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A review of best practices of rapid-cooling vitrification for oocytes and embryos: a committee opinion (2021)


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The focus of this paper is to review best practices for rapid-cooling cryopreservation of oocytes and embryos. The discussion of best practices includes the types of cryoprotectants and cryo devices typically used. Key performance indicators of rapid-cooling vitrification success are defined. (Fertil Steril® 2021;115:305-10. ©2020 by American Society for Reproductive Medicine.)

The focus of this paper is to review best practices for rapid-cooling cryopreservation of oocytes and embryos. The discussion of best practices includes the types of cryoprotectants and cryo devices typically used. Key performance indicators of rapid-cooling vitrification success are defined.

DEFINITIONS 

Embryo and Oocyte Warming. Commonly but incorrectly referred to as ‘‘thawing’’ or ‘‘rewarming,’’ refers to the relatively rapid increase in temperature of cells stored in liquid nitrogen to room temperature or above under defined laboratory conditions (1, 2). 

Rapid Cooling. A reduction in temperature, typically at rates of more than -2,500oC/min, before storage in liquid nitrogen at -196oC. Commonly referred to in the literature as ‘‘vitrification.’’ 

Slow Cooling. A gradual reduction in temperature, typically at rates of -0.1 to -3oC/min, to -30oC or lower before storage in liquid nitrogen at -196oC. Ice crystal formation occurs extracellularly. 

Slow Freezing. A misnomer for slow cooling that implies the presence of intracytoplasmic ice crystals, which ideally does not happen.

Vitrification. Formation of an amorphous solid or glass-like state (noncrystalline). Vitrification depends on cooling rate and solution composition and can occur with both slow and rapid cooling. 

HISTORY OF GAMETE AND EMBRYO CRYOPRESERVATION 

In 1985, a conventional embryo cryopreservation method using 1, 2-propanediol (PROH) as a cryoprotectant and a programmed slow-cooling method was reported (3). Successful pregnancies were achieved from slow-cooled human embryos but rarely achieved from slow-cooled human oocytes. The first report of a successful pregnancy using cryopreserved oocytes with a slow-cooling and rapid-warming method was in 1986 (4). Additional studies of oocyte cryopreservation were reported, but the overall efficiency of these protocols remained low. The technique came under scrutiny when it was suggested that cryopreserved oocytes showed higher levels of chromosomal anomalies compared with fresh oocytes (5–7), thus tempering enthusiasm for the technology. In the 9 years that followed the first reported pregnancy, a total of only five births from cryopreserved-warmed oocytes were reported (8–10). Subsequent research determined that there was no increase in aneuploidy after oocyte cryopreservation (11, 12). In 1998, the first baby was born from a cryopreserved immature oocyte (13). 
 
Improvements of culture media and laboratory techniques led to a resurgence of research toward improving oocyte and blastocyst cryopreservation (14–18). In 1998, a landmark publication described the use of an open pulled straw (OPS) and ethylene glycol and dimethyl sulfoxide (DMSO) that allowed for a minimum volume of 1–2 μL of medium to be used for cryopreservation of bovine ova (19). The combination of cryoprotectants, rapid-cooling rate (greater than -10,000oC/min), and small volume with an ‘‘open’’ device allowed the cells to survive plunging into liquid nitrogen from room temperature. Rapidly cooled–warmed oocyte survival rates of >90% were seen and live births reported (20–22). 
 

PRINCIPLES OF VITRIfiCATION 

The idea of vitrification, achieving a glass-like state, was first described in 1860, and then again in 1937 (23). It was not until nearly 50 years later, in 1985, that rapid cooling from above-zero temperatures was described as a potential alternative to slow cooling (24). As the temperature of a liquid decreases to below the glass-transition temperature, molecules remain in the disordered pattern of a liquid. However, the physical properties become more similar to those of a rigid solid. Molecules become locked in place as though the liquid were frozen in time. The resulting ‘‘solid liquid’’ is called a glass or an amorphous glass-like state. 
 
Vitrification can be described in an equation with four variables: cooling rate, warming rate, viscosity, and sample volume (25). Current rapid-cooling vitrification procedures involve exposure of cells suspended in very small volumes to relatively high concentrations of cryoprotectant(s) for brief periods of time to avoid chemical toxicity, followed by rapid cooling in liquid nitrogen. The high osmolarity of the vitrification solutions rapidly dehydrates the cell, and submersion into liquid nitrogen quickly solidifies the cell so that the remaining intracellular water does not have time to form damaging ice crystals. The cell undergoes a temperature transition from room temperature to -196oC in <2 seconds, resulting in extremely fast rates of cooling (greater than -10,000oC/min) (26). To facilitate rapid heat transfer and reduce chemical toxicity, minimal volumes and small, open cryopreservation devices are used. The warming rate is at least, if not more, important than the cooling rate, as elegantly demonstrated in experiments using mouse oocytes (27). The investigators concluded that the lethality of a slow warming rate is a consequence of allowing time for the development and growth of small intracellular ice crystals by recrystallization. 
 
Even though there are numerous rapid-cooling solutions and methodologies available that work well, these are largely modifications of the DMSO-based protocol described in 1998. During the same period, another vitrification system that did not use DMSO as the main permeable cryoprotectant was also developed (28). The different cryopreservation devices used to store the cells are all microvolume devices that will hold 1–3 μL of medium along with several cells. They all have roughly the same rapid-cooling rates (greater than -10,000 to -50,000oC/min) when plunged into liquid nitrogen from room temperature. A list of key papers describing the evolution of rapid-cooling vitrification are listed in Supplemental Table 1 (4, 13, 19, 24, 28–40).

Supplemental Table 1. Vitrification bibliography summary.


Reference Significance
Rall et al. 1985 (24) First application of rapid cooling for mouse embryos
Chen. 1986 (4) First pregnancy after human oocyte cryopreservation
Schiewe et al. 1991 (29) First sheep embryo rapid cooling
Tucker et al. 1998 (13) Cryopreservation of immature oocyte, in vitro IVM, subsequent birth
Vajta et al. 1998 (19) Bovine rapid cooling in open pulled straws
Kuleshova et al. 1999 (30) First birth after human oocyte rapid cooling
Mukaida et al. 2001 (31) First delivery after human blastocyst rapid cooling
Stachecki et al. 2004 (32) Overview of oocyte cryopreservation
Kuwayama. 2007 (33) Description of Cryotop method of oocyte rapid cooling
Chang et al. 2008 (34) Pregnancies after oocyte rapid cooling and blastocyst re–rapid cooling
Stachecki et al. 2008 (28) Description of novel blastocyst rapid-cooling system
Stachecki et al. 2008 (35) Description of S3 rapid cooling system
Gook. 2011 (36) Overview of oocyte cryopreservation
Cobo et al. 2012 (37) Cohort study of cleavage-stage and blastocyst rapid cooling outcomes
Schiewe et al. 2015 (38) Description of novel cryopreservation device microSecure
de Munck et al. 2017 (39) Overview of human oocyte cryobiology
Reinzi et al. 2017 (40) Meta-analysis and overview of assisted reproductive technology cryopreservation

Note: This is not a comprehensive list.

OOCYTE RAPID-COOLING VITRIfiCATION BEST PRACTICES 

Cryoprotectants 

The two sets of solutions discussed here, DMSO based and non–DMSO based, are not in opposition; rather, they represent two separate and very different systems to obtain vitrified oocytes and embryos. To date, efficient rapid-cooling vitrification of MII oocytes has been achieved using both DMSO and non-DMSO solutions in a number of mammalian species, including mice, rabbit, bovine, porcine, felid, and humans, in combination with various commonly used devices, many of which are listed in Supplemental Table 2 (available online at www.fertstert.org). When done properly, both systems have very good results. Both represent current and best viable options for vitrification of human oocytes and embryos. There are differences between the systems (volumes, cooling rate, etc.). The major difference is the fact that DMSO is present or absent, because this changes the dynamics of the system; the absence of DMSO allows for the use of slower cooling rates, large volumes, and different carriers. The potential toxicity of cryoprotectants, including DMSO, is widely published on (24, 41). 
 
Outcomes following rapid-cooling vitrification are closely related to the skills of the operators who perform the procedure. Therefore, a well-trained team is mandatory to succeed and to obtain consistent results. A strict quality-control program must be applied to the application of rapid-cooling vitrification, which includes controlling learning curves, analysis of the operator’s outcomes, vendor lots of solutions used, etc. (42, 43). 

Cryopreservation Devices 

At least 30 different carrier tools have been described, and at least 15 versions are commercially available (Vajta 2015). Most of them are slightly modified versions of the initially introduced carrier tools, such as the OPS (19), Cryoloop (44, 45), and Cryotop (33). All of these systems are open systems in their original form. Some closed systems are the results of the modifications of these open systems (46). Less commonly used devices include electron microscope grids, drops into liquid nitrogen, and gel loading tips, to name a few. 
 
The Cryotop system, developed in Japan, was mass-marketed and became a best-selling microvolume storage device (36). Owing to its technically challenging nature, the Cryotop system, in conjunction with the ethylene glycol/DMSO/sucrose process, was originally slow to gain widespread acceptance for oocyte rapid-cooling vitrification (47, 48). However, after minor methodologic modifications, two large comparative studies established its place in oocyte cryopreservation history (49, 50). Most laboratories using rapid-cooling vitrification solutions also use open devices for both cooling and storage in liquid nitrogen (39). This is due to the faster rates of cooling. 
 
The possibility of viral contamination of the liquid nitrogen has been suggested after the experimental spiking of liquid nitrogen storage vessels with high viral titers (51). Furthermore, the contamination of vitrification carriers immersed in liquid nitrogen with high microbial and fungal contamination levels has been demonstrated (52). However, there are no published reports of actual cross-contamination of cryopreserved embryos when open storage containers are used (26). Microbial contamination of liquid nitrogen has been reported, but again, there is no evidence that an embryo has been contaminated by direct contact with the liquid nitrogen (53). With these studies in mind, the concerns about contamination during liquid nitrogen storage remain theoretical, even more so when considering the fact that slow-cooled embryos have been stored in cryovials for a number of years, allowing liquid-nitrogen leakage during long-term storage (54). In reproductive biology, including mammalian and human assisted reproduction, no disease transmission caused by liquid nitrogen–mediated cross-contamination, or other cryopreservation-related source, has been reported (46). There are few commercially available vitrification devices that completely meet the requirement of sterility, and reported data are still limited (46). Straightforward procedures can be performed to minimize any potential contamination risk during vitrification, warming, shipping, and cryo storage using open or semiclosed carriers (55). 
 
There is a reluctance to vitrify using closed devices because of the hypothetical reduction in cooling rates, which may be produced in closed systems owing to thermoisolation and may increase the possibility of ice crystal formation during the cooling process and of recrystallization on warming (56). A systematic review and meta-analysis including seven studies reporting survival, implantation, clinical pregnancy, or live birth rates after closed or open rapid-cooling vitrification of blastocysts was published (57). There were no statistically significant differences in survival rates (risk ratio [RR] 1.00, 95% confidence interval [CI] 0.98–1.02), implantation rates (RR 1.02, 95% CI 0.93–1.11), clinical pregnancy rates (RR 0.99, 95% CI 0.89–1.10), or live birth rates (RR 0.77, 95% CI 0.58–1.03) between closed and open rapid-cooling vitrification. Although there was no statistically significant difference, the trend toward lower live birth rates with closed rapid-cooling vitrification than with open rapid-cooling vitrification is potentially concerning. The closed systems commonly in use are listed in Supplemental Table 2.

Supplemental Table 2. List of cryopreservation (cryo) devices in routine use (2020)


Cryotop Open cryo device
Cryolock Open cryo device
Vitriguard Open cryo device
Cryotec Open cryo device
Rapid-i Semiopen cryo device
Cryo Bio High Security Vitrification System (HSV) Closed cryo device
0.25 cc cryo straw Closed cryo device
microSecure Closed cryo device

KEY PERFORMANCE INDICATORS 

With this background as context, practical recommendations to optimize patient outcomes with oocyte rapid-cooling vitrification include the following:
 
  • A structured program for training and proficiency in oocyte cryopreservation should be developed. Similarly to other technically sensitive assisted reproductive technologies, operator metrics to demonstrate acquisition of competence should be recorded and evaluated, including the number of oocytes vitrified and the percentage surviving warming without evidence of damage. Meaningful benchmarks for proficiency must be determined and applied in operator evaluations. 
  • Because the number of oocytes at retrieval may vary widely among patients and stimulation cycles, a plan for cryopreservation will be prudent to develop in advance. This can include decisions concerning whether to cryopreserve all oocytes or only mature oocytes and how to distribute the oocytes, i.e., how many oocytes will be loaded in or on each cryo device. 
  • A validated technique with specific cryopreservation and warming solution formulations and cryopreservation devices should be used for oocyte vitrification. Composition of cryopreservation and warming solutions should be those associated with optimal outcomes. 
  • Handling of the oocytes and timing of the vitrification and warming procedures should be associated with optimal outcomes. 
  • The technical proficiency of the embryologists involved should be continually monitored through competency assessment and quality management system audits. 
  • A database should be maintained, allowing tracking and analysis of outcome parameters from the oocyte cryopreservation program, including such variables as:  

    1. total number of oocytes retrieved. 
    2. number of oocytes cryopreserved and stratified by maturational status. 
    3. number of oocytes warmed. 
    4. number of oocytes survived and inseminated by intracytoplasmic sperm injection. 
    5. number of oocytes fertilized. 
    6. number of embryos acquiring a developmental and quality stage consistent with transfer or cryopreservation. 
    7. number of embryos transferred. 
    8. number of embryos cryopreserved. 
    9. implantation rate. 
    10. clinical pregnancy rate. 
    11. live birth rate. 
    12. number of embryos or blastocysts warmed and transferred for vitrified-warmed embryo transfer (FET) cycles. 
    13. FET cycle outcome data. 
    14. clinically important information on the pregnancy/delivery/neonates. 
  • The same tenets that comprise a total quality management program in cryopreservation management should apply to cryopreserved oocytes. Best practices for management of cryopreserved tissues, including minimum standards and requirements for critical cryostorage, have been published (58).


EMBRYO AND BLASTOCYST RAPID-COOLING VITRIfiCATION 

Cleavage-stage embryos and blastocysts on day 5–7 can be rapidly cooled by the same, or slightly modified, protocols used to vitrify oocytes. Reports indicate that artificial shrinkage of the large blastocele of a day-5–7 blastocyst might lessen cryoinjury during both cooling and warming phases (59). Manual puncture of the trophectoderm by needle or laser before rapid-cooling vitrification has been demonstrated to improve survival rates of rapid-cooled blastocysts and results in a higher percentage of high-quality and hatching blastocysts, but not necessarily in improved implantation rates. 
A study published in 2016 demonstrated that transfer of rapid-cooled day-3 and day-5 embryos did not adversely affect the neonatal health of offspring compared with transfer of fresh embryos. Furthermore, neonatal outcomes were not different after transfer of rapid-cooled blastocysts compared with rapid-cooled cleavage-stage embryos (60). There are many other studies that support the effectiveness of modern rapid-ooling vitrification techniques for both oocytes and embryos. 


FUTURE PERSPECTIVES 

Vitrification via rapid cooling is strongly recommended as standard of care for cryopreservation of human oocytes and embryos. Rapid-cooling strategies are being developed for ovarian tissue and for sperm, particularly for patients with oligospermia or for patients with either nonobstructive or obstructive azoospermia for whom testicular sperm must be cryopreserved in very low numbers.


SUMMARY 

  • Implementation of quality-control measures are necessary to obtain consistent high-quality results with rapid-cooling vitrification of oocytes and embryos. 
  • Selection of optimized protocols, along with operator training, will result in increases in efficiency, consistency, reliability, and safety. 
  • Current vitrification systems work well, with survival rates of embryos approaching 100% and pregnancy rates that are similar to if not better than fresh transfer rates. 
 
Acknowledgments: This report was developed under the direction of the Practice Committee of the American Society for Reproductive Medicine (ASRM) in collaboration with the Society of Reproductive Biologists and Technologists (SRBT) as a service to its members and other practicing clinicians. Although this document reflects appropriate management of a problem encountered in the practice of reproductive medicine, it is not intended to be the only approved standard of practice or to dictate an exclusive course of treatment. Other plans of management may be appropriate, taking into account the needs of the individual patient, available resources, and institutional or clinical practice limitations. The Practice Committees and the Boards of Directors of ASRM and SRBT have approved this report. 
 
This document was reviewed by ASRM members, and their input was considered in the preparation of the final document. The following members of the ASRM Practice Committee participated in the development of this document: Alan Penzias, M.D., Kristin Bendikson, M.D., Tommaso Falcone, M.D., Karl Hansen, M.D., Ph.D., Micah Hill, D.O., William Hurd, M.D., M.P.H., Sangita Jindal, Ph.D., Suleena Kalra, M.D., M.S.C.E., Jennifer Mersereau, M.D., Catherine Racowsky, Ph.D., Robert Rebar, M.D., Richard Reindollar, M.D., Anne Steiner, M.D., M.P.H.: Dale Stovall, M.D., and Cigdem Tanrikut, M.D. All Committee members disclosed commercial and financial relationships with manufacturers or distributors of goods or services used to treat patients. Members of the Committee who were found to have conflicts of interest based on the relationships disclosed did not participate in the discussion or development of this document. The Practice Committee also acknowledges the special contribution of Sangita Jindal Ph.D., Kathryn Go, Ph.D., James Stachecki, Ph.D., and Zsolt Peter Nagy, M.D., Ph.D., in the preparation of this document. 


REFERENCES 

  1. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984;247(3 Pt 1):C125–42. 
  2. Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update 2003;9:583–605. 
  3. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985;44:645–51. 
  4. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;1: 884–6. 
  5. Johnson MH, Pickering SJ. The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte. Development 1987;100:313–24. 
  6. 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. 
  7. Sathananthan AH, Trounson A, Freemann L, Brady T. The effects of cooling human oocytes. Hum Reprod 1988;3:968–77. 
  8. Al-Hasani S, Diedrich K, van der Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod 1987;2:695–700. 
  9. Chen C. Pregnancies after human oocyte cryopreservation. Ann N Y Acad Sci 1988;541:541–9. 
  10. van Uem JF, Siebzehnrubl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet 1987;1:752–3. 
  11. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993;8:1101–9. 
  12. Gook DA, Osborn SM, Bourne H, Johnston WI. Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod 1994;9:684–91. 
  13. Tucker MJ, Wright G, Morton PC, Massey JB. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998;70: 578–9. 
  14. Levi Setti PE, Porcu E, Patrizio P, Vigiliano V, de Luca R, d’Aloja P, et al. Human oocyte cryopreservation with slow freezing versus vitrification. Results from the National Italian Registry data, 2007–2011. Fertil Steril 2014;102: 90–5.e2. 
  15. Fabbri R, Porcu E, Marsella T, Primavera MR, Rocchetta G, Ciotti PM, et al. Technical aspects of oocyte cryopreservation. Mol Cell Endocrinol 2000; 169:39–42. 
  16. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001;16:411–6. 
  17. Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, et al. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000;169:33–7. 
  18. Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004;82: 601–5. 
  19. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–8. 
  20. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80: 223–4. 
  21. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300–8. 
  22. Kyono K, Fuchinoue K, Yagi A, Nakajo Y, Yamashita A, Kumagai S. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte. Fertil Steril 2005;84:1017. 
  23. Luyet BE. The vitrification of organic colloids and protoplasm. Biodynamica 1937;1:1–14. 
  24. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 oC by vitrification. Nature 1985;313:573–5. 
  25. Panagiotidis Y, Vanderzwalmen P, Prapas Y, Kasapi E, Goudakou M, Papatheodorou A, et al. Open versus closed vitrification of blastocysts from an oocyte-donation programme: a prospective randomized study. Reprod Biomed Online 2013;26:470–6. 
  26. Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, et al. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod 2008;23:1976–82. 
  27. Seki S, Mazur P. The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure. Cryobiology 2009;59:75–82. 
  28. Stachecki JJ, Garrisi J, Sabino S, Caetano JP, Wiemer KE, Cohen J. A new safe, simple and successful vitrification method for bovine and human blastocysts. Reprod Biomed Online 2008;17:360–7. 
  29. Schiewe MC, Rall WF, Stuart LD, Wildt DE. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology 1991;36:279–93.
  30. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999;14:3077–9. 
  31. Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001;76:618–20. 
  32. Stachecki JJ, Cohen J. An overview of oocyte cryopreservation. Reprod Biomed Online 2004;9:152–63. 
  33. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 2007;67: 73–80. 
  34. Chang CC, Shapiro DB, Bernal DP, Wright G, Kort HI, Nagy ZP. Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrifi cation. Reprod Biomed Online 2008;16:346–9. 
  35. Stachecki JJ, Cohen J. S3 vitrification system: a novel approach to blastocyst freezing. J Clin Embryol 2008;11:5–14. 
  36. Gook DA. History of oocyte cryopreservation. Reprod Biomed Online 2011; 23:281–9. 
  37. Cobo A, de los Santos MJ, Castello D, Gamiz P, Campos P, Remohi J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril 2012;98:1138–46.e1. 
  38. Schiewe MC, Zozula S, Anderson RE, Fahy GM. Validation of microSecure vitrification (mS-VTF) for the effective cryopreservation of human embryos and oocytes. Cryobiology 2015;71:264–72. 
  39. de Munck N, Vajta G. Safety and efficiency of oocyte vitrification. Cryobiology 2017;78:119–27. 
  40. Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update 2017;23:139–55. 
  41. Fahy GM, Wowk B. Principles of Cryopreservation by Vitrification. Methods Mol Biol 2015;1257:21–82. 
  42. Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006;12:779–96. 
  43. Cobo A, Garrido N, Pellicer A, Remohi J. Six years’ experience in ovum donation using vitrified oocytes: report of cumulative outcomes, impact of storage time, and development of a predictive model for oocyte survival rate. Fertil Steril 2015;104:1426–34.e1–8. 
  44. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999;72:1073–8. 
  45. Lane M, Gardner DK. Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev 2001;58:342–7. 
  46. Vajta G, Rienzi L, Ubaldi FM. Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online 2015;30:325–33. 
  47. Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006;85: 108–11. 
  48. Selman H, Angelini A, Barnocchi N, Brusco GF, Pacchiarotti A, Aragona C. Ongoing pregnancies after vitrification of human oocytes using a combined solution of ethylene glycol and dimethyl sulfoxide. Fertil Steril 2006;86:997– 1000. 
  49. Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohi J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89:1657–64. 
  50. Antinori M, Licata E, Dani G, Cerusico F, Versaci C, Antinori S. Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries. Reprod Biomed Online 2007;14:72–9. 
  51. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000;40:110–6. 
  52. Parmegiani L, Accorsi A, Bernardi S, Amone A, Cognigni GE, filicori M. A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2). Fertil Steril 2012;98:870–5. 
  53. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–52. 
  54. Pomeroy KO, Harris S, Conaghan J, Papadakis M, Centola G, Basuray R, et al. Storage of cryopreserved reproductive tissues: evidence that cross-contamination of infectious agents is a negligible risk. Fertil Steril 2010; 94:1181–8. 
  55. Alikani M, Parmegiani L. Human reproductive cell cryopreservation, storage, handling, and transport: risks and risk management. Semin Reprod Med 2018;36:265–72. 
  56. Papatheodorou A, Vanderzwalmen P, Panagiotidis Y, Petousis S, Gullo G, Kasapi E, et al. How does closed system vitrification of human oocytes affect the clinical outcome? A prospective, observational, cohort, noninferiority trial in an oocyte donation program. Fertil Steril 2016;106:1348–55. 
  57. Youm HS, Choi JR, Oh D, Rho YH. Closed versus open vitrification for human blastocyst cryopreservation: a meta-analysis. Cryobiology 2017;77:64–70. 
  58. Practice Committee of the American Society for Reproductive Medicine. Revised minimum standards for practices offering assisted reproduction: a committee opinion. Fertil Steril 2020;113:536–41. 
  59. Mukaida T, Oka C. Vitrification of oocytes, embryos and blastocysts. Best Pract Res Clin Obstet Gynaecol 2012;26:789–803. 
  60. Belva F, Bonduelle M, Roelants M, Verheyen G, van Landuyt L. Neonatal health including congenital malformation risk of 1072 children born after vitrified embryo transfer. Hum Reprod 2016;31:1610–20. 

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EMR Shared Phrases/Template Library

This resource includes phrases shared by ASRM physician members to provide a template for individuals to create their own EMR phrases.

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ASRM Practice Documents

These guidelines have been developed by the ASRM Practice Committee to assist physicians with clinical decisions regarding the care of their patients.

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ASRM Ethics Opinions

Ethics Committee Reports are drafted by the members of the ASRM Ethics Committee on the tough ethical dilemmas of reproductive medicine.

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Practice Guidance

Coding Corner Q & A

The Coding Corner Q & A is a list of previously submitted and answered questions from ASRM members about coding. Answers are available to ASRM Members only.

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Practice Guidance

COVID-19 Resources

A compendium of ASRM resources concerning the Novel Corona virus (SARS-COV-2) and COVID-19.

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Patient Resources

ReproductiveFacts.org provides a wide range of information related to reproductive health and infertility through patient education fact sheets, infographics, videos, and other resources.

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Topic Resources

View more on the topic of embryo
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ASRM Files Amicus Brief in Texas Embryo Case

ASRM has filed an amicus curiae (friend of the court) brief in the case of Antoun v Antoun, which is pending before the Texas Supreme Court. 

View the Press Release
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IVF-assisted pregnancies constitute 2.5% of all births in 2022

In 2022, the number of babies born from IVF increased from 89,208 in 2021 to 91,771 in 2022. This means that 2.5% of births in the US are a result of ART.

View the Press Release
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Fertility and Sterility On Air - Unplugged: March 2024

Topics include: melatonin and implantation (4:38), whole-genome screening of embryos, and bioengineering assisted reproductive technology. Listen to the Episode
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Fertility and Sterility On Air - Live from PCRS 2024

Fertility & Sterility on Air brings you the highlights from the 2024 Annual Meeting of the Pacific Coast Reproductive Society. Listen to the Episode
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ASRM provides testimony to Senate Judiciary Committee on threats facing IVF

ASRM shared with the Senate Judiciary Committee the dangers to reproductive medicine nearly two years after the Dobbs decision.

View the Press Release
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What's New from the Fertility and Sterility Family of Journals

Here’s a peek at this month’s issues from our family of journals! As an ASRM Member, you can access all of our journals.
Read More about the newest articles
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Alabama Supreme Court Rules Frozen Embryos are “Unborn Children” and admonishes IVF’s “Wild West” treatment

Legally Speaking™ on presenting facts and reflecting on the impact and potential implications of  legal developments in ART. View the Column
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What's New from the Fertility and Sterility Family of Journals

Here’s a peek at this month’s issues from our family of journals! As an ASRM Member, you can access all of our journals. Not yet a member? Click here(no link) to learn more. Find More
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Shipping of frozen embryos

I have some infertility coverage, under which my insurance said they will cover frozen embryo shipping/transport from one facility to another.  View the Answer
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Clinical management of mosaic results from preimplantation genetic testing for aneuploidy of blastocysts: a committee opinion (2023)

This document incorporates studies about mosaic embryo transfer and provides evidence-based considerations for embryos with mosaic results on PGT-A. View the Committee Opinion
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Frozen Embryo Destruction and Potential Travel Restrictions for Surrogacy Arrangements

Legally Speaking™ focuses on the impact and the potential implications of legal developments on the assisted reproductive technologies. View the Column
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Journal Club Global: Transferencia de embriones frescos versus congelados: ¿Cuál es la mejor opción

Los resultados de nuevas técnicas de investigación clínica que utilizan información de bancos nacionales de vigilancia médica.   View the Video
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Defining embryo donation: an Ethics Committee opinion (2023)

The ethical appropriateness of patients donating embryos to other patients for  family building, or for research, is well established.
View the Committee Opinion
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Does the number of eggs being frozen matter?

There is currently only one CPT code for the cryopreservation of mature oocytes and embryos.  View the Answer
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Reproductive Tissue Storage

What are the CPT codes for the Storage of Reproductive Cells/Tissues? View the Answer
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ICSI and Embryo Biopsy

How to bill for ICSI or embryo biopsies that occur in different days?  View the Answer
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Lab RVUs

Is there a list of RVUs for embryology and andrology laboratory procedures, and if so, where can it be found? View the Answer
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Embryo Biopsy

Have any new codes been introduced for the lab portion of PGT? View the Answer
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Embryo Biopsy Embryologist Travel Costs

Can we bill insurance for the biopsy procedure? Can we bill for travel expenses? View the Answer
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Embryo Biopsy PGS Testing

What codes are appropriate for PGS testing? View the Answer
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Embryo Co-culture

Can codes 89250 and 89251 be billed on different days of the same cycle?  View the Answer
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Embryo Culture Denied As Experimental

We have received denials from insurance payers when billing CPT code 89251.  View the Answer
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Embryo Culture Less Than And More Than Four Days

When coding 89250 culture of oocytes/embryo <4 days, should that code be submitted to the insurance company for each of the days? View the Answer
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Embryo Freezing/Thawing

Our question refers to the CPT code 89258 “Cryopreservation; Embryo(s)” and 89352 “Thawing of Cryopreserved; Embryo”.  View the Answer
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Embryo Storage Fees For Multiple Cycles

We bill embryo storage 89342 for a year's storage.  View the Answer
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Embryo Thawing/Warming

Is it allowable to bill 89250 for the culture of embryos after thaw for a frozen embryo transfer (FET) cycle? View the Answer
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Gamete Thawing/Warming

Can patients be charged for each vial/straw of reproductive gametes or tissues thawed? View the Answer
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D&C Under Ultrasound Guidance

What are the CPT codes and ICD-10 codes for coding a surgical case for a patient with history of Stage B adenocarcinoma of the cervix ... View the Answer
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Assisted Hatching Billed With Embryo Biopsy

Do you know if both assisted hatching (89253) and embryo biopsy for PGS/PGD/CCS (89290/89291) can be billed during the same cycle?  View the Answer
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Assisted Zona Hatching

Can assisted hatching and embryo biopsy for PGT-A; PGT-M or PGT-SR be billed during the same cycle? View the Answer
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Billing For Cryopreservation Of Embryos Under The Male Partner

Can 89258 be billed under the male partner of a female patient? View the Answer
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Embryo Transfer

A summary of Embryo Transfer codes collected by the ASRM Coding Committee View the Coding Summary
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Colorado court balances religious and secular beliefs in frozen embryo divorce dispute

The day before the Dobbs decision, the Colorado Court of Appeals ruled on a divorcing couple’s disputed control over their frozen embryos. View the Legally Speaking
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Journal Club Global: Is PGT-P cutting edge or should we cut it out?

PGT for polygenic risk scoring (PGT-P) is a novel screening strategy of embryos for polygenic conditions and traits. View the Video
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Disposition of unclaimed embryos: an Ethics Committee opinion

Programs should create and enforce written policies addressing the designation, retention, and disposal of unclaimed embryos. View the Committee Opinion
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Ethics in embryo research: a position statement by the ASRM Ethics in Embryo Research Task Force and the ASRM Ethics Committee (2020)

Scientific research using human embryos advances human health and offspring well-being and provides vital insights into the mechanisms for reproduction and disease. Research involving human embryos is ethically acceptable if it is likely to provide significant new knowledge that may benefit human health, well-being of the offspring, or reproduction. View the Committee Opinion
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Guidance for Providers Caring for Women and Men Of Reproductive Age with Possible Zika Virus Exposure (Updated 2019)

This ASRM guidance specifically addresses Zika virus infection issues and concerns of individuals undergoing assisted reproductive technologies (ART). View the Guideline
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Blastocyst culture and transfer in clinically assisted reproduction: a committee opinion (2018)

The purposes of this document is to review the literature regarding the clinical application of blastocyst transfer. View the Committee Opinion
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Posthumous retrieval and use of gametes or embryos: an Ethics Committee opinion (2018)

Posthumous gamete (sperm or oocyte) retrieval or use for reproductive purposes is ethically justifiable if written documentation from the deceased authorizing the procedure is available. View the Committee Opinion
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Disclosure of medical errors involving gametes and embryos: an Ethics Committee opinion (2016)

Medical providers have an ethical duty to disclose clinically significant errors involving gametes and embryos as soon as they are discovered. Clinics also should have written policies in place for reducing and disclosing errors. View the Committee Document
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Recommended practices for the management of embryology, andrology, and endocrinology laboratories: a committee opinion (2014)

A general overview for good management practices within the endocrinology, andrology, and embryology laboratories in the United States. View the Recommendation
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Informed consent and the use of gametes and embryos for research: a committee opinion (2014)

The ethical conduct of human gamete and embryo research depends upon conscientious application of principles of informed consent. View the Committee Opinion
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ASRM EDGE Tool

Get the EDGE on your fellow Embryologists! As the grading of embryos varies within IVF laboratories and between laboratories, EDGE allows you to compare yourself against embryologists in the US and around the world. Learn more about the EDGE Tool

Topic Resources

View more on the topic of oocytes (eggs)
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Fertility and Sterility On Air - Microdose Interview: Dr. Papri Sarkar and Dr. Phillip Romanski

Dr. Papri Sarkar and Dr. Phillip Romanski, discuss their recent article "Optimal antimüllerian hormone levels in oocyte donors: a national database analysis." Listen to the Episode
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Reimbursement for cost of donor egg

My wife and I are going through a fertility treatment process, and we have purchased a donor egg out-of-pocket from a donor bank.  View the Answer
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Journal Club Global: IVM in Clinical Practice: An Idea Whose Time Has Come?

In vitro maturation (IVM) has the potential to make IVF cheaper, safer, and more widely accessible to patients with infertility. View the Video
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Does the number of eggs being frozen matter?

There is currently only one CPT code for the cryopreservation of mature oocytes and embryos.  View the Answer
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Journal Club Global - What is the optimal number of oocytes to reach a live-birth following IVF?

The optimal number of oocytes necessary to expect a live birth following in vitro fertilization remains unclear. View the Video
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Reproductive Tissue Storage

What are the CPT codes for the Storage of Reproductive Cells/Tissues? View the Answer
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Oocyte Denudation

Is there is a separate code for denudation of oocytes?  View the Answer
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Oocyte Preservation Consult

Our center performs oocyte preservation procedures for women looking to preserve their fertility. View the Answer
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Embryo Culture Less Than And More Than Four Days

When coding 89250 culture of oocytes/embryo <4 days, should that code be submitted to the insurance company for each of the days? View the Answer
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Gamete Thawing/Warming

Can patients be charged for each vial/straw of reproductive gametes or tissues thawed? View the Answer
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Donor Screening

Is there a specific CPT code used for Donor Physical Exams or would a practice just bill using the appropriate E&M Code?  View the Answer
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Journal Club Global: Should everyone freeze oocytes by age 33?

Oocyte cryopreservation is one of the fastest growing areas of reproductive medicine. View the Video
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Repetitive oocyte donation: a committee opinion (2020)

Donors should be advised of the number of cycles/donations that a given oocyte donor may undergo. View the Committee Opinion
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Posthumous retrieval and use of gametes or embryos: an Ethics Committee opinion (2018)

Posthumous gamete (sperm or oocyte) retrieval or use for reproductive purposes is ethically justifiable if written documentation from the deceased authorizing the procedure is available. View the Committee Opinion