Proliferative Capacities and Differentiation Potentials of Human Periodontal Ligament Stem Cells after Slow-freezing Cryopreservation

@#Introduction: The cryopreservation of periodontal ligament stem cells (PDLSCs) required a good combination of CPA composition as a step in the preparation of PDLSCs. This study aimed to analyze the proliferative capacities and differentiation potentials of PDLSCs after slow-freezing cryopreservation with CPA in different combinations. Methods: The fourth passage of the primary PDL cells were examined their fibroblast-like morphology and colony forming unit-fibroblast (CFU-F), and characterized by surface markers for mesenchymal stem cells using flow cytometry. PDLSCs were divided into two groups of freshly-PDLSCs (fPDLSCs) and cryopreserved-PDLSCs (cPDLSCs). The PDLSCs were cryopreserved using slow freezing method with CPA in different combinations: 1) 90%FBS+10%DMEM (FD-group), 2) 90%DMEM+10%DMSO (DDs-group), 3) 90%FBS+10%DMSO (FDs-group), and 4) 100% Cell Banker (CB-group) as positive control. The proliferation of fPDLSCs and cPDLSCs were evaluated by trypan blue dye exclusion method. The multipotency of cells was assessed by Oil Red O, Alizarin Red, and Alcian Blue staining. Results: The primary PDL cells had fibroblast-like morphology and CFU-F ability. They expressed more than 95% positive MSC surface markers of CD90, CD73, CD150, and CD44, but showed less than 2% hematopoietic cell markers of CD11b/CD19/CD34/CD45 and HLA-DR. The cPDLSCs viability of FDs-group was 81.5% and 80% in -80oC and LN2, respectively. The fPDLSCs and cPDLSCs proliferation and doubling time were no statistically significant difference (p>0.05). They could differentiate into adipogenic, osteogenic, and chondrogenic differentiation. Conclusion: The cPDLSCs could maintain their proliferative capacities and differentiation potentials after slow-freezing cryopreservation with 90%FBS+10%DMSO in -80oC.

Research progress of methods to promote the survival of cryopreserved ovarian tissue auto-transplantation / 器官移植

With the technology development of cancer treatment, the survival rate of patients with cancer has been significantly improved. However, chemotherapy and radiation therapy may lead to premature ovarian failure and infertility in young women with cancer. Cryopreserved ovarian tissue auto-transplantation is an effective method to preserve fertility of such female patients. At present, the biggest challenge of this technique is mass loss of follicles after transplantation. In this article, the influencing factors and improvement methods of survival of cryopreserved ovarian tissue auto-transplantation were reviewed.

Improving native human sperm freezing protection by using a modified vitrification method / 亚洲男科学杂志(英文版)

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice. However, it has been shown to have a negative impact on sperm function and structure. Vitrification as a successful alternative method has been proved to have better protective effects on human embryos, but vitrification of spermatozoa is still subject to low recovery rates. In this study, a modified vitrification method for native spermatozoa was developed. A total of 28 semen samples were included; each sample was divided into three equal parts and assigned to fresh, slow freezing, and vitrification groups. Sperm vitality, motility, morphology, DNA integrity, and acrosome reaction were assessed for each of the groups. The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing; vitrification achieves a higher recovery rate (P < 0.05), motility (P <0.05), morphology (P <0.05), and curve line velocity (P <0.05) than slow freezing. Furthermore, DNA fragmentation was decreased (P <0.05) and better acrosome protection (P <0.05) was exhibited in the spermatozoa after vitrification. Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster, indicating that spermatozoa are better preserved through vitrification. In conclusion, while both slow freezing and vitrification have negative effects on sperm function and structure, the vitrification protocol described here had a relatively better recovery rate (65.8%) and showed improved preservation of several sperm quality parameters compared with slow freezing.

Effect of Long-time Slow-frozen Storage on Epigenetic Changes of Histone in Cleavage-stage Embryos / 中山大学学报(医学科学版)

@#【Objective】To investigate the impact of long- term storage time on epigenetic modification of histone in human cleavage stage embryos.【Methods】According to the length of storage time，donated embryos after slow-freezing were divided into 3 groups ：6-year group ，9-year group ，and 12-year group ，while the control group consisted of donated fresh embryos. Immunocytochemistry was performed to compare the expression levels of HDAC1， H3K9ac， H3K4me3 ，and H3K9me3 among 4 groups. Transcription levels of HDAC1 ，SUV39H1 ，SETDB1 ，and KDM5A were analyzed through Single-Cell qRT-PCR.【Results】The relative abundances of HDAC1 and SUV39H1 mRNA showed no significant differences among 4 groups（P > 0.05）. SETDB1 exhibited a climbing pattern as storage time increased，but no significant difference was observed（P > 0.05）. There were no differences in H3K9 trimethylation and H3K9 methylation among 4 groups. However ，the expression level of KDM5A increased with the increasing storage time（P < 0.05）.【Conclusions】 Storage time did not affect the expression of deacetylase HDAC1，methylase SUV39H1 and SETDB1. H3K9ac/me3 and H3K4me3 also exhibited no significant difference as the storage time increases. However ，the increasing storage length might induce the elevating expression of demethylase KDM5A，which may be associated with inhibition of embryonic transcription.

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate / Congelação de embriões caprinos em diferentes estádios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta

The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.(AU)

Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.(AU)

Comparison between Slow Freezing and Vitrification in Terms of Ovarian Tissue Viability in a Bovine Model / Comparação da viabilidade do tecido ovariano após congelamento lento e vitrificação em modelo bovino

Abstract Objective To assess the viability of bovine ovarian tissue after cryopreservation through either slow freezing or vitrification, and to compare it to that of control tissue by performing morphological analyses. Methods The study included 20 bovine ovarian cortex fragments that were divided into control, vitrification, and slow freezing groups. Each group consisted of four fragments of the same ovary, two fixed without cultivation, and two fixed with cultivation. Tissues were evaluated based on follicular morphology immediately after heating and after 7 days of culture, and compared with the control group. Results A total of 240 fragments were analyzed, generating a sample of 1,344 follicles without cultivation and 552 with cultivation. When the non-cultivated samples were classified as non-atretic follicles, 572 were found in the control group, 289 in the vitrification group, and 373 in the slow freezing group, showing no significant differences. When classified as atretic, 46 follicles were found in the control group, 23 in the vitrification group, and 41 in the slow freezing group, also showing no statistical difference. In the post-culture sample, an evolution of the follicular stages could be observed. This finding was important to support that the follicles considered non-atretic in the non-cultivated group were actually viable in the morphological evaluation. Conclusion With no differences between the protocols, vitrification was shown to be an advanced and alternative method for patients who will undergo treatments that.

Resumo Objetivo avaliar a viabilidade do tecido ovariano bovino após a criopreservação, utilizando congelamento lento e vitrificação, e comparando com o tecido controle por meio de análises morfológicas. Métodos o estudo incluiufragmentos de córtex de vinte ovários bovinos divididos em grupos controle, vitrificação e congelamento lento. Cada grupo foi composto por quatro fragmentos do mesmo ovário, sendo dois fragmentos fixados sem cultivo e dois fragmentos fixados pós-cultivo. Os tecidos foram avaliados pela morfologia folicular logo após o aquecimento e após sete dias de cultivo, e comparados com o grupo controle. Resultados um total de 240 fragmentos foi analisado, gerando uma amostra de 1.344 folículos sem cultivo e 552 pós-cultivo. Quando a amostra sem cultivo teve seus folículos agrupados em não atrésicos, obtivemos 572 no grupo controle, 289 no vitrificação, e 373 no congelamento lento, não apresentando diferença estatística. Quando agrupados em atrésicos, o grupo controle apresentou 46 folículos, o vitrificação, 23, e o congelamento lento, 41, não apresentando também diferença estatística. Na amostra pós-cultivo, podemos observar uma evolução dos estágios foliculares: esse achado foi importante para sustentar que os folículos considerados não atrésicos na avaliação morfológica sem cultivo estavam realmente viáveis. Conclusão não havendo diferenças entre os protocolos, a vitrificação se mostra um avanço e um método alternativo para pacientes que irão se submeter a tratamentos que podem levar a uma falência ovariana, uma vez que a metodologia é mais barata, mais rápida e mais bem adaptável a uma rotina de um laboratório.

Comparison of vitrification and slow freezing for cryopreservation of cleavage stage embryos (Day 3) and its impact on clinical outcome.

Background: The objective of this retrospective study was to compare the efficacy of slow freezing and Vitrification for the cryopreservation of supernumerary cleavage stage embryos on day 3 after IVF and its impact on clinical outcome. Methods: 485 supernumerary embryos of IVF cycles (from Oct 2011 to Dec 2012) were cryopreserved by slow freezing method while 502 embryos (from Jan 2013 to April 2014) by Vitrification method. 362/485 and 230/502 embryos were thawed for FET cycles (65 patients in each group).After warming the survival rate, post warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups. Results: There were 65 frozen thawed cycles in each group. The percentage of excellent and good morphology embryos before cryopreservation were same in both the groups, but after thawing the results were significantly in favour of Vitrification as compared to Slow freezing. In Vitrification group versus Slow freezing group, the different outcomes were survival rate (96.95% vs. 69.06%, p-0.000), post warmed excellent morphology embryos (94.17% vs. 60.8%, p-0.000) clinical pregnancy rate (41.53% vs. 21.53%, p-0.043) and the implantation rate (14.41% vs. 7.01%, p-0.024). Conclusions: Vitrification is a promising alternate to the conventional slow freezing method in terms of not only excellent survival and post warmed excellent morphology embryo rate but also higher clinical pregnancy and implantation rate.

How to improve the pregnancy outcome by optimizing embryo cryopreservation protocol / 天津医药

Objective To investigate how to optimize the protocol of embryo cryopreservation to improve the success of frozen-thawed embryo transfer (FET), reduce multiple pregnancy rate and increase the cumulative pregnancy rate from one oocyte retrieval process. Methods The clinical data of 1 166 FET cycles were retrospectively analyzed and separated into different groups:445 for vitrification and 721 for slow-freezing. The vitrification group was divided into single embryo (28 cy?cles), double embryos (71 cycles) and triple embryos (346 cycles). 0-1 optimal embryo was called O0-1 group (235 cycles), 2 optimal embryos were called O2 group (80 cycles), 3 optimal embryos were called O3 group (130 cycles). The difference preg?nancy outcomes (implantation rate, clinical pregnancy, abortion rate and live-birth rate) were compared between groups. Results (1) There were significantly higher embryo survival rate(98.3%vs 73.1%), embryo recovery rate without damaging (83.3%vs 62.1%), implantation rate(36.8%vs 29.9%), clinical pregnancy(57.1%vs 44.0%) and live-birth rate(47.9%vs 34.5%) in vitrification group than those of slow freezing group(P0.05). Conclusion Vitrification technology can improve the clinical pregnancy and live-birth rate, and decrease multiple preg?nancy rate. Two optimal embryos in one tube are supposed to be the preferred method for embryo cryopreservation.

Criopreservación de embriones equinos y primer reporte de un potro de raza criolla colombiana nacido por transferencia de un embrión equino vitrificado / Cryopreservation of Equine Embryos and First Report of a Native Colombian Breed Born by Transfer of an Equine Vitrified Embryo / Criopreservação de embriões equinos e primeiro relatório de um potro de raça crioula colombiana, nascido por transferência de um embrião equino vitrificado

El objetivo de este artículo es informar sobre el éxito de un procedimiento de criopreservación de embriones equinos, a fin de conseguir una preñez viable. Se colectaron embriones equinos al día 6-6,5 (< 300 µm; n = 24) y se sometieron a dos técnicas de criopreservación: grupo 1 (n = 12), vitrificados exponiéndolos a una solución VS1 (Gli [1,4 M]) 5 min, VS2 (Gli [1,4 M] + EG [3,6 M]) y VS3 (Gli [3,4 M] + EG [4,6 M] 1 min. Se empacaron en pajillas de 0,25 ml y se sumergieron en nitrógeno líquido; grupo 2 (n = 12), congelación lenta: expuestos a una solución de congelación (1,8 M de EG + 0,1 M sucrosa) por 10 min, empacados en pajillas de 0,25 ml, llevados al congelador de embriones, exponiéndolos a una curva de congelación y sumergidos en nitrógeno líquido. Posterior a la descongelación, a los 24 embriones se les removió el crioprotector mediante un paso; fueron sumergidos en medio de cultivo DMEM/F12 + 10% de suero fetal bovino (SFB) e incubados bajo atmosfera controlada (5% CO2, 5% N2 90% O2) por 48 h. Se evaluó el desarrollo embrionario en el 75% de los embriones vitrificados (n = 4); el 20% de los embriones fueron sometidos a congelación lenta (n = 1). No se observaron diferencias significativas en los grupos respecto al desarrollo embrionario, pero sí mayor tendencia de supervivencia en los embriones vitrificados. Igualmente, uno de estos embriones vitrificados fue transferido a una receptora, se logró una preñez viable y el nacimiento de un potro vivo.

The aim of this paper is to report on the success of a cryopreservation procedure of equine embryos to achieve a viable pregnancy. Equine embryos were collected on day 6-6.5 (<300 µm, n = 24) and subjected to two cryopreservation techniques: group 1 (n = 12), vitrified, exposing them to a VS1 (Gli [1.4 M]) 5 min, VS2 (Gli [1.4 M] + EG [3.6 M]) and VS3 (Gli [3.4 M] + EG [4.6 M] 1 min solution. They were packed in 0.25 ml straws and immersed in liquid nitrogen; group 2 (n = 12), slow freezing: exposed to a freezing solution (1.8 M EG + 0.1 M sucrose) for 10 minutes, packed into 0.25 ml straws, brought to the embryos freezer, exposed to a freezing curve and immersed in liquid nitrogen. Following defrosting, cryoprotectants were removed from the 24 embryos in one step; they were submerged in culture medium DMEM/F12 + 10% of fetal bovine serum (FBS) and incubated under controlled atmosphere (5% CO2, 5% N2 90% O2) for 48 h. Embryonic development was evaluated in 75% of the vitrified embryos (n = 4); 20% of the embryos were subjected to slow freezing (n = 1). No significant difference was observed in the groups regarding embryonic development, but a greater survival tendency on the vitrified embryos was noted. Also, one of these vitrified embryos was transferred to a receiver, achieving a viable pregnancy and the birth of a living foal.

O objetivo deste artigo é informar sobre o sucesso de um procedimento de criopreservação de embriões equinos, a finalidade de conseguir uma prenhez viável. Coletaram-se embriões equinos ao dia 6-6,5 (< 300 µm; n = 24) e se submeteram a duas técnicas de criopreservação: grupo 1 (n = 12), vitrificados expondo-os à uma solução VS1 (Gli [1,4 M]) 5 min, VS2 (Gli [1,4 M] + EG [3,6 M]) e VS3 (Gli [3,4 M] + EG [4,6 M] 1 min. Se empacaram em tubos de 0,25 ml e se submergiram em nitrogênio líquido; grupo 2 (n = 12), congelação lenta: expostos a uma solução de congelação (1,8 M de EG + 0,1 M sacarosa) por 10 min, embalados em tubos de 0,25 ml, levados ao congelador de embriões, expondo-os a uma curva de congelação e submergidos em nitrogênio líquido. Posterior à descongelação, aos 24 embriões foi-lhes foi removido o crio protetor mediante um passo; foram submergidos em meio de cultivo DMEM/F12 + 10% de soro fetal bovino (SFB) e incubados bajo atmosfera controlada (5% CO2, 5% N2, 90% O2) por 48 h. Se avaliou o desenvolvimento embrionário no 75% dos embriões vitrificados (n = 4); o 20% de os embriões foram submetidos a congelação lenta (n = 1). Não se observaram diferenças significativas nos grupos com respeito ao desenvolvimento embrionário, mas sim uma maior tendência de sobrevivência nos embriões vitrificados. Igualmente, um destes embriões vitrificados foi transferido a uma receptora, obteve-se uma prenhez viável e o nascimento de um potro vivo.

In vitro development and gene expression of frozen-thawed 8-cell stage mouse embryos following slow freezing or vitrification / 대한생식의학회지

OBJECTIVE: This study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined. METHODS: Eight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes (Rbm3, Birc5, Sod1, Sod2, Cirbp, Caspase3, Trp53, Hsp70.1) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos. RESULTS: There were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers (107.0+/-21.0 vs. 115.0+/-19.7), or ICM cell numbers of blastocysts (11.3+/-5.2 vs. 11.1+/-3.7). Cell numbers of blastocysts were significantly (p<0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of CirbP and Hsp70.1 were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences. CONCLUSION: These results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

Criopreservação de oócitos / Oocytes cryopreservation

criopreservação de oócitos atualmente representa uma grande evolução em Reprodução Humana Assistida. Essa técnica consiste na conservação de células ou tecidos a temperaturas inferiores a -196¨¬C. A criopreservação de oócitos é um dos principais destaques, que surgiu com o objetivo de preservar a fertilidade feminina e, ainda, contornar as questões éticas e legais associadas ao congelamento de embriões. As técnicas de criopreservação vêm sendo aprimoradas, tendo sido observado um avanço notável nas taxas de fertilização obtidas a partir de oócitos congelados. Com base nisto, este estudo teve como objetivo realizar uma revisão bibliográfica sobre dois métodos de criopreservação (congelamento lento e vitrificação). Para isso, realizaram-se leitura e seleção de informações trabalhadas por outros autores em revistas científicas, sites de busca e livros específicos de reprodução humana assistida. Foram analisados: histórico da criopreservação de oócitos; indicações; crioprotetores; métodos de criopreservação e resultados das taxas de fertilização, gravidez e aborto segundo pesquisas já realizadas tanto com congelamento lento quanto com vitrificação de oócitos. Apesar dos resultados favoráveis à criopreservação oocitária, são necessárias mais pesquisas para que haja estabilização dos resultados e estabelecimento de uma técnica de criopreservação de oócitos humanos que seja universal e padronizada, podendo ser aplicada com sucesso nas clínicas de Reprodução Humana Assistida.

Cryopreservation of oocytes currently represents a major evolution in the human assisted reproduction. This technique consists of the conservation of cells or tissues in temperatures less than -196 ¢ªC. In view of the controversy in several cultures on legal and ethical issues associated with the freezing of embryos, the development of techniques that could solve this problem became necessary. The cryopreservation of oocytes is one of the highlights, aiming to preserve the fertility of women. The techniques of cryopreservation have been improving in search of quality and good results. Nowadays, we observe a progress in fertilization rates obtained from frozen oocytes. Our objective was to carry out a bibliographic review of two methods of cryopreservation (slow freezing and vitrification). In order to do that, we read and checked information provided by other authors in scientific journals, search engines and books on human assisted reproduction. We analyzed: history of oocytes cryopreservation; indications; cryoprotectors; methods and results of cryopreservation of fertilization, pregnancy and abortion rates, according to other surveys on slow freezing and oocyte vitrification. Despite the favorable results of oocytes cryopreservation, further studies are necessary to stabilize the results and to establish a technique which be universal for cryopreservation of human oocytes and can be successfully applied in human assisted reproduction clinics.

Comparison of viability of oral epithelial cells stored by different freezing methods / 대한치과보존학회지

This study examined the influence of the storage methods on the viability of oral epithelial cells using conventional cell freezing storage, slow freezing preservation, rapid freezing preservation, and slow freezing preservation with a pressure of 2 Mpa or 3 Mpa. The cell viability was evaluated by cell counting, WST-1 and the clonogenic capacity after 6 days of freezing storage. After 6 days, the frozen cells were thawed rapidly, and the cell counting, WST-1, and clonogenic capacity values were measured and compared. 1. The results from cell counting demonstrated that conventional cryopreservation, slow freezing under a 2 Mpa pressure and slow freezing under a 3 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05). 2. The results from the optical density by WST-1 demonstrated that slow freezing under a 2 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05). 3. The clonogenic capacity demonstrated that slow freezing under a 2 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05).

A comparison between conventional slow freezing and vitrification of mouse blastocysts using cryo-E.

Objective: To compare the efficiency of two cryopreservations between conventional slow freezing and vitrification of mouse blastocysts using cryo-E. Methods: ICR female mice (8 weeks) were superovulated with 5 IU/ml of pregnant mare serum gonadotrophin (PMSG), the successfulness of mating with males was verified by the presence of a vaginal plug. Blastocysts were obtained between 3.5 and 4.5 days per p.c. or 96-108 hours after hCG administration by flushing the uterus. Randomly selected blastocysts were simultaneously frozen by slow-rate freezing and a vitrification method. One month later, the embryos were thawed and cultured in the blastocyst medium (COOK; Sydney IVF, Australia). Results: Based on 250 slow freezing and 310 vitrified mouse blastocysts, vitrification resulted in a slightly higher survival and hatching rates than the slow-freezing method (83.9% VS 82.0%, and 68.8% VS 66.8%, respectively). Conclusion: Both slow freezing and vitrification of mouse blastocysts are useful methods for cryopreservation. These results showed that vitrification is better than slow freezing in terms of simplicity, duration, and cost-effectiveness.

The effectiveness of ethylene glycol as cryoprotectant in mouse embryo freezing with slow freezing method / 대한산부인과학회지

OBJECTIVE: We intended to know how the cryoprotectant ethylene glycol (EG) would affect the outcome of the embryo development when used in slow freezing method. And to know if there is any difference in the outcome of frozen-thawed embryos according to freezing methods and the timing. METHODS: We used 5-6 weeks old ICR female mice and T6 containing 0.4% BSA for basic culture media. The embryos at the developmental stages of 1-cell, 8-cell and blastocyst were cryopreserved respectively by slow freezing method using EG, propylene glycol (PROH), and glycerol as a cryoprotectant. We also compared the results of slow freezing and vitrification methods with the same cryoprotectant, EG. And finally, we evaluated the quality of blastocysts by counting the cell numbers in each group. RESULTS: The post-thaw embryo development were better in EG group when they were frozen at 1-cell and blastocyst stage (P<0.05). Although there were no differences in the recovery rate, the survival rate in vitrification group was significantly higher (P<0.05). Post-thaw embryo development to morula and blastocyst were better in vitrification group when frozen at 1-cell embryo (P<0.05), not at 8-cell and blastocyst group. The cell counts of blastocyst derived from 1-cell stage frozen EG group were significantly increased than that of PROH-glycerol groups (P<0.05), however, there was no difference between the two freezing methods. CONCLUSION: These results suggest that EG may be advantageous comparing with the conventional cryoprotectants, PROH and glycerol in slow freezing method for mouse embryo cryopreservation. In terms of freezing method, vitrification is better than slow freezing.

Development of Effective Cryopreservation Method for Mouse Oocytes / 대한불임학회지

OBJECTIVE: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. METHODS: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1,2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-beta-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). RESULTS: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. CONCLUSION: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

Effect of Ethylene Glycol (EG) and 1,2-Propanediol (PROH) on the Survival and the Development of Mouse and Human Embryosafter Slow Freezing/Rapid Thawing Protocol / 대한불임학회지

OBJECTIVE: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. METHODS: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG+0.2 M sucrose or 1.5 M PROH+0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. RESULTS: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. CONCLUSION: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

Establishment of Mouse Oocytes Cryopreservation Program / 대한산부인과학회잡지

OBJECTIVE: To establish the optimal cryopreservation method in mouse oocytes. METHODS: Firstly, mouse immature oocytes were exposed to various cryoprotectants, and then cryoprotectant with the best outcome was selected. Secondly, mouse immature oocytes were cryopreserved by either slow freezing and ultra-rapid thawing or vitrification. Finally, in mouse mature oocytes, the five different protocols were compared in their fertilization and hatching rates. RESULTS: 1) 1.5M 1,2-propanediol (PROH) and 1.5M PROH+0.1M sucrose had a higher rate of survival (73.1%, 81.9%) and in vitro maturation (28.2%, 30.1%). 2) Vitrification using 5.5M ethylene glycol (EG) showed significantly higher rate of survival and in vitro maturation, when compared with slow freezing and ultra-rapid thawing using 1.5M PROH+0.1M sucrose (65.9% vs 50.0%, 40.0% vs 28.2%, respectively). 3) In mouse mature oocytes, vitrification using 5.5M EG showed significantly higher survival rate, however, slow freezing and ultra-rapid thawing using 1.5M DMSO was superior to vitrification in view of fertilization rate. CONCLUSIONS: Vitrification showed better outcomes in mouse immature oocytes, but slow freezing and ultra-rapid thawing using 1.5M DMSO may be beneficial in mature oocytes.

Damage of Intracytoplasmic Meiotic Spindles in Cryopreserved-Thawed Mouse Oocytes / 대한산부인과학회잡지

OBJECTIVE: To investigate the extent of meiotic spindle damages in cryopreserved-thawed mouse mature oocytes. METHODS: After slow freezing and ultra-rapid thawing using 1.5M dimethylsulfoxide(DMSO), mouse mature oocytes were stained by anti-alpha tubulin monoclonal antibody. The meiotic spindle and chromosomes configuration were assessed using confocal microscope. The influence of time to post-hCG oocytes retrieval (i.e. 12 hrs vs 17 hrs) was also evaluated. RESULTS: The normal meiotic spindles were observed in 89.8% of post-hCG 12 hrs group, and 80.1% of post-hCG 17 hrs group, and these were significantly lower than that of each unfreezed control. Post-hCG 12 hrs group showed a significantly higher incidence of normal meiotic spindles, compared with post-hCG 17 hrs group. CONCLUSION: The extent of meiotic spindle damages was significantly increased after cryopreservation in mouse mature oocytes. We proposed that 12 hrs interval of post-hCG oocytes retrieval may be more beneficial.

Vitrification of Mouse Blastocyst Using Cryoloop / 대한불임학회지

OBJECTIVE: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst. METHODS: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-freezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of blastocysts in experimental groups. RESULTS: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower (X2-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%, 48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (+/-SD) cell number of blastocyst in the exposure only (89.2+/-11.5), EM grids (85.0+/-10.3) and cryoloop (89.0+/-11.0) groups, except slow-freezing group (79.0+/-10.0), were not significantly different from that of control group (93.1+/-13.9) 24 h after thawing (Student's t-test). CONCLUSION: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.