Methodologies for Cryopreservation of Mammalian Germline Cells and Tissues

Polash Chandra Karmakar,Sang-Eun Jung,Buom-Yong Ryu 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & developmental biology Vol.41 No.2

Until today, success in germline cells and tissue cryopreservation is limited mainly due to the poor understanding of the complex physiological processes can lead to cell damage during cryopreservation. Germline cells, from both male and female, have unique ability to differentiate into one or more cell lines and thus it becomes a crucial point to store them in subzero temperature with the minimal damage of their functional properties and maximum recovery of unchanged and viable cells when thawed. In the past three decades, a vast research has been performed using various different animal models which in fact have led to development of new methodologies and optimization of older one. However, successful use of animal model has provided the opportunity in research with human germline cells and tissues preservation, but not in all the cases. Therefore, the use of new cryo-protective chemicals and modified protocols have been often found in different groups of researchers based on the types, physical structures, utility and animal species of the specimens to be cryopreserved. This review discusses about the basics of different types of cryopreservation methodologies and commonly used optimized protocols and cryoprotectants for germline cells and tissues preservation.

난소기능저하 미혼여성의 난자동결보존시 한약복용을 병행한 증례보고

고지은,유명숙 대한한방부인과학회 2019 大韓韓方婦人科學會誌 Vol.32 No.2

Objectives: The aim of this case is to report the effects of herbal medicine on a single woman patient with a low level of AMH (anti-Müllerian Hormone) in progress of Oocyte Cryopreservation. Methods: A patient with a low level of AMH had symptom of secondary amenorrhea. For preparing oocyte cryopreservation after a long time of secondary amenorrhea, she was treated by twice a day herb medication for 10 months. And we observed the effects of treatments by improvement of symptoms and following up endometrium ultrasonography. After oocyte cryopreservation, for maintaining her menstruation, she was also treated by twice a day herb medication for two and a half months. Results: After treatments, symptom of amenorrhea was improved and the thickness of endometrium was increased as well as AMH in progress of oocyte cryopreservation. So 20 oocytes could be cryopreserved. Conclusions: This case shows that herbal medicine can be a concurrent method for a single woman patient with secondary amenorrhea in progress of oocyte cryopreservation. Objectives: The aim of this case is to report the effects of herbal medicine on a single woman patient with a low level of AMH (anti-Müllerian Hormone) in progress of Oocyte Cryopreservation. Methods: A patient with a low level of AMH had symptom of secondary amenorrhea. For preparing oocyte cryopreservation after a long time of secondary amenorrhea, she was treated by twice a day herb medication for 10 months. And we observed the effects of treatments by improvement of symptoms and following up endometrium ultrasonography. After oocyte cryopreservation, for maintaining her menstruation, she was also treated by twice a day herb medication for two and a half months. Results: After treatments, symptom of amenorrhea was improved and the thickness of endometrium was increased as well as AMH in progress of oocyte cryopreservation. So 20 oocytes could be cryopreserved. Conclusions: This case shows that herbal medicine can be a concurrent method for a single woman patient with secondary amenorrhea in progress of oocyte cryopreservation.

Cryopreservation of human adipose-derived stem cells for the clinical use

Seah Park,Jiyoung Kim,Hyun Mi Kang,Sujin Yun,Yeonhwa Song,Hye Jin Yang,A young Yoon,Sun Young Baek,Si Hyung Yoo,Sun Hee Kim,Haekwon Kim 한국발생생물학회 2011 한국발생생물학회 학술발표대회 Vol.30 No.-

Mesenchymal stem cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Clinical application of these cells for degenerative and age-related diseases has been accumulating. However, preparation of MSC before the onset of the diseases, it needs to develop the cryopreservation method. Most cryopreservation methods include fetal bovine serum (FBS) which is essential for effective cryopreservation. Yet it should not be used clinically because of the potential risk of infection. In the present study, we investigated whether human serum albumin (HSA), human serum (HS), and knockout serum replacement (KSR) can be used as an alternative of FBS for cryopreservation of human adipose derived stem cells (hADSC). Cells cryopreserved with 9% HSA showed much higher viability after thawing compared with cells frozen with 5% or 1% HSA. Cells cryopreserved with 90% HS or KSR exhibited greater viability than cells frozen with 25% and 5% HS or KSR, respectively. Viability of cells frozen with 9% HSA, 90% HS or 90% KSR was comparable to that with 90% FBS. Morphology and proliferation ability of these cells were not affected by cryopreservation when compared the freshly obtained cells. Cryopreserved hADSC expressed transcription factor genes including Oct3/4, Nanog, Nestin and Sox2, which are related to the self-renewal of stem cells. Flow cytometric analyses showed that both fresh and cryopreserved hADSC were positive for the antigens of HLA-ABC, CD44, CD73, CD90, and CD105, CD166, and negative for HLA-DR, CD31, and CD34. Similar to fresh cells, cryopreserved hADSC could differentiate into mesodermal lineages, adipogenic, osteogenic, or chondrogenic cells. These results suggest that 9% HSA, 90% HS or 90% KSR can be used to replace FBS during successful cryopreservation of hADSC.

간세포 장기 보존을 위한 적정 냉동보존에 관한 연구-2

정용희 ( Yong Hee Joung ),김병호 ( Byung Ho Kim ),이병욱 ( Byoung Wook Lee ),한요셉 ( Yo Seb Han ),동석호 ( Seok Ho Dong ),김효종 ( Hyo Jong Kim ),장영운 ( Young Woon Chang ),이정일 ( Joung Il Lee ),장린 ( Rin Chang ) 대한소화기학회 2003 대한소화기학회지 Vol.41 No.1

Background/Aims: The cryopreservation of primary hepatocytes could avoid unnecessary isolation of hepatocytes and supply them on demand. We compared two programs of computer-controlled freezing methods on the efficacy of hepatocyte cryopreservation. Methods: Rat hepatocytes were cryopreserved by computer-controlled freezing methods. In program I, the overall cooling rate was 1.33℃/min and single-step shock cooling was used to reduce the latent heat of fusion. In program II, the cooling rate of 2℃/min and two-step shock cooling were used. Two programs were compared in regard to hepatocyte viability and long-term cryopreservation and thawing temperature were also evaluated. Results: The hepatocyte viability showed the highest value of 74.5 2.5% when cryopreserved using the program II and at 2×106/mL cell concentration in cryopreservation medium. The hepatocyte attachment rate on culture was similar in every occasion, more or less 50%. The hepatocyte viability was improved by 10% when thawed at 40℃, compared to the value at 37℃ in the program I. The hepatocyte viability was decreased to 38.2 5.5% in the program I and 45.4 1.6% in the program II after long-term cryopreservation. Conclusions: The program II showed better survival of hepatocytes at 2×106/mL cell concentration. However, overall efficacy of hepatocyte cryopreservation was less than 35% and decreased more after long-term cryopreservation. Further studies are needed to develop a more effective program for hepatocyte cryopreservation. (Korean J Gastroenterol 2003;41:41-48)

Elimination of Potato Viruses (PLRV and PVY) by Cryopreservation of In Vitro Grown Shoot Tips of Potato (Solanum tuberosum L.)

Shambhu Prasad Dhital,Hak Tae Lim,Hira Kaji Manandhar 한국원예학회 2009 Horticulture, Environment, and Biotechnology Vol.50 No.3

Cryopreservation has been recognized as a practical and efficient tool for long-term storage of vegetatively propagated plants. This study was conducted to investigate the effect of sucrose concentration and hardening temperature on the survival of cryopreserved shoot tips and virus elimination through cryo-therapy. Virus-free in vitro plantlets of potato cultivars ‘Atlantic’ and ‘Superior’ were used for survival studies whereas naturally infected in vitro plantlets of potato genotype ‘F9-99’ by potato leaf roll virus (PLRV) and potato virus Y (PVY) were used for virus elimination by cryopreservation. Excised shoot tips were cryopreserved by vitrification using plant vitrification solution-2 (PVS-2). Nine-percentage sucrose concentration gave the highest survival of 41.7% in ‘Atlantic’ and 33.3% in ‘Superior’. The most optimum hardening temperature for the highest survival of ‘Atlantic’ (43.3%) and ‘Superior’ (32.3%) was 10℃. The virus status of the regenerated in vitro plantlets before and after treatment was tested by employing double antibody sandwich-enzymelinked immunosorbent assay (DAS-ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Virus status was subsequently detected following greenhouse acclimatization by both methods. In the case of virus elimination using different steps of cryopreservation, none of the steps were found effective before freezing. The conventional meristem tip culture resulted in 13.6% PLRV and 11.4% PVY free plantlets, while the cryoprotective treatment resulted in 36.8% PLRV free plantlets and 42.1% PVY free plantlets. Cryopreservation steps were not affected for virus elimination, whereas in the case of cryopreservation procedures, vitrification and cryopreservation were more effective than the encapsulation-vitrification and cryopreservation for elimination of PLRV and PVY from the in vitro plantlets. Leaf and tuber morphology of potato plants regenerated after cryopreservation (cryo-therapy) was similar to that of the control. Thus, this study demonstrated that PLRV and PVY can be successfully eliminated from the infected in vitro shoot tips of potato by cryo-therapy.

제대혈 단핵세포의 냉동 전ㆍ후의 염색체 핵형분석의 실험적 연구

구기영(Ki Young Ku),추미애(Mi Ae Chu),김지윤(Ji Yoon Kim),이건수(Kun Soo Lee) 대한의학유전학회 2008 대한의학유전학회지 Vol.5 No.1

목적 : 염색체 검사는 의학의 많은 영역과 혈액종양학 분야에서 중요한 역할을 하고 있다. 세포의 기능을 잘 유지하면서 장기간 보관할 수 있는 가장 효과적인 방법은 냉동보존(cryopreservation)으로 알려져 있으며, 이는 미래에 신기술을 적용할 수 있고 회귀연구를 가능하게 한다. 이에 본 연구에서는 제대혈에서 분리된 단핵세포의 냉동전과 해동후의 염색체 검사 결과를 비교하고자 한다. 방법: 실험에 대한 동의서를 획득한 제대혈 1례가 검사되었다. Ficoll-Hypaue로 단핵세포를 분리하였고, DMSO 등 냉동보존을 위한 전처리 후 프로그램 냉동기(Cryomed 1010, 미국)로 냉동하여 질소탱크(-196℃)에 3일간 보관하였고, 급속 냉동 후 염색체 검사를 시행하였다. 냉동전과 해동후의 염색체 핵형을 분석하였다. 결과: 1례의 제대혈은 3군으로 나누어, 냉동 전 배양과정 없던 CB-1군은 염색체 핵형 검사가 판독 불가능하였으며, 냉동 전 3일간 배양 후 냉동보관을 하였던 CB-2와 CB-3군은 냉동전과 해동후의 염색체 핵형이 판독 가능하였고, 서로 일치하였다. 결론 : 검체의 분포 불균형과 검체 수가 적다는 제한점이 있으나, 제대혈에서 냉동전과 해동후의 염색체 핵형이 일치하는 결과에서 제대혈 단핵세포의 유전적 안정성과 장기간 보관의 가능성을 예측할 수 있을 것으로 사료된다. Purpose : The ability to perform chromosome analysis of cryopreserved cord blood mononuclear cells is important for future retrospective studies. We compared the karyotypes of cryopreserved cells with cells before cryopreservation. Methods : One cord blood (CB) sample was obtained from normal healthy volunteer. Karyotype analysis was performed before cryopreservation. After mononuclear cell separation with Ficoll-Hypaque, the mononuclear cells were cryopreserved by programmed controlled-rate freezer and then transferred into the liquid nitrogen (-196℃) for 3 days. After rapid thawing, cytogenetic analysis was performed as the same method for each sample by different conditions. The samples were divided by three groups. The first group was no culture before cryopreservation, the second group was 72 hours culture before cryopreservation, but no 24 hours culture after thawing and the third group was 72 hours culture before cryopreservation and 24 hours culture after thawing. Results : The chromosome analysis was successful in the second and third groups of CB sample. Conclusion : The successful result from CB samples may suggest the usefulness of long-term cryopreservation for retrospective study in various clinical settings including hematologic malignancies.

제대혈 조혈모세포의 체외확장에 냉동보존이 미치는 영향

최재혁,강희정,김용길,오현아,이구,배성화,이재련,이경희,현명수,류헌모 대한조혈모세포이식학회 2002 대한조혈모세포이식학회지 Vol.7 No.1

연구배경: 제대혈 조혈모세포이식술은 많은 장점이 있지만 채혈할 수 있는 제대혈액의 양에 제한이 있어 아직 성인에서는 시행되지 못하고 있다. 이런 문제점을 극복하기 위해서는 냉동보존된 제대혈 조혈모세포를 해동한 후 체외확장을 통한 세포수의 증가가 필요하다. 이에 저자는 냉동보존 전후의 제대혈액의 단핵세포수, 세포생존율 및 세포 집락 형성능을 비교하여 제대혈 조혈모세포의 세포생존율과 체외확장에 냉동보존이 어떤 영향을 미치는지를 알아보기 위해서 본 연구를 시행하였다. 방법: 정상 산모에서 질식분만 후 제대에서 20 mL의 제대혈액을 채집하여 단핵세포를 분리하고 냉동보전 전후에 제대혈 단핵세포의 세포생존율을 계산하여 비교하였다. 냉동보존을 하지 않은 상태의 제대혈 단핵세포와 7일간 또는 28일간 냉동보존을 한 후 해동한 제대혈 단핵세포를 각각 성장인자가 첨가된 배지에서 2×10^(6)/mL개의 단핵세포를 섞은 뒤 35 mm Petri dish 에서 37℃, 100% 습윤, 5% CO₂ 조건의 배양기에서 14일간 배양한 후 세포 집락수와 모양을 관찰하고 냉동보존 전후의 세포 집락 형성능을 비교하였다. 결과: 6예의 산모에서 제대혈액을 채집하여 측정한 제대혈 단핵세포 수는 냉동보존 하기 전이 2.92±1.08×10^(6)/mL개였고 7일간 냉동보존 한 경우와 28일간 냉동보존을 한 경우는 각각 1.15±0.36×10^(6)/mL개와 1.42±0.42×10^(6)/mL개였다. 제대혈 단핵세포의 세포생존율은 냉동보존을 하지 않은 경우가 92±2.88%였고 7일간 냉동보존한 경우와 28일간 냉동보존을 한 경우는 각각 61±3.84%와 66±3.87%로 냉동보존이 세포생존율은 감소시키지만 냉동보존의 기간에 따른 세포생존율에는 큰 차이가 없었다(P=0.064). 제대혈 단핵세포의 집락수와 세포 집락 형성능은 냉동보존을 하지 않고 배양한 경우 총 집락수는 101.5±23.74였고 그중 CFU-GM 28.8±2.04 (29.5±5.80%), CFU-GEMM 21.5±5.79 (21.0±1.45%), BFU-E 24.8±5.00 (24.8±5.0%)였으며 CFU-G와 CFU-M을 제외한 집락수에서 CFU-GM, CFU-GEMM, BFU- E가 차지하는 비율은 각각 39.0±4.57%, 28.2±3.59%, 32.9±1.47%였고, 28일간 냉동보존을 한 후 해동하여 배양한 경우에는 총 집락수는 52.5±12.13이었으며 이 중 CFU-GM 14.6±2.73, CFU-GEMM 13.7±1.21, BFU-E 23.5±5.01이었으며 CFU-G와 CFU-M을 제외한 집락수에서 CFU-GM, CFU-GEMM, BFU-E가 차지하는 비율은 각각 28.2±2.13%, 26.7±3.00%, 45.1±3.55%로 냉동보존을 한 경우 집락수는 감소하였지만 세포 집락 형성능의 감소는 없었다. 결론: 냉동보존 자체가 제대혈 조혈모세포의 세포생존율은 감소시키지만 세포 집락 형성능에는 영향을 거의 주지 않으므로 냉동보존 후 제대혈 조혈모세포의 체외확장을 통해 성인에서도 성공적으로 제대혈 조혈모세포이식술을 시행할 수 있을 것이라고 생각된다. Background: Umbilical cord blood stem cell transplantation has many advantages over bone marrow transplantation or peripheral blood stem cell transplantation. But, there are some problems to be solved in order to be applied to adults. The main problem is limitation of volume, which can be collected from one placenta was only between 80 mL and 120 mL. To overcome this problem, The ex vivo expansion of cryopreserved umbilical cord blood stem cells is needed. The object of this study was to evaluate the effect of cryopreservation on ex vivo expansion potential and viability of umbilical cord blood stem cells. Methods: After normal delivery, cord blood was drawn from umbilical cord vein and was used to evaluate the mononuclear cell count, the cell viability and clonogenic capacity of cord blood stem cells before and after cryopreservation. Results: Before cryopreservation, the mononuclear cell count of umbilical cord blood was 2.92±1.08×10^(6)/mL, cell viability was 92±2.88%, total colony count was 101.5±23.74 and percentages of CFU-GM, CFU-GEMM, BFU-E were 29.5±5.80%, 21.0±1.45%, 24.8±5.0%, respectively. The mononu-clear cell count of umbilical cord blood cryopreserved for 28 days was 1.42±0.42×10^(6)/mL and cell viability was 66±3.87%. Total colony count of umbilical cord blood cryopreserved for 28 days was 52.5±12.13 and percentages of CFU-GM, CFU- GEMM, BFU-E were 28.0±3.45%, 27.2±6.52%, 45.3±4.99%. But, There were few colony count which could be observed after cryopreserving for 7 days. Conclusion: There was no difference of clonogenic capacity of umbilical cord blood stem cells before and after cryopreservation. The cell viability of umbilical cord blood stem cells was decreased after cryopreservation but there was no difference between umbilical cord blood cryopreserved for 7 days and 28 days. Therefore, it is possible that sufficient umbilical cord blood stem cells could be obtained by ex vivo expansion of cryopreserved umbilical cord blood in order to be used for adult patient.

Effect of lactoferrin on ram sperm motility after cryopreservation

Su Jie,Wang Caiyun,Song Yongli,Yang Yanyan,Cao Guifang 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.9

Objective: The objective of this study was to analyse the differentially abundant proteins caused by freeze–thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro. Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 μg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS). Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 μg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05). Conclusion: The LTF is an important protein during cryopreservation, and the addition of 10 μg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm. Objective: The objective of this study was to analyse the differentially abundant proteins caused by freeze–thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro.Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 μg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS).Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 μg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 μg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 μg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05).Conclusion: The LTF is an important protein during cryopreservation, and the addition of 10 μg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.

냉장보존 및 냉동보존이 성견 치주인대세포 생존능에 미치는 영향

이동은,정임희,엄유정,정의원,김창성,이승종,최성호 한국생체재료학회 2010 생체재료학회지 Vol.14 No.1

The purpose of this study was to evaluate the effects of cold preservation at 4 C and cryopreservation at -196 C on the viability of periodontal ligament cells in dog teeth using WST-1(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tet-razolio]-1,3-benzene disulfonate) assay. A total of 7 beagle dogs were used. Eight teeth of each group were divided into 7 experimental groups depending upon the preservation condition. The experimental groups were group 1 (immediate control), group 2-1 (cold preservation at 4 C for 3 days), group 2-2 (cold preservation at 4C for 1 week), group 2-3 (cold preservation at 4C for 2 week), group 3-1 cryopreservation for 3 days), group 3-2 (cryopreservation for 1week), group 3-3 (cryopreservation for 2 weeks). F-medium and 10% dimethylsulfoxide (DMSO) were used as preservation medium and cryoprotectant. For cryopreservation groups, thawing was performed in 37 C water bath, then WST-1 assay was processed. The values of optical density obtained by WST-1 were divided by the values of eosin staining for tissue volume standardization. In WST-1 assay, all cold preservation (4C) groups (group 2, 3, 4) showed significantly higher viability of periodontal ligament cells than cryopreservation group (group 5, 6, 7) (p < 0.05), but showed lower viability than immediate control group (p < 0.05). In cold preservation (4 C) groups, group 2 showed significantly higher viability than group 3 and 4 (p < 0.05). There was no significant difference between all cryopreservation groups (-196 C). From the results of this study, cold preservation method suggests the better efficacy for short term preservation of the teeth than cryopreservation.

Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation

Liu, Jiaen,Sills, E. Scott,Yang, Zhihong,Salem, Shala A.,Rahil, Tayyab,Collins, Gary S.,Liu, Xiaohong,Salem, Rifaat D. The Korean Society for Reproductive Medicine 2012 Clinical and Experimental Reproductive Medicine Vol.39 No.2

Objective: During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation. Methods: First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization d5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation. Results: Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis). Conclusion: While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.