 19 mm in diameter, hCG (Pregnyl; Scherin-Plough) 10,000 IU IM was administered. Oocytes were retrieved 35 hours after hCG injection. The endometrial preparation of the recipient was performed with estradiol as a transdermal therapeutic system; 2 mg (Estraderm TTS; Novartis, Istanbul, Turkey) was initiated on cycle day 2 and the dose was gradually increased every 2 days. Progesterone in oil 50 mg was added by cycle day 15, and embryos were transferred on cycle day 18. The couple was traveling abroad to where the clinic was located. The estimated arrival time was a few hours before the oocyte retrieval. However, the donor has administered hCG injection earlier than the scheduled time; therefore, the oocytes had to be retrieved approximately 12 hours earlier than the recipients’ arrival. At the time of treatment, the procedures regarding oocyte vitrification were not fully active in the clinic, because the initial experiments resulted in poor survival rates. The embryologists (O.C. and O.H.) considered refrigeration of the oocytes until insemination, because this could have slowed down their metabolism and prolong cell viability (5). A total of 20 oocytes were stripped from their cumulus cells 2 hours after retrieval; 17 displayed polar body extrusion and were distributed into a bicarbonate-buffer solution supplemented with synthetic serum substitute (Quinn’s Advantage Fertilization Medium; SAGE In-Vitro Fertilization, Trumbull, CT) in a four-well dish (0.8 mL/well) covered with mineral oil. The dish was subsequently placed into a conventional refrigerator (in air) of which the actual temperature recordings taken by a digital thermometer during the day of the event are given in Figure 1. Informed consent was obtained from the couple before the procedure. The husband’s operation was approximately 11 hours
FIGURE 1 The actual temperature values of the refrigerator recorded by a digital thermometer at the time of short-term storage of the oocyte. (Annual calibrations of this thermometer were performed against a standardized thermometer.) The left column represents the temperature values in degrees Celsius, and the horizontal line is the time course during the storage of the gametes (12 hours).
Coban. Birth after oocyte refrigeration. Fertil Steril 2010.
2769.e6
Coban et al.
Birth after oocyte refrigeration
after oocyte retrieval, and motile sperm were obtained. The sample was prepared by density gradient centrifugation and subsequent washing in a bicarbonate-buffer solution (SAGE, Trumbull, CT). The oocytes were removed from the refrigerator immediately before ICSI. The cytoplasm of the oocytes looked slightly blurry, although this condition did not affect the microinjection procedure. After ICSI, oocytes were taken into the Quinn’s Advantage Cleavage Medium supplemented with synthetic serum substitute (SAGE) and were incubated under 5.5% CO2 þ 5% O2. Embryo culture has been performed in microdrops (30 mL) under oil. At 16 hours, 14 oocytes displayed two pronuclei, after which culture media was replaced. Embryos were cultured for a total of 6 days in microdrops under oil. At day 3, the culture media (supplemented with synthetic serum substitute) has been replaced (Quinn’s Advantage Blastocyst Medium, SAGE, Trumbull, CT). At day 5, two grade 4BB blastocysts (10) were transferred to the patient, whose urinary bladder was full, under ultrasound guidance using a Wallace catheter. A total of six excess blastocysts were frozen. After 12 days, the result of pregnancy test was positive, and a gestational sac with a heartbeat was detected by ultrasound examination 1 month after transfer. The patient delivered a healthy boy (2,800 g) at 38 weeks’ gestation.
DISCUSSION In this case report, delaying ICSI up to 12 hours did not harm the outcome when the oocytes were refrigerated. Microinjection after a minimum of one hour of in vitro matured human oocytes up to 8 hours after extrusion of the first polar body resulted in high fertilization rates and good embryo quality (11). Prolonged incubation of metaphase II oocytes to a maximum of 8 hours did not compromise fertilization and pregnancy rates (12–15). The longest period of delay between retrieval and in vitro insemination without interfering outcome has been reported as 16 hours (16). These data contradict the potentially deleterious effect of aging to yield poor outcomes. Refrigeration of oocytes seems, at a minimum, not to harm embryo development and, subsequently, the outcome. Methods used to maintain embryos in a metabolically inhibited but viable state include cooling at refrigerated temperatures (short-term storage) (5). In several species, including rabbits (17), cows (18, 19), sheep (20) and mice (21–29), successful refrigeration of preimplantation embryos at 0–7 C has been reported. Although short-term storage of unfertilized oocytes is not a common practice, there have been many studies on the refrigeration of fertilized oocytes in several mouse strains (5, 22, 26). Initial attempts resulted in poor embryo development and low blastocyst yield (22, 26). A higher survival rate and better subsequent embryo development have been reported when oocytes have been refrigerated in bicarbonate buffer compared with bicarbonate-free media (21). Therefore, the use of the physiologic bicarbonate buffer solution could have contributed to the outcome by corresponding to the significantly reduced or inhibited metabolic demands of human oocytes during storage (30). Recently, blastocyst development up to 50% has been reported (5), and the difference between the results has been attributed to selection of mouse strains and/or use of improved media (5). Molecular assessment of fertilized mouse oocytes exposed to 4 C for up to 36 hours revealed temporary suppression of several events related to cell cycle progression and metabolism of the zygote (5). These events included DNA replication, cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene, and elevated mRNA Vol. 94, No. 7, December 2010
expression of zygotic gene activation-related genes (5). The disassembly of the meiotic spindle of mouse oocytes was reversible after cooling and rewarming (31). These findings indicate that metabolism of the zygote, which has a higher metabolic activity than an unfertilized oocyte, could be temporarily diminished by refrigeration. It is likely that successful outcome obtained from the present study on human oocytes resulted from prolonged survival of the cells because of lower metabolic activity, as has been observed in the previously mentioned studies.
Delivery of an infant after transfer of two blastocysts obtained from insemination of human oocytes refrigerated for 12 hours may offer an alternative for long-term storage of female gametes or prolonging the insemination period when the availability of the sperm is delayed. Although refrigeration of gametes is a simple procedure, future studies to assess survival and fertilization rates, as well as embryo development when compared with prolonged incubation and/or to cryopreservation of oocytes, are needed.
REFERENCES 1. Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:1014–9. 2. Fukuda A, Roudebush WE, Thatcher SS. Influences of in vitro oocyte aging on microfertilization in the mouse with reference to zona hardening. J Assist Reprod Genet 1992;9:378–83. 3. Saito H, Koike K, Saito T, Nohara M, Kawagoe S, Hiroi M. Aging changes in the alignment of chromosomes after human chorionic gonadotropin stimulation may be a possible cause of decreased fertility in mice. Horm Res 1993;39(Suppl 1):28–31. 4. Goud P, Goud A, Van Oostveldt P, Van der Elst J, Dhont M. Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity. Fertil Steril 1999;72:245–52. 5. Sakurai T, Kimura M, Sato M. Temporary developmental arrest after storage of fertilized mouse oocytes at 4 degrees C: effects on embryonic development, maternal mRNA processing and cell cycle. Mol Hum Reprod 2005;11:325–33. 6. Practice Committee opinion of the American Society of Reproductive Medicine. Essential elements of informed consent for elective oocyte cryopreservation: a Practice Committee opinion. Fertil Steril 2008;90:S134–5. 7. Cobo A, Bellver J, Domingo J, Perez S, Crespo J, Pellicer A, et al. New options in assisted reproduction technology: the Cryotop method of oocyte vitrification. Reprod Biomed Online 2008;17:68–72. 8. Fadini R, Brambillasca F, Renzini MM, Merola M, Comi R, De Ponti E, et al. Human oocyte cryopreservation: comparison between slow and ultrarapid methods. Reprod Biomed Online 2009;19:171–80. 9. Rienzi L, Romano S, Albricci L, Maggiulli R, Capalbo A, Baroni E, et al. Embryo development of
Fertility and Sterility
10. 11.
12.
13.
14.
15.
16.
17.
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