Sacs 276 128 80 34 41 559

Implantation Rate 25.5% 26.5% 24.8% 19.0% 28.1% 25.3%

Table 4. Retrospective outcome data at FCI from vitrified day 6 blastocysts blastocyst in

regards to the patients age between June 2007 till July 2011.

Table 3. Retrospective outcome data at FCI from vitrified day 5 blastocysts in regards to the

Embryos transferred (MEAN) 1.8 1.8 1.8 1.7 1.8 1.8 Positive Pregnancies/Transfer 43.0% 43.6% 39.2% 31.1% 50.0% 42.1% Clinical Pregnancies/Transfer 37% 38% 35% 29% 42% 36.5% Ongoing Pregnancies/Transfer 31% 30% 27% 20% 32% 29.2%

Embryos transferred (MEAN) 1.9 1.8 1.9 1.9 1.8 1.9 Positive Pregnancies/Transfer 56.1% 56.7% 56.1% 51.4% 54.5% 55.8% Clinical Pregnancies/Transfer 50% 49% 43% 43% 50% 48.3% Ongoing Pregnancies/Transfer 43% 37% 30% 30% 39% 38.8%

and 36.5% of in 1204 day 6 FETs.

patients age between June 2007 till July 2011.

not more than two blastocysts in a 1l drop on the Cryotop, the carrier was plunged into fresh clean liquid nitrogen (LN2). After loading the embryos, the Cryotop was capped under the LN2 to seal and protect the vitrified material prior to cryo storage. In contrast, after loading the HSV, the straw was heat sealed and then plunged in LN2, and stored the same way as the cryotop (Liebermann & Tucker, 2006; Liebermann, 2009, 2011).

To remove the cryoprotectants, blastocysts were warmed and diluted in a two step process. With the Cryotop or HSV submerged in LN2, the protective cap (Cryotop) or inner straw (HSV) were removed, and then both carriers with the blastocysts were removed from the LN2 and placed directly into a pre-warmed (~35-37oC) organ culture dish containing 1ml of 1.0M sucrose. Blastocysts were picked up directly from the Cryotop and placed in a fresh drop of 1.0M sucrose at 24°C. After 5min blastocysts were transferred to 0.5M sucrose solution. After an additional 5min, blastocysts were washed in the base medium and returned to the culture medium (SAGE Blastocyst Medium, Trumbull, CT, USA) until transfer.

Between January 2004 and July 2011 the *Fertility Centers of Illinois* "IVF Laboratory River North"(Chicago) has vitrified 13,568 blastocysts *without artificial shrinkage* before the cryopreservation procedure (Table 1). After 2562 frozen embryo transfers (FET) including day 5 and day 6 blastocysts with a mean age of the patients of 34.9 ± 5.1 years, to date we have seen a survival rate, implantation, and clinical pregnancy rate per transfer (cPR) of 97.3%, 30.2%, and 42.5%, respectively (Table 2). After 7 1/2years of vitrifying blastocysts the perinatal outcome is as follow: from 687 deliveries with vitrified blastocysts, 852 babies (422 boys and 430 girls) were born (Table 2). No abnormalities were recorded. The singleton, twin and triplet pregnancy rates were 71%, 27%, and 2%, respectively.


Table 1. Retrospective data from 3,712 patients (average age 33.8±4.9) with blastocyst cryopreservation by vitrification from January 2004 till July 2011.


Table 2. Retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) where vitrification (VIT) technology was applied from January 2004 till July 2011.

not more than two blastocysts in a 1l drop on the Cryotop, the carrier was plunged into fresh clean liquid nitrogen (LN2). After loading the embryos, the Cryotop was capped under the LN2 to seal and protect the vitrified material prior to cryo storage. In contrast, after loading the HSV, the straw was heat sealed and then plunged in LN2, and stored the same way as the

To remove the cryoprotectants, blastocysts were warmed and diluted in a two step process. With the Cryotop or HSV submerged in LN2, the protective cap (Cryotop) or inner straw (HSV) were removed, and then both carriers with the blastocysts were removed from the LN2 and placed directly into a pre-warmed (~35-37oC) organ culture dish containing 1ml of 1.0M sucrose. Blastocysts were picked up directly from the Cryotop and placed in a fresh drop of 1.0M sucrose at 24°C. After 5min blastocysts were transferred to 0.5M sucrose solution. After an additional 5min, blastocysts were washed in the base medium and returned to the culture

Between January 2004 and July 2011 the *Fertility Centers of Illinois* "IVF Laboratory River North"(Chicago) has vitrified 13,568 blastocysts *without artificial shrinkage* before the cryopreservation procedure (Table 1). After 2562 frozen embryo transfers (FET) including day 5 and day 6 blastocysts with a mean age of the patients of 34.9 ± 5.1 years, to date we have seen a survival rate, implantation, and clinical pregnancy rate per transfer (cPR) of 97.3%, 30.2%, and 42.5%, respectively (Table 2). After 7 1/2years of vitrifying blastocysts the perinatal outcome is as follow: from 687 deliveries with vitrified blastocysts, 852 babies (422 boys and 430 girls) were born (Table 2). No abnormalities were recorded. The singleton,

**Day of Development Day 5 Day 6 Day 7 Total** 

6988 (51%) 360

(3%) <sup>13568</sup>

(46%)

**Technique VIT** Patient's age (y) 34.9 5.1 No. of warmed cycles 2580 No. of transfers 2562 No. of blastocysts warmed 4965 No. of blastocysts survived (%) 4829 (97.3) No. of blastocysts transferred 4752 Mean no. of blastocysts transferred 1.8

No. of implantations (%) 1432 (30.2) No. of positive pregnancy/warm (%) 1255 (48.6) No. of positive pregnancy/VET (%) 1255 (49.0) No. of clinical pregnancy/warm (%) 1089 (42.2) No. of clinical pregnancy/VET (%) 1089 (42.5) Ongoing pregnancies/VET (%) 875 (34.2)

Table 2. Retrospective data from the blastocyst cryopreservation program (Fertility Centers of Illinois, Chicago) where vitrification (VIT) technology was applied from January 2004 till

No. of livebirths 852 (422 boys & 430 girls)

Table 1. Retrospective data from 3,712 patients (average age 33.8±4.9) with blastocyst

cryotop (Liebermann & Tucker, 2006; Liebermann, 2009, 2011).

medium (SAGE Blastocyst Medium, Trumbull, CT, USA) until transfer.

twin and triplet pregnancy rates were 71%, 27%, and 2%, respectively.

cryopreservation by vitrification from January 2004 till July 2011.

Number of Blastocysts vitrified (%) <sup>6220</sup>

July 2011.

When the vitrified-warmed blastocysts were divided into day 5 and day 6 groups, the following data was gather (Table 3 & 4). In 1265 FETs transferring day 5 blastocysts, the survival, implantation, and cPR were 97.6%, 34.8%, and 48.3% compared to 97.2%, 25.3%, and 36.5% of in 1204 day 6 FETs.


Table 3. Retrospective outcome data at FCI from vitrified day 5 blastocysts in regards to the patients age between June 2007 till July 2011.


Table 4. Retrospective outcome data at FCI from vitrified day 6 blastocysts blastocyst in regards to the patients age between June 2007 till July 2011.

Vitrification of Oocytes and Embryos 179

Furthermore, a vitrification solution with a mixture of 7.5% EG/DMSO, followed by a 15% EG/DMSO with 0.5M sucrose step is safe for clinical use, giving rise to healthy babies without abnormalities. Vitrification of blastocysts using and open or closed system (Cryotop or HSV) is effective for achieving high implantation and pregnancy rates as seen in fresh embryo transfers. Although the outcome in terms of implantation and clinical pregnancy is significantly different when comparing day 5 blastocyst to day 6 blastocysts, our 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 Saphiro *et al.* (2001) and Levens *et al*. (2008); they found that blastocyst development rate impacts outcome in slow cryopreserved

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 LN2) and the possible danger of contamination by bacteria, fungus or different strains of virus from LN2, can be avoided by moving forward to a closed system providing lower cooling rates, but without a negative

There are many potential advantages of vitrification in that it is an easy, cheap, fast and an apparently successful cryopreservation method; however, there is one issue that is still up for debate. It has been shown that fungi, bacteria and viruses are able to survive in liquid nitrogen (LN2) (Tedder *et al*, 1995; Fountain *et al,* 1997; Bielanski *et al,* 2000; 2003; Kyuwa *et al*, 2003; Letur-Konirsch *et al*, 2003). Given the direct exposure of the human cells as they are directly plunged into LN2 during the vitrification process, this therefore raises the question as to whether the LN2 has to be sterilized, as it may be a possible source of contamination for those cells. To this point there has been no fungal, viral or bacterial contamination that has been described from about 400 publications related to vitrification since the first report in 1985. Bielanski and colleagues (2000) demonstrated a viral transmission rate of 21 % to human embryos stored in open freezing containers under experimental conditions of extremely elevated viral presence; while in contrast all embryos stored in sealed freezing containers were free from contamination. Based on this observation they proposed that the sealing of freezing containers appears to prevent exposure to potential contaminants. Commercial systems to purify LN2 by filtration have been developed, however this technology to date has received little practical application in IVF laboratories that have active cryopreservation programs. While it is not totally clear that contamination is a real risk in everyday use of LN2, nevertheless it may be prudent to consider routine sterilization of LN2 when open carrier systems are used for vitrification, followed by a sealing of that system for cryo-storage. Further there are currently at least three 'closed' sealed vitrification systems that are commercially available, with FDA clearance, that represent successful alternatives to open systems for embryo vitrification

**7. Contamination of LN2: Open versus closed systems** 

blastocyst transfer cycles.

impact on the outcome.

(Liebermann, 2009, 2011)

In addition, in 1128 FET using aseptic vitrification, 2041 blastocysts were transferred with a survival, implantation, and cPR of 98.4%, 31.8%, and 44.2%, respectively (Table 5). After 4 1/2 years of vitrifying blastocysts using a closed system the perinatal outcome is as follow: 313 babies (165 boys and 148 girls) were born (Table 5). No abnormalities were recorded.


Table 5. Retrospective data from the blastocyst cryopreservation program (*Fertility Centers of Illinois, Chicago*) where aseptic vitrification (aVIT) technology was applied from June 2007 till July 2011.

Our data has shown that freezing at the blastocyst stage provides excellent survival, implantation and clinical pregnancy (Liebermann & Tucker, 2006; Liebermann, 2009, 2011). To achieve this data the following points should be considered: a) without a successful blastocyst vitrification storage program, extended culture should never be attempted, b) the blastocyst is composed of more cells and therefore better able to compensate for cryo-injury, c) the cells are smaller thus making cryoprotectant penetration faster, and d) on average fewer embryos per patient are cryo-stored, but each one when thawed, has a greater potential for implantation, often with an opportunity for an ET with a single blastocyst.

In addition, in 1128 FET using aseptic vitrification, 2041 blastocysts were transferred with a survival, implantation, and cPR of 98.4%, 31.8%, and 44.2%, respectively (Table 5). After 4 1/2 years of vitrifying blastocysts using a closed system the perinatal outcome is as follow: 313 babies (165 boys and 148 girls) were born (Table 5). No abnormalities were recorded.

**Technique aVIT** 

Patient's age (y) 34.5 5.0

No. of warmed cycles 1132 No. of transfers 1128

No. of blastocysts warmed 2102 No. of blastocysts survived (%) 2069 (98.4) No. of blastocysts transferred 2041

Mean no. of blastocysts transferred 1.8

No. of positive pregnancy/warm (%) 572 (50.7) No. of positive pregnancy/VET (%) 572 (53.9) No. of clinical pregnancy/warm (%) 499 (44.1) No. of clinical pregnancy/VET (%) 499 (44.2) Ongoing pregnancies/VET (%) 423 (37.5)

No. of implantations (%) 650 (31.8)

No. of livebirths 313 (165 boys & 148 girls)

Table 5. Retrospective data from the blastocyst cryopreservation program (*Fertility Centers of Illinois, Chicago*) where aseptic vitrification (aVIT) technology was applied from June 2007 till

Our data has shown that freezing at the blastocyst stage provides excellent survival, implantation and clinical pregnancy (Liebermann & Tucker, 2006; Liebermann, 2009, 2011). To achieve this data the following points should be considered: a) without a successful blastocyst vitrification storage program, extended culture should never be attempted, b) the blastocyst is composed of more cells and therefore better able to compensate for cryo-injury, c) the cells are smaller thus making cryoprotectant penetration faster, and d) on average fewer embryos per patient are cryo-stored, but each one when thawed, has a greater potential for implantation, often with an opportunity for

July 2011.

an ET with a single blastocyst.

Furthermore, a vitrification solution with a mixture of 7.5% EG/DMSO, followed by a 15% EG/DMSO with 0.5M sucrose step is safe for clinical use, giving rise to healthy babies without abnormalities. Vitrification of blastocysts using and open or closed system (Cryotop or HSV) is effective for achieving high implantation and pregnancy rates as seen in fresh embryo transfers. Although the outcome in terms of implantation and clinical pregnancy is significantly different when comparing day 5 blastocyst to day 6 blastocysts, our 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 Saphiro *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 LN2) and the possible danger of contamination by bacteria, fungus or different strains of virus from LN2, can be avoided by moving forward to a closed system providing lower cooling rates, but without a negative impact on the outcome.
