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Vitrification of human blastocysts: an update
Vol. 19 Suppl. 4 2009 Reproductive BioMedicine Online; www.rbmonline.com/Article/4328 on web 10 June 2009
Contribution from 11th World Congress on Controversies in Obstetrics, Gynecology and Infertility (COGI) 'Celebration - 30 years of IVF' and Serono Symposia International Foundation
Vitrification of human blastocysts: an update Juergen Liebermann Fertility Centers of Illinois, Chicago, IL, USA Correspondence: e-mail: [email protected] Juergen Liebermann obtained his PhD in Agricultural Science at the Technical University of Munich-Weihenstephan, Germany. Since then he has been directing IVF laboratories, currently being IVF Laboratory Director of the Fertility Centers of Illinois, performing 2000 oocyte retrievals per year. An author of many papers concerning reproductive medicine, Dr Liebermann with Michael Tucker has recently completed a book entitled Vitrification in Human Assisted Reproduction. In 2004 he qualified as a university lecturer in Experimental Reproductive Medicine at the Bavarian University of Wuerzburg, Germany. A member of SSR, ASRM, ESHRE, ASA, and ABB, his research interests include every aspect of embryology, and ultra-rapid cooling methods, especially vitrification.
Abstract Transfer of blastocyst-stage embryos has been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos. At this late stage of development, lower numbers of embryos can be transferred, resulting in less high-order multiple pregnancies and increased implantation rates. Between January 2004 and February 2009, 8449 blastocysts from 2453 patients were vitrified. After 1398 vitrified embryo transfers (VET) of both day-5 and day-6 blastocysts with a mean patient age of 34.6 ± 5.0 years, the study centre has seen a survival rate of 96.3% (2730/2835), an implantation rate of 29.4% and a clinical pregnancy rate per VET of 42.8% (599 pregnancies/1398 warmed embryo transfers). After more than 5 years of vitrifying blastocysts, the perinatal outcome was, from 348 deliveries with vitrified blastocysts, the births of 431 babies (202 boys and 229 girls). One of the benefits of blastocyst vitrification is that it can be undertaken on a more flexible basis by laboratory staff. Also, vitrification may allow individual blastocysts to be cryopreserved at their optimal stage of development and expansion. Keywords: aseptic vitrification, blastocyst transfer, clinical pregnancy, cryopreservation
©Published by Reproductive Healthcare Ltd., Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Vitrification of human blastocysts – J Liebermann
Introduction With approximately 700,000 babies born worldwide following cryopreservation, this technology has become well established and is a widely used routine procedure. Data from the Centres for Disease Control and Prevention (CDC) from 2001 to 2006 show that about 20% of all offspring born in the USA from IVF cycles came from oocyte and embryo cryopreservation. The CDC also compared data of live births per transfer using frozen and fresh embryos (26.6 versus 34.4%), clearly showing that cryopreservation is an important step in maximizing the efficiency of an IVF cycle. However, it must be remembered that clinical success with cryopreservation seems to be highly variable from laboratory to laboratory and may depend on many factors, including patient age, stimulation protocol, quality of embryos selected for cryopreservation (scoring system), developmental stage at cryopreservation, media formulation including type of cryoprotectant agent used, parameters of freezing/cooling and thawing/warming, cryopreservation protocol (traditional slow or vitrification).
Vitrification technique At present, most IVF centres are still using traditional slow-freezing techniques because they have the longest clinical track record and provide a greater comfort level amongst embryologists. In addition, traditional embryo cryopreservation can be seen as a highly positive contribution to overall patient treatment. However, the limitations of current slow-rate freezing methods have become more evident in the clinical arena (Oktay et al., 2006). One way to achieve an ice-crystal-free state is to establish a glassy or vitreous state with use of ultra-high cooling rates provided by vitrification protocols. The achievement is a state of suspended animation wherein molecular translational motions are arrested without structural reorganization of the liquid. Vitrification, initially reported by Rall and Fahy (1985) as a successful cryopreservation approach for mouse embryos, has taken a backseat to the much more widely adopted slow-freezing technology applied to both gametes and embryos in animal and human assisted reproduction. Vitrification protocols are relatively simple for the practitioner, potentially faster and inexpensive. It relies on the placement of the cell in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed cryo-straws or vials. The importance in use of a very small volume, also referred to ‘minimal volume approach’, using the cryotop technology was first described and published by Kuwayama et al. (2005a) and Kuwayama (2007). Although some problems remain to be fully addressed with vitrification as a routine cryopreservation technique, vitrification is showing much promise as a viable alternative to conventional freezing technology. Trying to convince embryologists to convert from the slow-freeze protocols with which they are familiar is perhaps one of the greatest hurdles that remains in the acceptance and use of vitrification. Nevertheless, when (not if!) IVF programmes overcome this fear of the unknown and take on the challenge of a short learning curve with vitrification, then vitrification RBMOnline®
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protocols will become more and more clinically established. Today, vitrification techniques are already starting to enter the mainstream of human assisted reproduction treatment (Liebermann et al., 2002, 2003).
The advantages of blastocyst cryopreservation With recent increased confidence in growth-stage-sequenced culture media, blastocyst culture for fresh transfers has become more routine. The reasons for this development are: (i) freezing zygotes limits the selection of embryos for fresh transfer and again after thawing: the same handicap is true for cleavage-stage freezing because most of the preembryos arrest at the time of genome activation, which is far before blastocyst formation; (ii) blastocyst culture is becoming more common because it has been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos and because, at this late stage of development, lower numbers of embryos can be transferred in fresh cycles, resulting in fewer high-order multiple pregnancies: cryopreserved blastocysts also show increased pregnancy rates as well as increased implantation per thawed embryo transferred; (iii) most domestic species of commercial value (cattle, pig, horse) have their embryos frozen at this stage, demonstrating a proven track record of blastocyst freezing; (iv) at approximately 120 h (day 5) into development, the healthy human embryo should be at the blastocyst stage, which is comprised of some 50– 150 cells of which about 20–30% make up the inner cell mass, the remainder being the trophectoderm: the higher cell number allows better compensation for cryo-injuries, which results in greater viability and faster recovery, and the cytoplasmic volume of the cells is lower and the surface-to-volume ratio is higher, which makes the penetration of the cryoprotectant faster; and (v) on average, fewer embryos per patient are vitrified, but the vitrified blastocysts show greater potential for implantation after being thawed. As demonstrated through these findings, blastocyst-stage cryopreservation is superior to cryopreservation of an embryo at an earlier developmental stage, thus making a successful blastocyst cryopreservation programme increasingly relevant. Furthermore, it seems to be that day-3 morphology can only predict at an approximately 48% rate, those embryos that will eventually form blastocysts suitable for use on day 5/6 (Graham et al., 2000). Therefore, selection is the key to extended culture, since the less viable embryos will tend to arrest in development early on, ‘selecting’ themselves as non-candidates for fresh transfer or cryopreservation. This may seem like a waste of embryos; nevertheless, the net result is that chances of achieving a pregnancy are potentially improved. Today it is known that not all embryos that form blastocysts are viable or usable: i.e., there is a distinct difference between formation and utilization rates. Based on data from the Fertility Centers of Illinois (unpublished), blastocyst formation rate (e.g. 55–60%) is not necessarily equal to the usable blastocyst rate (closer to 35–40%). Before blastocyst transfer or cryopreservation, the following selection criteria should be considered: (i) expanded blastocyst growth rate in terms of cell numbers (trophectoderm RBMOnline®
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and inner cell mass) and grade of expansion on day 5 versus day 6 versus day 7; day 5 blastocysts reach a certain number of cells much earlier than blastocysts on day 6 or day 7 because of their speed of development and therefore should be preferentially considered for transfer; (ii) the overall cell number ≥60 cells, taking into consideration the passage of time, i.e., cell number relative to the number of cell cycles that the embryo has had time to undergo; and (iii) the original quality of early-stage embryo, i.e., pronuclear formation, blastomere regularity and mono-nucleation, and fragmentation. Knowing how to select potentially viable blastocysts is the key for successful outcomes (Tucker and Liebermann, 2003). To achieve this goal, the assessment of blastocyst quality must accommodate a measure of the two chief elements of the embryo at this stage: inner cell mass and trophectoderm. Before cryopreservation is considered, the cell count and integrity of the inner cell mass and trophectoderm need to be sufficient so as to indicate potential embryonic viability. Issues such as how early a blastocyst can be cryopreserved, or if blastocysts that are partially or totally hatched can be consistently cryopreserved, have yet to be fully answered. It seems that hatching and occasionally fully hatched blastocysts can be cryopreserved with inconsistent outcomes, and early cavitating blastocysts are better cultured to a later stage of expansion before attempting cryostorage (unpublished observations). As of today, successful vitrification of rhesus monkey blastocysts with the cryoloop has resulted in high survival (85% survived, 77% expanded and 71% hatched) (Yeoman et al., 2001). Vitrification of human blastocysts using the Cryoloop, hemi-straw system or Cryotop reports survival rates of 72–90%, clinical pregnancy rates of 37–53%, an implantation rate of 22–30% and live births (Mukaida et al., 2001, 2003a,b; Yokota et al., 2000, 2001; Reed et al., 2002; Vanderzwalmen et al., 2002, 2003; Son et al., 2003; Hiraoka et al., 2004; Huang et al., 2005; Kuwayama et al., 2005b; Takahashi et al., 2005; Liebermann and Tucker, 2006).
Materials and methods Both natural and hormone replacement cycles seem to provide comparable levels of receptivity in naturally cycling women, though they differ in level of convenience. The easiest way to calculate the day of transfer is to calculate the day of ovulation (whether in a natural or artificial transfer cycle), then warm and transfer all blastocysts on day 5 of development, counting ovulation day as day 0. Regardless of the day of cryopreservation of the embryo (whether day 5, 6 or 7), blastocysts should be treated when warming as if they had been vitrified on day 5 of development. Vitrification of blastocysts was undertaken utilizing an open system (Cryotop; Kitazato Bio Pharma Co, Fuji-shi, Japan) and a closed system (High Security Vitrification Kit, HSV; CryoBio System, L’Aigle, France) after a two-step loading with cryoprotectant agents at 24°C. Briefly, blastocysts were placed in equilibration solution, which is the base medium, HEPES-buffered human tubal fluid with 20% synthetic serum RBMOnline®
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supplement (SSS), containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulphoxide (DMSO). After 5–7 min, the blastocysts were washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG and 0.5 mol/l sucrose, for 45– 60 sec and transferred onto the Cryotop or HSV using a micropipette. Immediately after the loading of not more than two blastocysts in a 1 µl drop on the Cryotop, the carrier was plunged into fresh clean liquid nitrogen. After loading the embryos, the Cryotop was capped under liquid nitrogen to seal and protect the vitrified material prior to cryostorage. In contrast, after loading the HSV, the straw was heat sealed and then plunged in liquid nitrogen and stored the same way as the Cryotop. To remove the cryoprotectants, blastocysts were warmed and diluted in a two-step process. With the Cryotop or HSV submerged in liquid nitrogen, the protective cap (Cryotop) or inner straw (HSV) were removed and then both carriers with the blastocysts were removed from the liquid nitrogen and placed directly into a pre-warmed (~35–37°C) organ culture dish containing 1 ml of 1.0 mol/l sucrose. Blastocysts were picked up directly from the Cryotop and placed in a fresh drop of 1.0 mol/l sucrose at 24°C. After 5 min blastocysts were transferred to 0.5 mol/l sucrose solution. After an additional 5 min, blastocysts were washed in the base medium and returned to the culture medium (SAGE Blastocyst Medium, Trumbull, CT, USA) until transfer.
Results Between January 2004 and February 2009 at the Fertility Centers of Illinois IVF Laboratory River North (Chicago), 8449 blastocysts were vitrified without artificial shrinkage before the cryopreservation procedure (Table 1). After 1398 vitrified embryo transfers (VET), including day-5 and day-6 blastocysts with a mean age of the patients of 34.6 ± 5.0 years, the study centre has seen rates of survival, implantation and clinical pregnancy of 96.3%, 29.4% and 42.8%, respectively (Table 2). In addition, in 277 VET using aseptic vitrification, 543 blastocysts were transferred to give rates of survival, implantation and clinical pregnancy of 96.8%, 30.6% and 44.8%, respectively (Table 3). After 5 years of vitrifying blastocysts the perinatal outcome was, from 348 deliveries with vitrified blastocysts, the births of 431 babies (202 boys and 229 girls) (Table 2). No abnormalities were recorded. When the vitrified–warmed blastocysts were divided into day-5 and day-6 groups, the following data were gathered (Table 4). In 678 VET transferring day-5 blastocysts, the rates of survival, implantation and clinical pregnancy were 96.3%, 33.9% and 49.9% compared with 96.3%, 24.9% and 36.3% for day-6 blastocysts. As shown in Table 4, implantation and clinical pregnancy rates occurring in the day-5 blastocysts group were significantly higher than in the day-6 blastocyst group (chi-squared test; P < 0.05, P < 0.01 respectively). If day-5 versus day-6 outcomes using a closed system are compared, the following differences in the rate of survival, implantation, and clinical pregnancy are observed: 97.2%, 38.5%, 55.5% versus 96.4%, 22.4% and 34.3%, respectively (Table 5). As shown in Table 5, implantation and clinical RBMOnline®
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pregnancy rates occurring in the day-5 blastocyst group were significantly higher than the day-6 blastocyst group (chi-squared test; P < 0.01 for any comparison).
Discussion These data show that vitrifying at the blastocyst stage provides excellent survival, implantation and clinical pregnancy. To achieve this data, the following points should be considered: (i) without a successful blastocyst vitrification storage programme, extended culture should never be attempted; (ii) the blastocyst is composed of more cells and therefore is better able to compensate for cryo-injury; (iii) the cells are smaller thus making cryoprotectant penetration faster; and (iv) on average, fewer embryos per patient are cryo-stored but each one when warmed has a greater potential for implantation, often with an opportunity for an embryo transfer with a single blastocyst. Furthermore, a vitrification solution with a mixture of 7.5% EG/DMSO, followed by a 15% EG/DMSO with 0.5 mol/l sucrose step is safe for clinical use, giving rise to healthy babies without abnormalities. Vitrification of blastocysts using an open or closed system (Cryotop or HSV) is effective for achieving high implantation and pregnancy rates as seen in fresh embryo transfers. Both carriers require a short learning curve in handling, but in the long run they are easy to use. After warming more than 3390 blastocysts using both carriers, not one single blastocyst was lost during the vitrification steps of cooling and warming. Although the outcome in terms of implantation and clinical pregnancy is significantly different when comparing day-5 to day-6 blastocysts, these data should encourage cryopreservation of day-6 blastocysts as well. Based on the data presented, it is clear that the vitrification of day-6 blastocysts is of clinical value since it can result in live births. This observation is confirmed by Shapiro et al. (2001) and Levens et al. (2008); they found that blastocyst development rate impacts outcome in slow cryopreserved blastocyst transfer cycles. In conclusion, vitrification of human blastocysts is a viable and feasible alternative to traditional slow-freezing methods. The key to this success lies in the more optimal timing of embryo cryopreservation, e.g., individual blastocysts may be cryopreserved at their optimal stage of development and expansion. In addition, the repeatedly discussed topic of using open systems (direct contact between cells and liquid nitrogen) and the possible danger of contamination by bacteria, fungus or different strains of virus from liquid nitrogen, can be avoided by moving forward to a closed system which provides lower cooling rates, but without negative impact on the outcome.
Acknowledgements The author wants to thank the Fertility Centers of Illinois including their medical directors Dr Angelina Beltsos and Dr Kevin Lederer and the IVF laboratory staff at River North Elissa Pelts, Jill Matthews, Sara Sanchez, RBMOnline®
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Amanda Erman, Rebecca Brohammer, Yuri Wagner and Andrew Barker, whose clinical skills and invaluable contributions and support, facilitated the clinical application of routine vitrification within that laboratory.
References Graham J, Han T, Porter R et al. 2000 Day 3 morphology is a poor predictor of blastocyst quality in extended culture. Fertility and Sterility 74, 495–497. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K 2004 Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction 19, 2884–2888. Huang CC, Lee TH, Chen SU et al. 2005 Successful pregnancy following blastocyst cryopreservation using super- cooling ultra-rapid vitrification. Human Reproduction 20, 122–128. Levens ED, Whitcomb BW, Henessy S et al. 2008 Blastocyst development rate impacts outcome in cryopreserved blastocyst transfer cycles. Fertility and Sterility 90, 2138–2143. Kuwayama M 2007 Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 67, 73–80. Kuwayama M, Vajta G, Kato O, Leibo SP 2005a Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive BioMedicine Online 11, 300– 308. Kuwayama M, Vajta G, Ieda S, Kato O 2005b Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reproductive BioMedicine Online 11, 608–614. Liebermann J, Tucker MJ 2006 Comparison of vitrification versus conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertility and Sterility 86, 20–26. Liebermann J, Dietl J, Vanderzwalmen P, Tucker MJ 2003 Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? Reproductive BioMedicine Online 7, 623–633. Liebermann J, Nawroth F, Isachenko V et al. 2002 Potential importance of vitrification in reproductive medicine. Biology of Reproduction 67, 1671–1680. Mukaida T, Takahashi K, Kasai M 2003a Blastocyst cryopreservation: ultrarapid vitrification using Cryoloop technique. Reproductive BioMedicine Online 6, 221–225. Mukaida T, Nakamura S, Tomiyama T et al. 2003b Vitrification of human blastocysts using Cryoloops: clinical outcome of 223 cycles. Human Reproduction 18, 384–391. Mukaida T, Nakamura S, Tomiyama T et al. 2001 Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertility and Sterility 76, 618–623. Oktay K, Cil AP 2006 Efficiency of oocyte cryopreservation: a meta-analysis. Fertility and Sterility 86, 70–80. Rall WF, Fahy GM 1985 Ice-free cryopreservation of mouse embryos at –196 degress C by vitrification. Nature 313, 573–575. Reed ML, Lane M, Gardner DK et al. 2002 Vitrification of human blastocysts using the Cryoloop method: successful clinical application and birth of offspring. Journal of Assisted Reproduction and Genetics 19, 304–306. Shapiro B, Richter K, Harris D, Daneshmand ST 2001 A comparison of day 5 and 6 blastocysts transfers. Fertility and Sterility 75, 1126–1130. Son WY, Yoon SH, Yoon HJ et al. 2003 Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction 18, 137–139. Takahashi K, Mukaida T, Goto T, Oka C 2005 Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertility and Sterility 84, 88–92. Tucker MJ, Liebermann J 2003 Morphological scoring of human embryos and its relevance to blastocyst transfer. In: Patrizio P, Tucker MJ, Guelman V (eds) Color Atlas of Human Assisted Reproduction: Laboratory and Clinical Insight. Lippincott, Williams and Wilkins, Philadelphia, 99–108. Vanderzwalmen P, Bertin G, Debauche C et al. 2003 Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction 18, 1504–1511. RBMOnline®
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Vanderzwalmen P, Bertin G, Debauche Ch et al. 2002 Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction 17, 744–751. Yeoman RR, Gerami-Naini B, Mitalipov S et al. 2001 Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts. Human Reproduction 16, 1965–1969. Yokota Y, Sato S, Yokota M et al. 2001 Birth of a healthy baby following vitrification of human blastocysts. Fertility and Sterility 75, 1027–1029. Yokota Y, Sato S, Yokota M et al. 2000 Successful pregnancy following blastocyst vitrification. Human Reproduction 15, 1802–1803. Declaration: The authors report no financial or commercial conflicts of interest. Received 31 March 2009; refereed 22 May 2009; accepted 8 June 2009.
Table 1. Retrospective data from 2453 patients (average age 33.6 ± 4.9) with blastocyst cryopreservation by vitrification from January 2004 until February 2009. Day of development
Day 5
Day 6
Day 7
No. of blastocysts vitrified (%)
3567 (42)
4576 (54) 306 (4)
Total 8449
Table 2. Retrospective data from the blastocyst cryopreservation programme at Fertility Centers, where vitrification technologies were applied from January 2004 until February 2009. Parameter
Vitrification technology
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing pregnancies from VET (%) Live births
34.6 ± 5.0 1411 1398 2835 2730 (96.3) 2708 1.9 796 (29.4) 695 (49.3) 695 (49.7) 599 (42.5) 599 (42.8) 508 (36.3) 431 (202 boys and 229 girls)
Values are numbers unless otherwise described. VET = vitrified embryo transfer.
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Table 3. Retrospective data from the blastocyst cryopreservation programme at Fertility Centers, where aseptic vitrification technology was applied from June 2007 until February 2009. Parameter
Aseptic vitrification technology
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing pregnancies from VET (%) Live births
34.0 ± 4.4 279 277 563 545 (96.8) 543 1.9 166 (30.6) 147 (52.7) 147 (53.1) 124 (44.4) 124 (44.8) 122 (44.0) 37 (18 boys and 19 girls)
Values are numbers unless otherwise described. VET = vitrified embryo transfer.
Table 4. A comparison of retrospective data from the blastocyst cryopreservation programme at Fertility Centers of vitrified day 5 and day 6 from January 2004 until February 2009. Day of development
Day 5
Day 6
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing/delivered pregnancies from VET (%) Live births
34.5 ± 5.2 680 678 1426 1373 (96.3) 1357 2.0 460 (33.9)a 396 (58.2)b 396 (58.4)b 338 (49.7)b 338 (49.9)b 288 (42.5) 255
34.6 ± 4.9 731 720 1409 1357 (96.3) 1351 1.8 336 (24.9)a 299 (40.9)b 299 (41.5)b 261 (35.7)b 261 (36.3)b 220 (30.6) 176
Values are numbers unless otherwise described. VET = vitrified embryo transfer. a,b Values within a row with the same superscript are significantly different (P < 0.05 and P < 0.01 respectively).
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Table 5. A comparison of retrospective data from the blastocyst cryopreservation programme at Fertility Centers of aseptic vitrified day-5 and day-6 blastocysts from June 2007 until February 2009. Parameter
Day 5
Day 6
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing/delivered pregnancies from VET (%) Live births
34.1 ± 4.72 137 137 285 277 (97.2) 275 2.0 106 (38.5)a 89 (65.0)a 89 (65.0)a 76 (55.5)a 76 (55.5)a 75 (54.7) 24
34.0 ± 4.2 142 140 278 268 (96.4) 268 1.9 60 (22.4)a 58 (40.8)a 58 (41.4)a 48 (33.8)a 48 (34.3)a 47 (33.6) 13
Values are numbers unless otherwise described. VET = vitrified embryo transfer. a Values within a row with the same superscript are significantly different (P < 0.01).
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Contribution from 11th World Congress on Controversies in Obstetrics, Gynecology and Infertility (COGI) 'Celebration - 30 years of IVF' and Serono Symposia International Foundation
Vitrification of human blastocysts: an update Juergen Liebermann Fertility Centers of Illinois, Chicago, IL, USA Correspondence: e-mail: [email protected] Juergen Liebermann obtained his PhD in Agricultural Science at the Technical University of Munich-Weihenstephan, Germany. Since then he has been directing IVF laboratories, currently being IVF Laboratory Director of the Fertility Centers of Illinois, performing 2000 oocyte retrievals per year. An author of many papers concerning reproductive medicine, Dr Liebermann with Michael Tucker has recently completed a book entitled Vitrification in Human Assisted Reproduction. In 2004 he qualified as a university lecturer in Experimental Reproductive Medicine at the Bavarian University of Wuerzburg, Germany. A member of SSR, ASRM, ESHRE, ASA, and ABB, his research interests include every aspect of embryology, and ultra-rapid cooling methods, especially vitrification.
Abstract Transfer of blastocyst-stage embryos has been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos. At this late stage of development, lower numbers of embryos can be transferred, resulting in less high-order multiple pregnancies and increased implantation rates. Between January 2004 and February 2009, 8449 blastocysts from 2453 patients were vitrified. After 1398 vitrified embryo transfers (VET) of both day-5 and day-6 blastocysts with a mean patient age of 34.6 ± 5.0 years, the study centre has seen a survival rate of 96.3% (2730/2835), an implantation rate of 29.4% and a clinical pregnancy rate per VET of 42.8% (599 pregnancies/1398 warmed embryo transfers). After more than 5 years of vitrifying blastocysts, the perinatal outcome was, from 348 deliveries with vitrified blastocysts, the births of 431 babies (202 boys and 229 girls). One of the benefits of blastocyst vitrification is that it can be undertaken on a more flexible basis by laboratory staff. Also, vitrification may allow individual blastocysts to be cryopreserved at their optimal stage of development and expansion. Keywords: aseptic vitrification, blastocyst transfer, clinical pregnancy, cryopreservation
©Published by Reproductive Healthcare Ltd., Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Vitrification of human blastocysts – J Liebermann
Introduction With approximately 700,000 babies born worldwide following cryopreservation, this technology has become well established and is a widely used routine procedure. Data from the Centres for Disease Control and Prevention (CDC) from 2001 to 2006 show that about 20% of all offspring born in the USA from IVF cycles came from oocyte and embryo cryopreservation. The CDC also compared data of live births per transfer using frozen and fresh embryos (26.6 versus 34.4%), clearly showing that cryopreservation is an important step in maximizing the efficiency of an IVF cycle. However, it must be remembered that clinical success with cryopreservation seems to be highly variable from laboratory to laboratory and may depend on many factors, including patient age, stimulation protocol, quality of embryos selected for cryopreservation (scoring system), developmental stage at cryopreservation, media formulation including type of cryoprotectant agent used, parameters of freezing/cooling and thawing/warming, cryopreservation protocol (traditional slow or vitrification).
Vitrification technique At present, most IVF centres are still using traditional slow-freezing techniques because they have the longest clinical track record and provide a greater comfort level amongst embryologists. In addition, traditional embryo cryopreservation can be seen as a highly positive contribution to overall patient treatment. However, the limitations of current slow-rate freezing methods have become more evident in the clinical arena (Oktay et al., 2006). One way to achieve an ice-crystal-free state is to establish a glassy or vitreous state with use of ultra-high cooling rates provided by vitrification protocols. The achievement is a state of suspended animation wherein molecular translational motions are arrested without structural reorganization of the liquid. Vitrification, initially reported by Rall and Fahy (1985) as a successful cryopreservation approach for mouse embryos, has taken a backseat to the much more widely adopted slow-freezing technology applied to both gametes and embryos in animal and human assisted reproduction. Vitrification protocols are relatively simple for the practitioner, potentially faster and inexpensive. It relies on the placement of the cell in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed cryo-straws or vials. The importance in use of a very small volume, also referred to ‘minimal volume approach’, using the cryotop technology was first described and published by Kuwayama et al. (2005a) and Kuwayama (2007). Although some problems remain to be fully addressed with vitrification as a routine cryopreservation technique, vitrification is showing much promise as a viable alternative to conventional freezing technology. Trying to convince embryologists to convert from the slow-freeze protocols with which they are familiar is perhaps one of the greatest hurdles that remains in the acceptance and use of vitrification. Nevertheless, when (not if!) IVF programmes overcome this fear of the unknown and take on the challenge of a short learning curve with vitrification, then vitrification RBMOnline®
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Vitrification of human blastocysts – J Liebermann
protocols will become more and more clinically established. Today, vitrification techniques are already starting to enter the mainstream of human assisted reproduction treatment (Liebermann et al., 2002, 2003).
The advantages of blastocyst cryopreservation With recent increased confidence in growth-stage-sequenced culture media, blastocyst culture for fresh transfers has become more routine. The reasons for this development are: (i) freezing zygotes limits the selection of embryos for fresh transfer and again after thawing: the same handicap is true for cleavage-stage freezing because most of the preembryos arrest at the time of genome activation, which is far before blastocyst formation; (ii) blastocyst culture is becoming more common because it has been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos and because, at this late stage of development, lower numbers of embryos can be transferred in fresh cycles, resulting in fewer high-order multiple pregnancies: cryopreserved blastocysts also show increased pregnancy rates as well as increased implantation per thawed embryo transferred; (iii) most domestic species of commercial value (cattle, pig, horse) have their embryos frozen at this stage, demonstrating a proven track record of blastocyst freezing; (iv) at approximately 120 h (day 5) into development, the healthy human embryo should be at the blastocyst stage, which is comprised of some 50– 150 cells of which about 20–30% make up the inner cell mass, the remainder being the trophectoderm: the higher cell number allows better compensation for cryo-injuries, which results in greater viability and faster recovery, and the cytoplasmic volume of the cells is lower and the surface-to-volume ratio is higher, which makes the penetration of the cryoprotectant faster; and (v) on average, fewer embryos per patient are vitrified, but the vitrified blastocysts show greater potential for implantation after being thawed. As demonstrated through these findings, blastocyst-stage cryopreservation is superior to cryopreservation of an embryo at an earlier developmental stage, thus making a successful blastocyst cryopreservation programme increasingly relevant. Furthermore, it seems to be that day-3 morphology can only predict at an approximately 48% rate, those embryos that will eventually form blastocysts suitable for use on day 5/6 (Graham et al., 2000). Therefore, selection is the key to extended culture, since the less viable embryos will tend to arrest in development early on, ‘selecting’ themselves as non-candidates for fresh transfer or cryopreservation. This may seem like a waste of embryos; nevertheless, the net result is that chances of achieving a pregnancy are potentially improved. Today it is known that not all embryos that form blastocysts are viable or usable: i.e., there is a distinct difference between formation and utilization rates. Based on data from the Fertility Centers of Illinois (unpublished), blastocyst formation rate (e.g. 55–60%) is not necessarily equal to the usable blastocyst rate (closer to 35–40%). Before blastocyst transfer or cryopreservation, the following selection criteria should be considered: (i) expanded blastocyst growth rate in terms of cell numbers (trophectoderm RBMOnline®
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and inner cell mass) and grade of expansion on day 5 versus day 6 versus day 7; day 5 blastocysts reach a certain number of cells much earlier than blastocysts on day 6 or day 7 because of their speed of development and therefore should be preferentially considered for transfer; (ii) the overall cell number ≥60 cells, taking into consideration the passage of time, i.e., cell number relative to the number of cell cycles that the embryo has had time to undergo; and (iii) the original quality of early-stage embryo, i.e., pronuclear formation, blastomere regularity and mono-nucleation, and fragmentation. Knowing how to select potentially viable blastocysts is the key for successful outcomes (Tucker and Liebermann, 2003). To achieve this goal, the assessment of blastocyst quality must accommodate a measure of the two chief elements of the embryo at this stage: inner cell mass and trophectoderm. Before cryopreservation is considered, the cell count and integrity of the inner cell mass and trophectoderm need to be sufficient so as to indicate potential embryonic viability. Issues such as how early a blastocyst can be cryopreserved, or if blastocysts that are partially or totally hatched can be consistently cryopreserved, have yet to be fully answered. It seems that hatching and occasionally fully hatched blastocysts can be cryopreserved with inconsistent outcomes, and early cavitating blastocysts are better cultured to a later stage of expansion before attempting cryostorage (unpublished observations). As of today, successful vitrification of rhesus monkey blastocysts with the cryoloop has resulted in high survival (85% survived, 77% expanded and 71% hatched) (Yeoman et al., 2001). Vitrification of human blastocysts using the Cryoloop, hemi-straw system or Cryotop reports survival rates of 72–90%, clinical pregnancy rates of 37–53%, an implantation rate of 22–30% and live births (Mukaida et al., 2001, 2003a,b; Yokota et al., 2000, 2001; Reed et al., 2002; Vanderzwalmen et al., 2002, 2003; Son et al., 2003; Hiraoka et al., 2004; Huang et al., 2005; Kuwayama et al., 2005b; Takahashi et al., 2005; Liebermann and Tucker, 2006).
Materials and methods Both natural and hormone replacement cycles seem to provide comparable levels of receptivity in naturally cycling women, though they differ in level of convenience. The easiest way to calculate the day of transfer is to calculate the day of ovulation (whether in a natural or artificial transfer cycle), then warm and transfer all blastocysts on day 5 of development, counting ovulation day as day 0. Regardless of the day of cryopreservation of the embryo (whether day 5, 6 or 7), blastocysts should be treated when warming as if they had been vitrified on day 5 of development. Vitrification of blastocysts was undertaken utilizing an open system (Cryotop; Kitazato Bio Pharma Co, Fuji-shi, Japan) and a closed system (High Security Vitrification Kit, HSV; CryoBio System, L’Aigle, France) after a two-step loading with cryoprotectant agents at 24°C. Briefly, blastocysts were placed in equilibration solution, which is the base medium, HEPES-buffered human tubal fluid with 20% synthetic serum RBMOnline®
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supplement (SSS), containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulphoxide (DMSO). After 5–7 min, the blastocysts were washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG and 0.5 mol/l sucrose, for 45– 60 sec and transferred onto the Cryotop or HSV using a micropipette. Immediately after the loading of not more than two blastocysts in a 1 µl drop on the Cryotop, the carrier was plunged into fresh clean liquid nitrogen. After loading the embryos, the Cryotop was capped under liquid nitrogen to seal and protect the vitrified material prior to cryostorage. In contrast, after loading the HSV, the straw was heat sealed and then plunged in liquid nitrogen and stored the same way as the Cryotop. To remove the cryoprotectants, blastocysts were warmed and diluted in a two-step process. With the Cryotop or HSV submerged in liquid nitrogen, the protective cap (Cryotop) or inner straw (HSV) were removed and then both carriers with the blastocysts were removed from the liquid nitrogen and placed directly into a pre-warmed (~35–37°C) organ culture dish containing 1 ml of 1.0 mol/l sucrose. Blastocysts were picked up directly from the Cryotop and placed in a fresh drop of 1.0 mol/l sucrose at 24°C. After 5 min blastocysts were transferred to 0.5 mol/l sucrose solution. After an additional 5 min, blastocysts were washed in the base medium and returned to the culture medium (SAGE Blastocyst Medium, Trumbull, CT, USA) until transfer.
Results Between January 2004 and February 2009 at the Fertility Centers of Illinois IVF Laboratory River North (Chicago), 8449 blastocysts were vitrified without artificial shrinkage before the cryopreservation procedure (Table 1). After 1398 vitrified embryo transfers (VET), including day-5 and day-6 blastocysts with a mean age of the patients of 34.6 ± 5.0 years, the study centre has seen rates of survival, implantation and clinical pregnancy of 96.3%, 29.4% and 42.8%, respectively (Table 2). In addition, in 277 VET using aseptic vitrification, 543 blastocysts were transferred to give rates of survival, implantation and clinical pregnancy of 96.8%, 30.6% and 44.8%, respectively (Table 3). After 5 years of vitrifying blastocysts the perinatal outcome was, from 348 deliveries with vitrified blastocysts, the births of 431 babies (202 boys and 229 girls) (Table 2). No abnormalities were recorded. When the vitrified–warmed blastocysts were divided into day-5 and day-6 groups, the following data were gathered (Table 4). In 678 VET transferring day-5 blastocysts, the rates of survival, implantation and clinical pregnancy were 96.3%, 33.9% and 49.9% compared with 96.3%, 24.9% and 36.3% for day-6 blastocysts. As shown in Table 4, implantation and clinical pregnancy rates occurring in the day-5 blastocysts group were significantly higher than in the day-6 blastocyst group (chi-squared test; P < 0.05, P < 0.01 respectively). If day-5 versus day-6 outcomes using a closed system are compared, the following differences in the rate of survival, implantation, and clinical pregnancy are observed: 97.2%, 38.5%, 55.5% versus 96.4%, 22.4% and 34.3%, respectively (Table 5). As shown in Table 5, implantation and clinical RBMOnline®
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pregnancy rates occurring in the day-5 blastocyst group were significantly higher than the day-6 blastocyst group (chi-squared test; P < 0.01 for any comparison).
Discussion These data show that vitrifying at the blastocyst stage provides excellent survival, implantation and clinical pregnancy. To achieve this data, the following points should be considered: (i) without a successful blastocyst vitrification storage programme, extended culture should never be attempted; (ii) the blastocyst is composed of more cells and therefore is better able to compensate for cryo-injury; (iii) the cells are smaller thus making cryoprotectant penetration faster; and (iv) on average, fewer embryos per patient are cryo-stored but each one when warmed has a greater potential for implantation, often with an opportunity for an embryo transfer with a single blastocyst. Furthermore, a vitrification solution with a mixture of 7.5% EG/DMSO, followed by a 15% EG/DMSO with 0.5 mol/l sucrose step is safe for clinical use, giving rise to healthy babies without abnormalities. Vitrification of blastocysts using an open or closed system (Cryotop or HSV) is effective for achieving high implantation and pregnancy rates as seen in fresh embryo transfers. Both carriers require a short learning curve in handling, but in the long run they are easy to use. After warming more than 3390 blastocysts using both carriers, not one single blastocyst was lost during the vitrification steps of cooling and warming. Although the outcome in terms of implantation and clinical pregnancy is significantly different when comparing day-5 to day-6 blastocysts, these data should encourage cryopreservation of day-6 blastocysts as well. Based on the data presented, it is clear that the vitrification of day-6 blastocysts is of clinical value since it can result in live births. This observation is confirmed by Shapiro et al. (2001) and Levens et al. (2008); they found that blastocyst development rate impacts outcome in slow cryopreserved blastocyst transfer cycles. In conclusion, vitrification of human blastocysts is a viable and feasible alternative to traditional slow-freezing methods. The key to this success lies in the more optimal timing of embryo cryopreservation, e.g., individual blastocysts may be cryopreserved at their optimal stage of development and expansion. In addition, the repeatedly discussed topic of using open systems (direct contact between cells and liquid nitrogen) and the possible danger of contamination by bacteria, fungus or different strains of virus from liquid nitrogen, can be avoided by moving forward to a closed system which provides lower cooling rates, but without negative impact on the outcome.
Acknowledgements The author wants to thank the Fertility Centers of Illinois including their medical directors Dr Angelina Beltsos and Dr Kevin Lederer and the IVF laboratory staff at River North Elissa Pelts, Jill Matthews, Sara Sanchez, RBMOnline®
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Amanda Erman, Rebecca Brohammer, Yuri Wagner and Andrew Barker, whose clinical skills and invaluable contributions and support, facilitated the clinical application of routine vitrification within that laboratory.
References Graham J, Han T, Porter R et al. 2000 Day 3 morphology is a poor predictor of blastocyst quality in extended culture. Fertility and Sterility 74, 495–497. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K 2004 Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction 19, 2884–2888. Huang CC, Lee TH, Chen SU et al. 2005 Successful pregnancy following blastocyst cryopreservation using super- cooling ultra-rapid vitrification. Human Reproduction 20, 122–128. Levens ED, Whitcomb BW, Henessy S et al. 2008 Blastocyst development rate impacts outcome in cryopreserved blastocyst transfer cycles. Fertility and Sterility 90, 2138–2143. Kuwayama M 2007 Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 67, 73–80. Kuwayama M, Vajta G, Kato O, Leibo SP 2005a Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive BioMedicine Online 11, 300– 308. Kuwayama M, Vajta G, Ieda S, Kato O 2005b Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reproductive BioMedicine Online 11, 608–614. Liebermann J, Tucker MJ 2006 Comparison of vitrification versus conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertility and Sterility 86, 20–26. Liebermann J, Dietl J, Vanderzwalmen P, Tucker MJ 2003 Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? Reproductive BioMedicine Online 7, 623–633. Liebermann J, Nawroth F, Isachenko V et al. 2002 Potential importance of vitrification in reproductive medicine. Biology of Reproduction 67, 1671–1680. Mukaida T, Takahashi K, Kasai M 2003a Blastocyst cryopreservation: ultrarapid vitrification using Cryoloop technique. Reproductive BioMedicine Online 6, 221–225. Mukaida T, Nakamura S, Tomiyama T et al. 2003b Vitrification of human blastocysts using Cryoloops: clinical outcome of 223 cycles. Human Reproduction 18, 384–391. Mukaida T, Nakamura S, Tomiyama T et al. 2001 Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertility and Sterility 76, 618–623. Oktay K, Cil AP 2006 Efficiency of oocyte cryopreservation: a meta-analysis. Fertility and Sterility 86, 70–80. Rall WF, Fahy GM 1985 Ice-free cryopreservation of mouse embryos at –196 degress C by vitrification. Nature 313, 573–575. Reed ML, Lane M, Gardner DK et al. 2002 Vitrification of human blastocysts using the Cryoloop method: successful clinical application and birth of offspring. Journal of Assisted Reproduction and Genetics 19, 304–306. Shapiro B, Richter K, Harris D, Daneshmand ST 2001 A comparison of day 5 and 6 blastocysts transfers. Fertility and Sterility 75, 1126–1130. Son WY, Yoon SH, Yoon HJ et al. 2003 Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction 18, 137–139. Takahashi K, Mukaida T, Goto T, Oka C 2005 Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertility and Sterility 84, 88–92. Tucker MJ, Liebermann J 2003 Morphological scoring of human embryos and its relevance to blastocyst transfer. In: Patrizio P, Tucker MJ, Guelman V (eds) Color Atlas of Human Assisted Reproduction: Laboratory and Clinical Insight. Lippincott, Williams and Wilkins, Philadelphia, 99–108. Vanderzwalmen P, Bertin G, Debauche C et al. 2003 Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction 18, 1504–1511. RBMOnline®
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Vanderzwalmen P, Bertin G, Debauche Ch et al. 2002 Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction 17, 744–751. Yeoman RR, Gerami-Naini B, Mitalipov S et al. 2001 Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts. Human Reproduction 16, 1965–1969. Yokota Y, Sato S, Yokota M et al. 2001 Birth of a healthy baby following vitrification of human blastocysts. Fertility and Sterility 75, 1027–1029. Yokota Y, Sato S, Yokota M et al. 2000 Successful pregnancy following blastocyst vitrification. Human Reproduction 15, 1802–1803. Declaration: The authors report no financial or commercial conflicts of interest. Received 31 March 2009; refereed 22 May 2009; accepted 8 June 2009.
Table 1. Retrospective data from 2453 patients (average age 33.6 ± 4.9) with blastocyst cryopreservation by vitrification from January 2004 until February 2009. Day of development
Day 5
Day 6
Day 7
No. of blastocysts vitrified (%)
3567 (42)
4576 (54) 306 (4)
Total 8449
Table 2. Retrospective data from the blastocyst cryopreservation programme at Fertility Centers, where vitrification technologies were applied from January 2004 until February 2009. Parameter
Vitrification technology
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing pregnancies from VET (%) Live births
34.6 ± 5.0 1411 1398 2835 2730 (96.3) 2708 1.9 796 (29.4) 695 (49.3) 695 (49.7) 599 (42.5) 599 (42.8) 508 (36.3) 431 (202 boys and 229 girls)
Values are numbers unless otherwise described. VET = vitrified embryo transfer.
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Table 3. Retrospective data from the blastocyst cryopreservation programme at Fertility Centers, where aseptic vitrification technology was applied from June 2007 until February 2009. Parameter
Aseptic vitrification technology
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing pregnancies from VET (%) Live births
34.0 ± 4.4 279 277 563 545 (96.8) 543 1.9 166 (30.6) 147 (52.7) 147 (53.1) 124 (44.4) 124 (44.8) 122 (44.0) 37 (18 boys and 19 girls)
Values are numbers unless otherwise described. VET = vitrified embryo transfer.
Table 4. A comparison of retrospective data from the blastocyst cryopreservation programme at Fertility Centers of vitrified day 5 and day 6 from January 2004 until February 2009. Day of development
Day 5
Day 6
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing/delivered pregnancies from VET (%) Live births
34.5 ± 5.2 680 678 1426 1373 (96.3) 1357 2.0 460 (33.9)a 396 (58.2)b 396 (58.4)b 338 (49.7)b 338 (49.9)b 288 (42.5) 255
34.6 ± 4.9 731 720 1409 1357 (96.3) 1351 1.8 336 (24.9)a 299 (40.9)b 299 (41.5)b 261 (35.7)b 261 (36.3)b 220 (30.6) 176
Values are numbers unless otherwise described. VET = vitrified embryo transfer. a,b Values within a row with the same superscript are significantly different (P < 0.05 and P < 0.01 respectively).
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Table 5. A comparison of retrospective data from the blastocyst cryopreservation programme at Fertility Centers of aseptic vitrified day-5 and day-6 blastocysts from June 2007 until February 2009. Parameter
Day 5
Day 6
Patient’s age (years) Warmed cycles Transfers Blastocysts warmed Blastocysts survived (%) Blastocysts transferred Blastocysts transferred (mean) Implantations (%) Positive pregnancies from warming (%) Positive pregnancies from VET (%) Clinical pregnancies from warming (%) Clinical pregnancies from VET (%) Ongoing/delivered pregnancies from VET (%) Live births
34.1 ± 4.72 137 137 285 277 (97.2) 275 2.0 106 (38.5)a 89 (65.0)a 89 (65.0)a 76 (55.5)a 76 (55.5)a 75 (54.7) 24
34.0 ± 4.2 142 140 278 268 (96.4) 268 1.9 60 (22.4)a 58 (40.8)a 58 (41.4)a 48 (33.8)a 48 (34.3)a 47 (33.6) 13
Values are numbers unless otherwise described. VET = vitrified embryo transfer. a Values within a row with the same superscript are significantly different (P < 0.01).
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