Vitrification of oocytes

DOI: 10.1111/j.1744-4667.2011.00078.x The Obstetrician & Gynaecologist http://onlinetog.org 2012;14:45 49 SAC review Vitrification of oocytes This article was commissioned by the Scientific Advisory Committee (SAC). Maureen J Wood PhD Honorary Research Fellow, Department of Obstetrics and Gynaecology, University of Aberdeen, Aberdeen Maternity Hospital, Foresterhill, Aberdeen AB25 2ZL, UK Correspondence: Maureen J Wood. Email: maureen.wood@abdn.ac.uk Key content Vitrified oocytes can retain a capacity for fertilisation and development which is similar to that of non-cryopreserved sibling oocytes. More than 450 births have resulted from assisted conception with vitrified oocytes. Controlled studies are required before the efficiency of vitrification can be fully assessed. Aspects of methodology and safety still need to be resolved before vitrification is accepted unreservedly as part of conventional assisted conception. Learning objectives To understand what is meant by vitrification and how it is achieved in practice. To learn about the advantages and drawbacks of oocyte vitrification. To have an overview of the success of assisted conception with vitrified oocytes. To appreciate where questions remain to be answered about the methodology, safety and clinical outcome of vitrification. Ethical issues Should assisted conception be offered with donor oocytes that have not undergone full quarantine similar to that required for donated sperm and embryos? Keywords assisted conception / congenital anomalies / oocyte cryopreservation / oocyte donation / pregnancy rates Pleasecitethis paper as:wood MJ. Vitrification of oocytes. The Obstetrician & Gynaecologist 2012;14:45 49. Introduction Oocyte cryopreservation has practical, legal and ethical advantages compared with embryo storage. It allows oocytes to be quarantined before donation, as is already mandatory for sperm and embryos, it simplifies the management of a donation programme and it ensures the most efficient distribution of the few donated oocytes available. Women at risk of early ovarian failure can store oocytes so that they can retain the possibility of bearing their own genetic children. This is vital for women without a partner and eliminates the problems associated with withdrawal of consent when embryos are stored. More contentiously, women could store oocytes to delay childbearing without there being a medical indication. During routine assisted conception cycles, oocytes can be cryopreserved as an emergency procedure or to avoid producing supernumerary embryos. The first successful pregnancy conceived by in vitro fertilisation (IVF) of a frozen oocyte, 1 in 1986, was followed by a decade with little progress. More recently, modification of the freezing medium and universal fertilisation by intracytoplasmic sperm injection (ICSI) have combined to increase the numbers of embryos available for transfer, resultinginthebirthof>532 children conceived from frozen oocytes worldwide. 2 Vitrification is an alternative to freezing for oocyte cryopreservation. The first birth from assisted conception with vitrified oocytes occurred in 1999 3 and >450 subsequent 2, 4 17 births have been reported. While most of us are familiar with gamete and embryo freezing, vitrification is less well understood. This article provides an introduction to the principles and practice of vitrification; outlines its advantages and drawbacks; and summarises the clinical outcome of assisted conception with vitrified in vivo-matured oocytes. What is vitrification? Vitrification is the process in which a solution and the cells within it solidify during cooling to form a transparent, amorphous glass. Ice does not form during cooling and, equally importantly for cell survival, the sample remains ice-free during warming. The main drawback of vitrification in practical applications is the requirement to suspend the oocytes in a concentrated solution of antifreeze (cryoprotectants). This puts them at risk of chemical injury and osmotic damage as water and cryoprotectants enter and leave the cell. Several strategies are designed to limit injury, including: C 2012 Royal College of Obstetricians and Gynaecologists 45

Vitrification of oocytes stepwise increases in cryoprotectant concentration before cooling precisely timed exposure to the concentrated vitrification solution controlled removal of the cryoprotectants after warming. The strategy that has had the greatest impact on the advancement of vitrification is that of increasing the cooling rate to >20 000 C/minute to obtain vitrification with the lowest possible concentration of cryoprotectants. Such high rates of cooling are achieved by suspending the oocyte in a tiny volume (often <0.1 μl) of the vitrification solution, then placing the sample in direct contact with liquid nitrogen at 196 C. Vitrified samples are warmed rapidly to avoid intracellular crystallisation, although the effect of warming rates on survival remains to be examined systematically. A variety of vitrification solutions and novel containers are in use; 3, 6, 16, 18 20 their relative effectiveness for human oocytes hasnotbeencomparedincontrolledtrials. Vitrification in practice Vitrification is said to be simpler, more efficient and cheaper than conventional freezing: all of which is true under certain circumstances. Opinions differ about the simplicity of vitrification. Exposure to the solutions must be timed precisely, and loading and storing some of the novel containers is more demanding than for conventional straws. An experienced embryologist should have no difficulty in mastering the techniques, although some have reported anecdotally a considerable learning curve 10, 16, 21 and noticeable technician effects. Vitrification is quicker than freezing for a small group of oocytes collected from a single donor, whereas freezing may be more efficient when oocytes are to be stored for several women onthesameday.however,thisefficiencymustbesetagainstthe flexibility of vitrification, which allows each group of oocytes to be stored at a precise time after administration of human chorionic gonadotrophin; thus ensuring sufficient time after warming for the oocyte cytoskeleton to repolymerise before sperm injection within the optimal window for fertilisation. In clinical practice ICSI is the preferred method for the fertilisation of vitrified oocytes, although its superiority over IVF for such oocytes has not been demonstrated in controlled trials. Vitrification does not require an expensive controlled rate freezer but may incur the costs of additional embryological support, highly purified liquid nitrogen and, possibly, sophisticated vapour phase storage equipment. Vitrification consumables are more expensive than their counterparts for freezing. The safety of vitrification for oocytes and any children conceived Oocyte vitrification in tiny volumes of solution in open containers has safety implications that must be addressed. Cooling oocytes in open containers risks contaminating them with pathogenic bacteria and viruses found in liquid nitrogen. 22 Containers that are heat-sealed before cooling are safest 22 and have been used successfully for oocyte vitrification. 16, 23 Many clinics, however, favour open containers, for which cooling in highly purified liquid nitrogen 7, 22 is recommended. After cooling, the container can be hermetically sealed within a secondary container before storage in conventional liquid nitrogen. 24 Alternatively, open containers can be stored in nitrogen vapour 7 to lower the risk of contamination. It has not yet been demonstrated whether the samples remain uncontaminated after prolonged vapour phase storage. Tiny vitrified samples are at considerably greater risk than conventional frozen ones of being warmed accidentally to lethally high temperatures (above 130 C). Strict procedures are vital to avoid any uncontrolled rise in temperature when handling vitrified samples during audit of storage Dewars and when samples are being retrieved from storage or transported to another clinic. Most low-temperature biologists would prefer the safety of keeping vitrified samples immersed in liquid nitrogen rather than in the vapour phase, even though oocytes remain viable for at least 10 months when stored in vapour in a sophisticated system with strict temperature control. 7 It remains to be confirmed whether vitrified oocytes will survive, in either liquid or vapour phase nitrogen, the prolonged storage required by some women. We cannot yet be certain that there are no short or long-term implications for the children conceived from vitrified oocytes; the same is true for frozen oocytes. Encouragingly, among 392 children developed from vitrified oocytes, only six birth anomalies were identified; an incidence that does not differ from that in natural conception. 2 How successful is oocyte vitrification? Most of the literature on the clinical application of oocyte vitrification is in the form of case reports and retrospective analyses of data. Prospective studies providing a sound evidence base for practice are scarce. 13, 16, 25, 26 Overall, high rates of survival, fertilisation and cleavage of the resultant zygotes are reported for oocytes vitrified after collection from donors or during routine assisted conception cycles (Table 1). 25, 26 In controlled comparisons, vitrified oocytes from donors and infertile women 13 were as competent to develop in vitro as their fresh sibling oocytes. It is more difficult to assess the outcome of transferring embryos derived from vitrified oocytes because the data, 46 C 2012 Royal College of Obstetricians and Gynaecologists

Wood Table 1. clinics a Survival, fertilisation and cleavage in vitrified oocytes collected from donors and patients undergoing assisted conception in 11 separate Oocytes from donors 7, 12, 25, 26 and fertile women with tubal ligation 10 Oocytes from assisted conception 6, 8, 13, 14, 16 18, 21 cycles No. of women from whom oocytes were collected Median (IQR) 37 (21.8 45.5) 46 (39.5 62) Range 10 295 12 120 Mean age (years) Median (IQR) 27.1 (26.7 27.7) b 34 (32.5 35.4) Range 26 31.7 31.5 35.8 No. of oocytes warmed Median (IQR) 429.5 (272 470.8) 307.5 (229 377.5) Range 153 3286 124 487 % oocyte survival c Median (IQR) 94 (90 95) 84 (78.5 91.8) Range 81 97 75 99 % fertilisation d Median (IQR) 73.5 (72.3 75.5) 79 (76.5 82) Range 72 87 73 93 % cleaved zygotes e Median (IQR) 95 (94.3 95.8) 91.5 (91 95.8) f Range 90 98 84 98 a Three separate studies from one clinic are cited. b Mean age missing from one donor study. c Percentage of oocytes with normal morphology after warming. d Percentage of injected oocytes with two pronuclei 18 hours post insemination. e Percentage of fertilised oocytes undergoing at least one cleavage division. f Cleavage data were missing from two citations. IQR = interquartile range particularly the details of live births, are often incomplete. Implantation and pregnancy rates may be distorted by factors such as patient age and variations in practice, including the developmental stage and numbers of embryos transferred. In some clinics there are restrictions on the numbers of oocytes injected and all embryos produced are transferred; in others, large groups of oocytes are injected, the best embryos selected for transfer and the supernumerary embryos cryopreserved for future use or discarded. The overview in Table 2 takes no account of these factors or the different vitrification procedures used. Most authors report pregnancies or implantations per embryo transferred or per oocyte warmed. Two controlled studies show no difference in ongoing pregnancy rates after thetransferofembryosdevelopedfromfreshandvitrified oocytes. 13, 26 The births of 392 children conceived from vitrified oocytes were verified in 2009 2 and since then at least 67 more have been reported, with many pregnancies ongoing. 4 17 It will be possible to evaluate fully the efficiency of vitrification only when, in line with current clinical practice, we have live birth data collected from controlled studies. The question most frequently asked: should we freeze or should we vitrify oocytes? remains unanswered. The first prospective randomised comparison of oocyte freezing and oocyte vitrification in a clinical setting 16 suggests that survival, fertilisation, subsequent embryo development and establishment of pregnancy are all significantly improved in cycles using vitrified oocytes. However, the authors themselves point out that, in hindsight, the freezing protocol in the trial may not have been optimal to conserve oocyte viability. Conclusions Vitrification is a promising approach to oocyte storage which can conserve oocyte competence for fertilisation and development at a level similar to that of freshly collected oocytes. Viable pregnancies have been conceived from the embryos derived from vitrified oocytes, but to date there have been too few live births to allow a realistic assessment of the efficiency of oocyte vitrification. A number of outstanding issues regarding methodology, safety of storage and the longterm health of the children born will be resolved only as oocyte vitrification becomes more widespread and trials are designed to provide the evidence base required in modern assisted conception. It is vital that procedures do not become set in stone before they are fully optimised and that protocols and outcomes, especially live births, are fully documented and published. C 2012 Royal College of Obstetricians and Gynaecologists 47

Vitrification of oocytes Table 2. Outcomes of transferring embryos developed in vitro from vitrified oocytes in 11 separate clinics a Oocytes from donors 7, 12, 25, 26 and fertile women with tubal ligation 10 Oocytes from assisted conception 6, 8, 13, 14, 16 18, 21 cycles No. of oocyte recipients or women having autologous transfer Mean (IQR) 37 (22.5 45.5) 46 (39.5 62) Range 19 295 12 120 Mean age (years) Median (IQR) 40.4 (39.8 40.9) 34 (32.5 35.4) Range 31.7 41.1 31.5 35.8 No. of embryo transfers Median (IQR) 33 (20.8 44.5) 46.5 (38.8 69) Range 19 267 11 120 No. of embryos transferred Median (IQR) 70 (50 87.8) 139 (104.3 187.3) Range 47 513 27 295 Mean no. of embryos transferred per recipient Median (IQR) 2 (1.8 2.3) 2.5 (2.4 3.2) Range 1.7 2.7 1.9 3.5 % pregnancy per embryo transfer Median (IQR) 59.5 (55.8 71.5) 38.5 (32.8 43) Range 54 80 18 73 % implantation per embryo transferred Median (IQR) 40.5 (40 44) 15.5 (13.8 19.3) Range 34 55 9 52 % implantation per oocyte warmed b Median (IQR) 7.5 (6.5 7.6) c 9.2 (5.6 10.4) Range 6.3 21.3 3.5 15.3 a Three separate studies from one clinic are cited. b The supernumerary embryos produced and cryopreserved for future use have been deducted from the total number of oocytes warmed in this calculation. c Value unobtainable from one citation IQR = interquartile range Acknowledgements I am grateful to Professor Brian Lieberman for his constructive comments on this manuscript and to Dr Jill Mollison for her advice on the presentation of data. References 1 Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;327:884 6 [http://dx.doi.org/10.1016/s0140-6736(86)90989 -X]. 2 Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online 2009;18:769 76 [http://dx.doi.org/ 10.1016/S1472-6483(10)60025-9]. 3 Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes. Hum Reprod 1999;14:3077 9 [http://dx.doi.org/10.1093/ humrep/14.12.3077]. 4 Chang C-C, Shapiro DB, Bernal DP, Wright G, Kort HI, Nagy ZP. Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrification. Reprod Biomed Online 2008;16:346 9 [http:// dx.doi.org/10.1016/s1472-6483(10)60594-9]. 5 Chen GA, Cai XY, Lian Y, Zheng XY, Qiao J, Chen XN, et al. Normal birth from cryopreserved embryos after intracytoplasmic sperm injection of frozen semen into vitrified human oocytes. Hum Fertil 2008;11:49 51 [http://dx.doi.org/10.1080/14647270701510025]. 6 Chian R-C, Huang JYJ, Gilbert L, Son W-Y, Holzer H, Cui SJ, et al. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril 2009;91:2391 8 [http://dx.doi.org/ 10.1016/j.fertnstert.2008.04.014]. 7 Cobo A, Romero JLl, Pérez S, de los Santos MJ, Meseguer M, Remohí J. Storage of human oocytes in the vapour phase of nitrogen. Fertil Steril 2010;94:1903 7 [http://dx.doi.org/10.1016/ j.fertnstert.2009.10.042]. 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 [http://dx.doi.org/10.1016/s1472-6483(10)60069-7]. 9 Keskintepe L, Agca Y, Sher G, Keskintepe M, Maassarani G. High survival rate of metaphase II human oocytes after first polar body biopsy and vitrification: determining the effect of previtrification conditions. Fertil Steril 2009;92:1706 15 [http://dx.doi.org/10.1016/ j.fertnstert.2008.08.114]. 10 Kim TJ, Laufer LR, Hong SW. Vitrification of oocytes produces high pregnancy rates when carried out in fertile women. Fertil Steril 2010;93:467 74 [http://dx.doi.org/10.1016/j.fertnstert. 2008.12.094]. 11 Liow SL, Foong LC, Chen NQ, Yip WY, Khaw CL, Kumar J, et al. Live birth from vitrified warmed human oocytes fertilized with frozen thawed testicular spermatozoa. Reprod Biomed Online 2009;19:198 201 [http://dx.doi.org/10.1016/s1472-6483(10)60072-7]. 48 C 2012 Royal College of Obstetricians and Gynaecologists

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