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Eggs come in from the cold
Forum: Science & Society
Special Focus: Fertility and pregnancy
Eggs come in from the cold Roger G. Gosden1,2* and Lucinda L. Veeck Gosden1,2* 1 2
Williamsburg, Virginia, USA Weill Cornell Medical College, New York, NY, USA
Eggs can be ‘forever’. A longstanding goal in assisted reproductive technology (ART) has been realized at last, namely, cryopreservation of oocytes by vitrification technology. This breakthrough heralds benefits for infertility treatment, fertility preservation, and even postponement of reproduction but, as so often with ARTs, new waves of technology draw ethical and societal concerns in their wake.
Little frosties The development of low-temperature storage (cryopreservation) of oocytes was arguably the major breakthrough in ARTs in the past decade. But why did such a desirable technology not emerge earlier? A twin pregnancy from frozen oocytes was reported as long ago as 1986 [1] using well-established principles and protocols from embryo cryopreservation [2]. Cells can avoid freeze-injury after partial dehydration in a cryoprotective agent (CPA) such as 1,2-propanediol, provided they are slowly cooled and rapidly rewarmed to avoid ice formation which is invariably lethal. Unfortunately, it proved hard to repeat the early result, despite embryo banking which has been routine for nearly thirty years (Figure 1). Oocytes are more vulnerable because of their large size and, being single cells, survival is strictly all-or-none. In addition, chilling hardens the zona pellucida by triggering cortical granule exocytosis prematurely, and the metaphase spindle depolymerizes, allowing chromatids to float in the cytoplasm, which raises the risks of aneuploidy during meiosis. Besides these problems, oocyte survival was often so poor (<50%) that it was hard to justify banking. Occasional success in the 1990s was attributed to the introduction of sperm injection (ICSI) to bypass the zona, as well as better dehydration from higher sugar concentrations, and substitution of 1,2-propanediol for dimethyl sulfoxide (DMSO) as the primary CPA [3–5]. In 2004, a new Italian law boosted research by placing a limit of three on the number of oocytes that could be inseminated in a given treatment cycle, and banning embryo cryopreservation. Although success was often elusive, oocyte freezing modestly bolstered cumulative pregnancy rates and provided data for more reliable estimates of efficiency. But of more than 40 000 oocytes thawed between 2005 to 2007 in 193 clinics, the survival rate was a mere 51% and the pregnanCorresponding author: Gosden, R.G. ([email protected]) Keywords: assisted reproductive technology; cryopreservation; embryo; fertility; oocyte. * Retired.
498
cy rate (12.5%) was only half of that of fresh oocytes [6]. The findings implied that almost 100 thawed oocytes were needed to create a single baby, so the field remained encumbered by a cloud of doubts. Nevertheless, a debate was emerging between proponents of the ‘standard’ slow cooling methods versus others advocating vitrification technology, which had already shown promise in farm animals after being pioneered with mouse embryos [7]. Vitrification has now won the argument and is being rapidly adopted worldwide for human oocytes and embryos. In glass Vitrification technology is based on fundamentally different principles. Instead of dehydrating cells during slow cooling, water is replaced by CPAs before ultra-rapid cooling. The CPAs are similar, usually ethylene glycol and DMSO, both good glass-formers, plus sucrose/trehalose, but at much higher total concentrations than previously. When a minute volume of this medium is plunged in liquid nitrogen (LN2) a stable vitreous solid is formed without ice crystals. As with any glass, it must be handled with care to avoid fractures. In practice, oocytes are equilibrated in two or more steps to minimize osmotic stress before loading on a ‘cryotool’, such as a thin plastic platter or loop or straw (Box 1) [8]. It seems at odds with our technological era, so dominated by microelectronics, satellite technology, and genomics that an unsophisticated technology can carry sway in any field, but the protocol has been finely-tuned and requires skilful execution. In expert hands survival rates approach 100%, and implantation, pregnancy, and live-birth rates are remarkably high after oocyte vitrification [9,10]. Worldwide, over 2000 babies have been conceived, and obstetric followup, although limited, has been reassuring so far [11]. Because most ART clinics in the USA offer oocyte banking [12], warnings from professional organizations that the technique is still ‘experimental’ seem out-of-date. Despite justified enthusiasm for vitrification, some supposed advantages need to be qualified. Each pair of oocytes takes 15 min to be processed, but the time taken for a large batch of, say, 20 or more from one patient is comparable to the slow-freezing method (3–4 h). Furthermore, the higher concentrations of CPAs (3- to 4-fold) raise concern about toxicity, although this is moderated by rapid processing and using two CPAs of half strength. There is also continuing discussion at professional and regulatory levels about the safety of ‘open’ systems, in which specimens are plunged directly into non-sterile LN2, although it is
Forum: Science & Society
(a)
Trends in Endocrinology and Metabolism October 2012, Vol. 23, No. 10
50 µm
(b)
(c) TRENDS in Endocrinology & Metabolism
Figure 1. Comparison of (a) human metaphase II oocyte, (b) eight-cell embryo, and (c) blastocyst.
acknowledged that barrier methods sometimes leak, as well as being somewhat less effective for cryopreservation. Reassuringly, there are no reported cases of contamination from LN2 in the history of ARTs. Although some clinics sterilize LN2 by filtration/UV light, we tested whether a pure non-reactive compound can serve as a secondary coolant in LN2. The most promising candidate was perfluoropropane, but it has handling difficulties. Perhaps the most underestimated limitation of the technology is its dependence on operator skill. Oocytes floating in viscous media can be lost, and each step in the protocol has little leeway for guaranteeing safe and effective cellular preservation. Success rates are likely to be variable between centers until sufficient expertise is more widespread from routine practice. Advancing technology may eventually address this problem by automating cryopreservation in a ‘chip’, but meantime let the buyer beware. But who are the buyers? Bank depositors First, and perhaps foremost, oocyte banking offers hope for young women needing to preserve fertility before sterilizing treatment for cancer and some non-malignant diseases, as well as for other causes of premature menopause. Until
Box 1. Representative protocol for oocyte vitrification based on Kuwayama’s Cryotop method [8] Pairs of fresh oocytes are transferred at room temperature from wash medium to equilibration medium containing 7.5% ethylene glycol + 7.5% DMSO in buffered salt solution containing protein. They are transferred to vitrification medium containing 15% ethylene glycol + 15% DMSO + 0.5 M sucrose/trehalose. More steps may be added to minimize osmotic stress, but for only 1–2 min per step. Transfer to the cryotool. In the case of the Cryotop or its analogs, excess medium is drained from the tip of the tool to leave a film of 0.1 ml. The cryotool is plunged into liquid nitrogen (LN2), cooling the specimen at >20 000 8C/min. The specimen, now vitrified on the cryotool, is stored in a protective sleeve in LN2 or LN2 vapor. Specimens are rewarmed very rapidly by plunging the cryotool into rehydration medium containing 0.5 M sucrose/trehalose followed by medium at normal osmolality.
recently, doubts about this technology encouraged ovarian tissue banking, which will probably remain a backup strategy, except where oocyte harvesting is not an option, for example in prepubertal children [13]. This progress is most welcome, but it will be a long time until sufficient data are available to estimate success and any complications reliably because such patients will bank their oocytes until they return to good health and are ready to start a family. Because oocyte donation is common (12% of ARTs in 2005, according to the Centers for Disease Control), it provides an earlier opportunity to gauge success with vitrification. Sometimes, there is even a chance to compare fresh and cryopreserved oocytes after randomization from the same cohort, using the same semen. In a report from a large research center there was no difference between fertilization, embryo development, and pregnancy rates in the two arms, indicating that vitrified oocytes retain excellent quality [10]. Banking also provides a benefit that has long served sperm donation by enabling oocytes to be quarantined until donors are retested for transmissible diseases. Oocyte vitrification will gradually replace embryo cryopreservation, a procedure which is fraught with issues concerning the disposition of surplus embryos or the action to be taken when one of the gamete partners dies or they divorce. Oocyte banking should therefore help to ease lingering opposition to ARTs. Considering the trend towards single blastocyst transfer, we imagine treatment evolving towards vitrification of an entire cohort of oocytes, except for one that is freshly fertilized, with subsequent rewarming of singletons in turn for as many conception attempts as are needed. This will give women greater reproductive autonomy at the same time as dodging multiple pregnancy. When frozen sperm are unavailable, banking also avoids wasting oocytes if the male partner fails to produce a semen sample or sperm cannot be retrieved from testicular biopsies. Finally, this technology is increasingly sought-after as an elective procedure, commonly, and somewhat pejoratively, known as ‘social egg banking’. In most developed countries, and increasingly elsewhere, rising educational and professional expectations and achievements push women into postponing a family until their late 30 s or 40 s, after the window of optimal fertility has closed [14]. 499
Forum: Science & Society For other women reasons can be the breakdown of a conjugal relationship or not having found the ‘right’ partner. Standard IVF technology cannot reverse the problem of reproductive aging, except by oocyte/embryo donation, but banking oocytes when young, although expensive, provides some reassurance, if not insurance, against infertility later in life [15]. Given the straits of modern life, women should not be censured for hedging their bets in this way. Even so, we suspect that most deposits in egg banks will never be drawn, as is the case with semen banking, but the female gamete, being so much more highly prized, could then be sold on the donor market or for stem cell technology. At any rate, cryopreservation will encourage commercial tendencies in ART, and so stocks in ‘oocyte futures’ look rosy. Acknowledgments We thank Dr Nikica Zaninovic for discussion.
References 1 Chen, C. (1986) Pregnancy after human oocyte cryopreservation. Lancet 1, 884–886 2 Whittingham, D.G. et al. (1972) Survival of mouse embryos frozen to – 196 degrees and –269 degrees C. Science 178, 411–414 3 Porcu, E. et al. (1997) Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil. Steril. 68, 724–726 4 Tucker, M.J. et al. (1998) Clinical application of human egg cryopreservation. Hum. Reprod. 13, 3156–3159
500
Trends in Endocrinology and Metabolism October 2012, Vol. 23, No. 10 5 Borini, A. et al. (2006) Cumulative pregnancy rates resulting from the use of fresh and frozen oocytes: 7 years experience. Reprod. Biomed. Online 12, 481–486 6 Scaravelli, G. et al. (2010) Analysis of oocyte cryopreservation in assisted reproduction: the Italian National Register data from 2005 to 2007. Reprod. Biomed. Online 21, 496–500 7 Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at –196 degrees C by vitrification. Nature 313, 573–575 8 Kuwayama, M. et al. (2005) Highly efficient vitrification method for the cryopreservation of human oocytes. Reprod. Biomed. Online 11, 300–308 9 Nagy, Z.P. et al. (2009) The efficiency and safety of human oocyte vitrification. Semin. Reprod. Med. 27, 450–455 10 Cobo, A. et al. (2011) Oocyte cryopreservation for donor banking. Reprod. Biomed. Online 23, 341–346 11 Noyes, N. et al. (2009) Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod. Biomed. Online 18, 769–776 12 Rudick, B. et al. (2010) The status of oocyte cryopreservation in the United States. Fertil. Steril. 94, 2642–2646 13 Gosden, R.G. (2009) Fertility preservation: definition, history and prospect. Semin. Reprod. Med. 27, 433–437 14 Wyndham, N. et al. (2012) A persistent misperception: assisted reproductive technology can reverse the ‘aged biological clock’. Fertil. Steril. 97, 1044–1047 15 Lockwood, G.M. (2011) Social egg freezing: the prospect of reproductive ‘immortality’ or a dangerous delusion? Reprod. Biomed. Online 23, 334–340 1043-2760/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2012.05.010 Trends in Endocrinology and Metabolism, October 2012, Vol. 23, No. 10
Special Focus: Fertility and pregnancy
Eggs come in from the cold Roger G. Gosden1,2* and Lucinda L. Veeck Gosden1,2* 1 2
Williamsburg, Virginia, USA Weill Cornell Medical College, New York, NY, USA
Eggs can be ‘forever’. A longstanding goal in assisted reproductive technology (ART) has been realized at last, namely, cryopreservation of oocytes by vitrification technology. This breakthrough heralds benefits for infertility treatment, fertility preservation, and even postponement of reproduction but, as so often with ARTs, new waves of technology draw ethical and societal concerns in their wake.
Little frosties The development of low-temperature storage (cryopreservation) of oocytes was arguably the major breakthrough in ARTs in the past decade. But why did such a desirable technology not emerge earlier? A twin pregnancy from frozen oocytes was reported as long ago as 1986 [1] using well-established principles and protocols from embryo cryopreservation [2]. Cells can avoid freeze-injury after partial dehydration in a cryoprotective agent (CPA) such as 1,2-propanediol, provided they are slowly cooled and rapidly rewarmed to avoid ice formation which is invariably lethal. Unfortunately, it proved hard to repeat the early result, despite embryo banking which has been routine for nearly thirty years (Figure 1). Oocytes are more vulnerable because of their large size and, being single cells, survival is strictly all-or-none. In addition, chilling hardens the zona pellucida by triggering cortical granule exocytosis prematurely, and the metaphase spindle depolymerizes, allowing chromatids to float in the cytoplasm, which raises the risks of aneuploidy during meiosis. Besides these problems, oocyte survival was often so poor (<50%) that it was hard to justify banking. Occasional success in the 1990s was attributed to the introduction of sperm injection (ICSI) to bypass the zona, as well as better dehydration from higher sugar concentrations, and substitution of 1,2-propanediol for dimethyl sulfoxide (DMSO) as the primary CPA [3–5]. In 2004, a new Italian law boosted research by placing a limit of three on the number of oocytes that could be inseminated in a given treatment cycle, and banning embryo cryopreservation. Although success was often elusive, oocyte freezing modestly bolstered cumulative pregnancy rates and provided data for more reliable estimates of efficiency. But of more than 40 000 oocytes thawed between 2005 to 2007 in 193 clinics, the survival rate was a mere 51% and the pregnanCorresponding author: Gosden, R.G. ([email protected]) Keywords: assisted reproductive technology; cryopreservation; embryo; fertility; oocyte. * Retired.
498
cy rate (12.5%) was only half of that of fresh oocytes [6]. The findings implied that almost 100 thawed oocytes were needed to create a single baby, so the field remained encumbered by a cloud of doubts. Nevertheless, a debate was emerging between proponents of the ‘standard’ slow cooling methods versus others advocating vitrification technology, which had already shown promise in farm animals after being pioneered with mouse embryos [7]. Vitrification has now won the argument and is being rapidly adopted worldwide for human oocytes and embryos. In glass Vitrification technology is based on fundamentally different principles. Instead of dehydrating cells during slow cooling, water is replaced by CPAs before ultra-rapid cooling. The CPAs are similar, usually ethylene glycol and DMSO, both good glass-formers, plus sucrose/trehalose, but at much higher total concentrations than previously. When a minute volume of this medium is plunged in liquid nitrogen (LN2) a stable vitreous solid is formed without ice crystals. As with any glass, it must be handled with care to avoid fractures. In practice, oocytes are equilibrated in two or more steps to minimize osmotic stress before loading on a ‘cryotool’, such as a thin plastic platter or loop or straw (Box 1) [8]. It seems at odds with our technological era, so dominated by microelectronics, satellite technology, and genomics that an unsophisticated technology can carry sway in any field, but the protocol has been finely-tuned and requires skilful execution. In expert hands survival rates approach 100%, and implantation, pregnancy, and live-birth rates are remarkably high after oocyte vitrification [9,10]. Worldwide, over 2000 babies have been conceived, and obstetric followup, although limited, has been reassuring so far [11]. Because most ART clinics in the USA offer oocyte banking [12], warnings from professional organizations that the technique is still ‘experimental’ seem out-of-date. Despite justified enthusiasm for vitrification, some supposed advantages need to be qualified. Each pair of oocytes takes 15 min to be processed, but the time taken for a large batch of, say, 20 or more from one patient is comparable to the slow-freezing method (3–4 h). Furthermore, the higher concentrations of CPAs (3- to 4-fold) raise concern about toxicity, although this is moderated by rapid processing and using two CPAs of half strength. There is also continuing discussion at professional and regulatory levels about the safety of ‘open’ systems, in which specimens are plunged directly into non-sterile LN2, although it is
Forum: Science & Society
(a)
Trends in Endocrinology and Metabolism October 2012, Vol. 23, No. 10
50 µm
(b)
(c) TRENDS in Endocrinology & Metabolism
Figure 1. Comparison of (a) human metaphase II oocyte, (b) eight-cell embryo, and (c) blastocyst.
acknowledged that barrier methods sometimes leak, as well as being somewhat less effective for cryopreservation. Reassuringly, there are no reported cases of contamination from LN2 in the history of ARTs. Although some clinics sterilize LN2 by filtration/UV light, we tested whether a pure non-reactive compound can serve as a secondary coolant in LN2. The most promising candidate was perfluoropropane, but it has handling difficulties. Perhaps the most underestimated limitation of the technology is its dependence on operator skill. Oocytes floating in viscous media can be lost, and each step in the protocol has little leeway for guaranteeing safe and effective cellular preservation. Success rates are likely to be variable between centers until sufficient expertise is more widespread from routine practice. Advancing technology may eventually address this problem by automating cryopreservation in a ‘chip’, but meantime let the buyer beware. But who are the buyers? Bank depositors First, and perhaps foremost, oocyte banking offers hope for young women needing to preserve fertility before sterilizing treatment for cancer and some non-malignant diseases, as well as for other causes of premature menopause. Until
Box 1. Representative protocol for oocyte vitrification based on Kuwayama’s Cryotop method [8] Pairs of fresh oocytes are transferred at room temperature from wash medium to equilibration medium containing 7.5% ethylene glycol + 7.5% DMSO in buffered salt solution containing protein. They are transferred to vitrification medium containing 15% ethylene glycol + 15% DMSO + 0.5 M sucrose/trehalose. More steps may be added to minimize osmotic stress, but for only 1–2 min per step. Transfer to the cryotool. In the case of the Cryotop or its analogs, excess medium is drained from the tip of the tool to leave a film of 0.1 ml. The cryotool is plunged into liquid nitrogen (LN2), cooling the specimen at >20 000 8C/min. The specimen, now vitrified on the cryotool, is stored in a protective sleeve in LN2 or LN2 vapor. Specimens are rewarmed very rapidly by plunging the cryotool into rehydration medium containing 0.5 M sucrose/trehalose followed by medium at normal osmolality.
recently, doubts about this technology encouraged ovarian tissue banking, which will probably remain a backup strategy, except where oocyte harvesting is not an option, for example in prepubertal children [13]. This progress is most welcome, but it will be a long time until sufficient data are available to estimate success and any complications reliably because such patients will bank their oocytes until they return to good health and are ready to start a family. Because oocyte donation is common (12% of ARTs in 2005, according to the Centers for Disease Control), it provides an earlier opportunity to gauge success with vitrification. Sometimes, there is even a chance to compare fresh and cryopreserved oocytes after randomization from the same cohort, using the same semen. In a report from a large research center there was no difference between fertilization, embryo development, and pregnancy rates in the two arms, indicating that vitrified oocytes retain excellent quality [10]. Banking also provides a benefit that has long served sperm donation by enabling oocytes to be quarantined until donors are retested for transmissible diseases. Oocyte vitrification will gradually replace embryo cryopreservation, a procedure which is fraught with issues concerning the disposition of surplus embryos or the action to be taken when one of the gamete partners dies or they divorce. Oocyte banking should therefore help to ease lingering opposition to ARTs. Considering the trend towards single blastocyst transfer, we imagine treatment evolving towards vitrification of an entire cohort of oocytes, except for one that is freshly fertilized, with subsequent rewarming of singletons in turn for as many conception attempts as are needed. This will give women greater reproductive autonomy at the same time as dodging multiple pregnancy. When frozen sperm are unavailable, banking also avoids wasting oocytes if the male partner fails to produce a semen sample or sperm cannot be retrieved from testicular biopsies. Finally, this technology is increasingly sought-after as an elective procedure, commonly, and somewhat pejoratively, known as ‘social egg banking’. In most developed countries, and increasingly elsewhere, rising educational and professional expectations and achievements push women into postponing a family until their late 30 s or 40 s, after the window of optimal fertility has closed [14]. 499
Forum: Science & Society For other women reasons can be the breakdown of a conjugal relationship or not having found the ‘right’ partner. Standard IVF technology cannot reverse the problem of reproductive aging, except by oocyte/embryo donation, but banking oocytes when young, although expensive, provides some reassurance, if not insurance, against infertility later in life [15]. Given the straits of modern life, women should not be censured for hedging their bets in this way. Even so, we suspect that most deposits in egg banks will never be drawn, as is the case with semen banking, but the female gamete, being so much more highly prized, could then be sold on the donor market or for stem cell technology. At any rate, cryopreservation will encourage commercial tendencies in ART, and so stocks in ‘oocyte futures’ look rosy. Acknowledgments We thank Dr Nikica Zaninovic for discussion.
References 1 Chen, C. (1986) Pregnancy after human oocyte cryopreservation. Lancet 1, 884–886 2 Whittingham, D.G. et al. (1972) Survival of mouse embryos frozen to – 196 degrees and –269 degrees C. Science 178, 411–414 3 Porcu, E. et al. (1997) Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil. Steril. 68, 724–726 4 Tucker, M.J. et al. (1998) Clinical application of human egg cryopreservation. Hum. Reprod. 13, 3156–3159
500
Trends in Endocrinology and Metabolism October 2012, Vol. 23, No. 10 5 Borini, A. et al. (2006) Cumulative pregnancy rates resulting from the use of fresh and frozen oocytes: 7 years experience. Reprod. Biomed. Online 12, 481–486 6 Scaravelli, G. et al. (2010) Analysis of oocyte cryopreservation in assisted reproduction: the Italian National Register data from 2005 to 2007. Reprod. Biomed. Online 21, 496–500 7 Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at –196 degrees C by vitrification. Nature 313, 573–575 8 Kuwayama, M. et al. (2005) Highly efficient vitrification method for the cryopreservation of human oocytes. Reprod. Biomed. Online 11, 300–308 9 Nagy, Z.P. et al. (2009) The efficiency and safety of human oocyte vitrification. Semin. Reprod. Med. 27, 450–455 10 Cobo, A. et al. (2011) Oocyte cryopreservation for donor banking. Reprod. Biomed. Online 23, 341–346 11 Noyes, N. et al. (2009) Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod. Biomed. Online 18, 769–776 12 Rudick, B. et al. (2010) The status of oocyte cryopreservation in the United States. Fertil. Steril. 94, 2642–2646 13 Gosden, R.G. (2009) Fertility preservation: definition, history and prospect. Semin. Reprod. Med. 27, 433–437 14 Wyndham, N. et al. (2012) A persistent misperception: assisted reproductive technology can reverse the ‘aged biological clock’. Fertil. Steril. 97, 1044–1047 15 Lockwood, G.M. (2011) Social egg freezing: the prospect of reproductive ‘immortality’ or a dangerous delusion? Reprod. Biomed. Online 23, 334–340 1043-2760/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2012.05.010 Trends in Endocrinology and Metabolism, October 2012, Vol. 23, No. 10