近端輸卵管阻塞可用超音波引導疏通率達˙成
懷孕率達26%

Int J Fertil Steril. 2016 Jan-Mar;9(4):497-505. Epub 2015 Dec 23.

Comparison of Pregnancy Outcome between Ultrasound- Guided Tubal Recanalization and Office-Based Microhysteroscopic Ostial Dilatation in Patients with Proximal Blocked Tubes.

Seyam EM1, Hassan MM1, Tawfeek Mohamed Sayed Gad M1, Mahmoud HS2, Ibrahim MG2.

Author information

Abstract

BACKGROUND:

The current research to the best of my knowledge is the first to compare the pregnancy outcome between ultrasound-guided tubal recanalization (UGTR) using a special fallopian tubal catheter, and office-based micrhysteroscopic ostial dilatation (MHOD) using the same tubal catherter in infertile women with previously diagnosed bilateral proximal tubal obstruction (PTO).

MATERIALS AND METHODS:

This prospective study reported the pregnancy outcomes for 200 women in private infertility care center in Arafa hospital in Fayoum and in El Minya University Hospital in the period between January 2010 and October 2013 treated as outpatients for their bilateral PTO after the routine hysterosalpingography (HSG). A Cook's catheter, special fallopian tubal catheter, were used to recanalize the blocked tubes in 100 women (group A) under UGTR, and the same Cook's tubal catheter was used through 2mm microhysteroscope to cannulate both ostia using MHOD in another 100 women (group B). Pregnancy outcome was determined after the procedures for a 12-month period follow-up.

RESULTS:

The number of the recanalization of PTO was not significantly different between two groups. As of the 200 blocked fallopian tubes in group A, 140 tubes (70%) were successfully recanalized by passing the ultrasound-guided special cannula, while 150 tubes (75%) were successfully recanalized in group B, using the same tubal catheter through a 2mm microhysteroscope. The cumulative pregnancy rate after the two procedures was not statistically different between two groups. It was 25.9% in group A, while it was 26.3% in group B, after a 12-month period follow-up.

延長培養至第7天並無法有效下降囊胚染色體異常率

Int J Fertil Steril. 2016 Jan-Mar;9(4):563-73. Epub 2015 Dec 23.

The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells.

Bazrgar M1, Gourabi H2, Eftekhari-Yazdi P3, Vazirinasab H2, Fakhri M2, Hassani F3, Chehrazi M4, Valojerdi MR3.

Author information

Abstract

BACKGROUND:

A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies.

MATERIALS AND METHODS:

In this cohort study, we used fluorescence in situ hybridi- zation (FISH) to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD) on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization.

RESULTS:

Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%), while 2(6.7%) were diploid, and 2(6.7%) were triploid. Of those with mosaicism, 23(88.5%) were determined to be diploid-aneuploid and 3(11.5%) were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05); however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality.

CONCLUSION:

Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

卵丘細胞較少(Fig a)之卵子異常率較高應優先考慮施行ICSI

異常現象包括: ZP外觀異常薄, 外圍粗燥(Fig c), perivitelline space過小&PB無法分辨(Fig c), 細胞質無彈性(fig e)----應施行ICSI

GV>M2過程中perivitelline space 會擴大

perivitelline space過大過小卵子較易異常



Representative images of the abnormal and normal oocytes and embryos under light inverted microscopy. a The abnormal oocyte–corona-cumulus complex with less granular cumulus around the abnormal oocyte, and (b) the normal one with much granular cumulus surrounded. c The denuded oocyte with narrow perivitelline space (PVS), heterogeous zona pellucida (ZP) with less smooth outer edge, and hard to distinguished polar body compared with (d) the normal oocyte. e No resistance during ICSI being performed and no crease left after ICSI for abnormal oocyte compared with (f) the normal oocyte. g, h The morphology of two pronuclear (2PN) zygotes from abnormal and normal oocytes looked similar without consideration of PVS. i, k The cleaved embryos at 4–8 cell stage without clear blastomere interval for abnormal oocytes and (j, l) normal 4–8-cell embryos Original magnification: 200×


http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947078/

卵細胞培養液中含有卵丘細胞可增進卵子受孕後形成囊胚之比率

cortical granule在卵成熟過程中(GV>M2)會逐漸聚集於卵子細胞外圍, 並在第一隻精蟲突破卵子後會並釋放(exocytosis)出防止其他精蟲再次多重受精之物質於perivitelline space

去除卵丘細胞後會造成cortical granule過早exocytosis, ZP變硬, 造成無法正常受精而需藉由ICSI受精

https://www.animalsciencepublications.org/publications/jas/articles/88/8/2604

Porcine oocytes denuded before maturation can develop to the blastocyst stage if provided a cumulous cell-derived coculture system

ABSTRACT

The physiological role of cumulus cells (CC) surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. However, removal of CC from oocytes is inevitable for some embryo manipulation techniques, such as germinal vesicle (GV) transfer, somatic cell haploidization, and oocyte cryopreservation. The present study was designed to determine an optimal method to culture porcine denuded oocytes (DO). The results indicated CC from cumulus-oocyte complexes at the GV stage (GVCC) or at the metaphase II stage, and mural granulosa cells could not improve the maturation of DO. However, GVCC could enhance the development of matured porcine DO after fertilization; the percentage of blastocysts was increased from 1.1 to 17.2% (P < 0.05), and the relative value of the x-axis and y-axis of spindles was also increased (P < 0.05). Coculture with GVCC had no effect on the distribution of mitochondria and cortical granules. The results contribute to our understanding of the mechanisms by which CC promote oocyte maturation and contribute to optimization of protocols for in vitro maturation of DO.

原始卵&卵丘細胞在卵細胞成熟過程中需要適量glucose提供其營養來源

Ann Biomed Eng. 2011 Oct;39(10):2654-67. doi: 10.1007/s10439-011-0353-y. Epub 2011 Jul 16.

Estimation of glucose uptake by ovarian follicular cells.

Clark AR1, Stokes YM, Thompson JG.

Author information

Abstract

In vitro maturation (IVM) of mammalian oocytes provides an alternative to traditional in vitro fertilization techniques for clinical treatment of infertility or animal breeding. IVM involves the collection of oocytes from the ovary prior to ovulation, with maturation occurring in a laboratory environment. The success of IVM is highly sensitive to the in vitro nutrient environment. The nurse cells surrounding the oocyte, known as cumulus cells, regulate this environment and removal of these cells reduces the ability of the oocyte to develop following insemination. Determining the nature of the interaction between the oocyte and cumulus cells, collectively called the cumulus-oocyte complex (COC), is a difficult task experimentally. Here we use a combination of experimental and mathematical techniques to investigate glucose transport within bovine COCs and find quantitative estimates of the glucose uptake rates of the oocyte and cumulus cells. Surprisingly, our modeling shows the rate of uptake of glucose by the oocyte to increase and then decrease with concentration, a result that needs further experimental investigation but which supports the expectation that high and low glucose concentrations are detrimental to oocyte development. The methodology described is suitable for use across species and for investigating the transport of other important nutrients within the COC.

IVF overnight    VS.     IVF 6-8h
-----IVF 6-8h 活產率較高

Cochrane Database Syst Rev. 2013 Apr 30;4:CD009391. doi: 10.1002/14651858.CD009391.pub2.

Brief co-incubation of sperm and oocytes for in vitro fertilization techniques.

Huang Z1, Li J, Wang L, Yan J, Shi Y, Li S.

Author information

Abstract

BACKGROUND:

The in vitro fertilization (IVF) technique is commonly used and is the only treatment option for a proportion of infertile couples. To obtain better outcomes of IVF, it is important to enhance embryo quality by optimizing IVF techniques. In IVF procedures, oocytes and sperm are routinely co-incubated overnight, which may expose oocytes and zygotes to suboptimal culture conditions with increased reactive oxygen species (ROS) produced by sperm in this long term culture. As an attempt to avoid possible detrimental effects on the oocytes from long exposure to sperm, the brief co-incubation insemination protocol was developed. However, despite a number of studies in this area, it is unclear whether brief co-incubation improves the IVF outcomes compared with the standard overnight insemination protocol.

OBJECTIVES:

This Cochrane review aimed to determine whether brief co-incubation of sperm and oocytes improves outcomes compared with the standard overnight insemination protocol for women undergoing IVF.

SEARCH METHODS:

We searched the Cochrane Menstrual Disorders and Subfertility Group Register (14 June 2012), Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, 1st quarter), MEDLINE (1948 to 14 June 2012), EMBASE (1989 to 14 June 2012), PsycINFO (1806 to 14 June 2012) and CINAHL (1980 to 26 July 2012). In addition, we searched trials registers, reference lists of articles, conference proceedings (American Society for Reproductive Medicine (ASRM), European Society of Human Reproduction and Embryology (ESHRE)) and contacted experts in the field.

SELECTION CRITERIA:

We included randomized controlled trials (RCTs) comparing brief co-incubation of gametes with the standard overnight insemination protocol.

DATA COLLECTION AND ANALYSIS:

Two review authors independently assessed studies for inclusion and trial quality, and extracted data. Disagreements were resolved by discussion with a third author. Statistical analysis was performed using RevMan software.

MAIN RESULTS:

Eight RCTs with 733 women in total that compared brief co-incubation and the standard insemination protocol were included. Live birth was not reported in the included studies. For ongoing pregnancy rate, there were 127 ongoing pregnancies in two trials including 426 women. The low quality evidence showed that brief co-incubation was associated with an increased ongoing pregnancy rate compared to the standard insemination protocol (pooled odds ratio (OR) 2.42, 95% confidence interval (CI) 1.55 to 3.77; P < 0.0001, I(2) = 0%). Measuring clinical pregnancy rate, there were 93 clinical pregnancies in three trials including 372 women. The low quality evidence showed that brief co-incubation was associated with a significantly higher clinical pregnancy rate than the overnight insemination protocol (pooled OR 2.36, 95% CI 1.45 to 3.85; P = 0.0006, I(2) = 0%). For the miscarriage rate, there were six miscarriages in one trial including 167 women. This low quality evidence suggested no significant difference in the odds of miscarriage between brief co-incubation and standard insemination (OR 1.98, 95% CI 0.35 to 11.09; P = 0.44).

AUTHORS' CONCLUSIONS:

This review has provided evidence that brief co-incubation of sperm and oocytes may improve the ongoing pregnancy and clinical pregnancy rates for infertile women undergoing IVF cycles. More RCTs are required to assess whether brief co-incubation would contribute to a higher live birth rate and a lower miscarriage rate compared to the standard overnight insemination protocol.

使用自體濾泡液+EG+sucrose製備冷凍液可達類似commercial 冷凍液效果
卵保留corona radiata 可冷凍&解凍施行IVF受精

Reprod Fertil Dev. 2014 Aug;26(7):1001-6. doi: 10.1071/RD13161.

Human oocyte vitrification with corona radiata, in autologous follicular fluid supplemented with ethylene glycol, preserves conventional IVF potential: birth of four healthy babies.

Tong XH1, Wu LM2, Jin RT2, Luan HB2, Liu YS2.

Author information

Abstract

The aim of this study was to examine the effects of vitrification with autologous follicular fluid (AFF) supplemented with ethylene glycol (EG) and sucrose on human oocytes with corona radiata. A total of 182 human oocytes with corona radiata from fifteen infertile patients were vitrified using either equilibration solutions (ES) and vitrification solution (VS) consisting of AFF, EG and sucrose (AFF group, n=67) or commercial ES and VS (control group, n=115). All oocytes were thawed in the next cycle, with surviving oocytes being inseminated by conventional IVF. The clinical outcome of vitrified-warmed oocytes by both vitrification methods was analysed retrospectively. In the AFF group, six patients received embryo transfer, with three couples taking four healthy babies home. In the control group, nine patients received embryo transfer, with four couples taking five healthy babies home. There was no significant difference in the survival rate (91.0 vs 92.2%), two pronuclei (2PN) fertilisation rate (73.8 vs 73.6%), cleavage rate (100 vs 100%), top-quality embryo rate (62.2 vs 59.2%), clinical pregnancy rate (50.0 vs 44.4%), implantation rate (33.3 vs 25%) or take-home baby rate (50.0 vs 44.4%) between the AFF group and the control group, respectively. These results show that AFF supplemented with EG and sucrose is an efficient, cost-effective cryoprotectant for human oocyte cryopreservation. A corona radiata on vitrified-warmed oocytes retains theoocytes' fertilisation capability in conventional IVF.

PMID:

 

23910960

 

[PubMed - indexed for MEDLINE]

不去除卵丘細胞之corona radiata之卵子經玻璃化冷凍解凍後仍可經IVF達到受孕

Hum Reprod. 2012 Nov;27(11):3208-14. doi: 10.1093/humrep/des295. Epub 2012 Aug 27.

Fertilization rates are improved after IVF if the corona radiata is left intact in vitrified-warmed human oocytes.

Tong XH1, Wu LM, Jin RT, Luo LH, Luan HB, Liu YS.

Author information

Abstract

BACKGROUND:

Before human MII oocytes are vitrified they are usually denuded from their cumulus cells. In this study we wanted to investigate the effects of an intact corona radiata on the vitrification and fertilization of human oocytes.

METHODS:

The study comprised two different parts. In Part 1, 36 MII stage oocytes, from 6 patients, were randomly assigned into a control group, a group of vitrified-warmed oocytes without a corona radiata and a group of vitrified-warmed oocytes with an intact corona radiata. In each group of 12, 6 oocytes were used for evaluation of the zona pellucida solubility (hardening) and another 6 oocytes were used for the analysis of their ultrastructure. In addition, six polyspermically fertilized oocytes were used as positive controls for zona pellucida hardening. In Part 2, 16 patients in total produced 107 fresh and 98 vitrified-warmed oocytes, with or without an intact corona radiata. All oocytes were fertilized via conventional IVF and embryos were transferred according to our standard ET routines. The oocyte survival and fertilization rates, embryo quality and pregnancy and implantation rates were evaluated.

RESULTS:

There were no differences in oocyte survival, zona pellucida solubility (hardening) or the number of cortical granules between the vitrified-warmed and fresh oocytes. There were also no differences in the zona pellucida solubility and the number of cortical granules between vitrified-warmed oocytes with or without an intact corona radiata. However, the oocytes with an intact corona radiata had a higher fertilization rate after conventional IVF insemination. No differences were seen in the survival and cleavage rates, the percentage of high-quality embryos or the clinical outcome.

CONCLUSIONS:

Zona hardening and ultrastructural damage do not seem to occur in vitrified human oocytes. An intact corona radiata in vitrified-warmed oocytes retains their fertilization capacity in conventional IVF, but does not improve the embryo quality. Poor fertilizing capacities of vitrified-warmed oocytes without an intact corona radiata seem to have been due to the complete removal of the cumulus cells.

卵丘細胞與卵細胞核成熟過程有關
卵子成熟前(GV>M1)去除卵丘細胞之卵子可使細胞核較靠近1PB
進而提高去核率

Jpn J Vet Res. 2012 Nov;60(4):191-203.

Removal of cumulus cells before oocyte nuclear maturation enhances enucleation rates without affecting the developmental competence of porcine cloned embryos.

Jeon RH1, Maeng GH, Lee WJ, Kim TH, Lee YM, Lee JH, Kumar BM, Lee SL, Rho GJ.

Author information

Abstract

The present study compared the efficiency of somatic cell nuclear transfer (SCNT) using porcine oocytes that were denuded of their cumulus cells at different maturation time. In pre-denuded group, the cumulus cells from cumulus-oocyte complexes (COCs) were removed at 29 hr post in vitro maturation (hpm) and followed by further culture for 12 hr. In control group, as a commonly followed procedure, cumulus cells were removed from COCs at 41 hpm. The majority of porcine oocytes at 29 hpm were observed in metaphase of the first meiotic division (MI). At 41 hpm, no significant (P>0.05) differences were observed in nuclear maturation and mitochondrial distribution of oocytes between pre-denuded and control groups. However, in pre-denuded group oocytes, metaphase II (MII) plate and spindle were located closely as 'adjacent' to the first polar body (PB1), resulting in an increased enucleation rates than in control group oocytes by blind enucleation method. Following SCNT and parthenogenesis (PA) using pre-denuded group and control group oocytes, no significant (P>0.05) differences were observed with respect to the development, total cell number, incidence of apoptosis and the expression profile of developmentally important genes (Pou5f1, Dnmt1, Dnmt3a, Igf2r, Bax, Bcl2 and Glut1) at the blastocyst stage. In conclusion, the removal of cumulus cells at 29 hpm in porcine oocytes increased the enucleation rates through proper positioning of PB1 without compromising the quality of SCNT embryos during preimplantation development. Hence, this could be a valuable strategy to improve the SCNT efficiency in a porcine model.

IVF 4h 去除卵丘細胞易造成多重受精(3PN)

J Assist Reprod Genet. 2013 Aug;30(8):1049-53. doi: 10.1007/s10815-013-0049-3. Epub 2013 Jul 19.

Effect of cumulus cell removal 4 h post-insemination on fertilization and embryo quality: a prospective randomized sibling-oocyte study.

Xue Y1, Tong X, Jiang L, Zhu H, Yang L, Zhang S.

Author information

Abstract

PURPOSE:

The study was designed to evaluate whether cumulus cell removal 4 h post-insemination could influence fertilization and embryo quality.

METHODS:

The study included 61couples undergoing standard long down regulation protocol from July 2011 to May 2012. Sibling oocytes of each patient were randomly assigned to either the 4 h group or the 20 group. For the 4 h group, cumulus cells were removed 4 h after gamete coincubation; for the 20 group, cumulus cells removal was performed 20 h after insemination. Fertilization rate, embryo quality, pregnancy rate and implantation rate were assessed.

RESULTS:

A total of 801 sibling cumulus-oocyte complexes (COCs) were randomized to the 4 h group (421 COCs) or 20 h group (380 COCs). There was no difference in the two pronuclei, one pronucleus and grade 1-2 embryo rate. Three pronuclei rate was significantly higher in the 4 h group compared to the 20 h group (12.6 % vs. 8.2 %, P = 0.041). Comparison of embryo transfer cycles in which either embryos from the 4 h group or 20 h group were transferred did not reveal any statistically significant differences in pregnancy or implantation rates.

CONCLUSION:

The results of the present study indicate that cumulus cell removal 4 h post-insemination may increase the percentage of tripronuclear zygotes. However, normal fertilization rate, embryo development, clinical pregnancy rate and implantation rates are not influenced by the timing of cumulus cell removal.

IVF 6h去除卵丘細胞 (vs 18-20h)容易造成胚胎多重受精, 品質下降, 囊胚率下降


file:///C:/Documents%20and%20Settings/xph/My%20Documents/Downloads/ARSci_2015080710223625.pdf

胚胎生長狀態與速度可能與胚胎性別有關 (75%關連性)
女性胚胎較晚分裂
女性胚胎較晚形成桑椹體
女性胚胎較慢形成囊胚期胚胎,
女性胚胎囊胚期胚胎膨脹較慢

J Assist Reprod Genet. 2016 Mar 1. [Epub ahead of print]

Time-lapse variables and embryo gender: a retrospective analysis of 81 live births obtained following minimal stimulation and single embryo transfer.

Bodri D1, Kawachiya S2, Sugimoto T2, Yao Serna J2, Kato R2, Matsumoto T2.

Author information

Abstract

PURPOSE:

The purpose of this study was to determine which morphokinetic variables are related to embryo gender in a cohort of consecutive live births obtained through single blastocyst transfer following mild ovarian stimulation.

METHODS:

Eighty-one live births (49 % of them females) from successfully treated, consecutive infertile patients (maternal age 36.9 ± 3.8 years, range 28-46) who underwent minimal ovarian stimulation, prolonged embryo culture in a time-lapse monitoring (TLM) incubator and elective single blastocyst transfers during 2012-2014. Early (PNf, t2-t9, cc2a, b, s2, s3) and late (tM, tSB, tfullB, texpB1, and texpB2) morphokinetic variables were scored according to published consensus criteria and were normalized to the time of pronuclear fading. For each variable, the ranges with the highest proportion of female embryos (optimal range) were determined by detailed examination of histograms.

RESULTS:

Female embryo gender was associated both with late cleavage (t8), morula (tM), and blastocyst stage morphokinetic variables. The strongest associations (adjusted ORs, 7.0-7.8) were found for late, expanded stage blastocyst parameters; tfullB, texpB1, and texpB2. The proportion of female embryos was 69-71 and 25-26 % inside and outside of the optimal ranges, respectively. This allowed to predict 74-78 % of them, increasing their proportion by 57 % compared to the average.

CONCLUSIONS:

Although the sample size of our cohort was limited, our findings suggest that several expanded blastocyst stage morphokinetic parameters are associated with female embryo gender. If confirmed on a larger sample these could be potentially used to increase the proportion of female embryos among non-invasively selected blastocysts following single embryo transfer.

slush nitrogen 半固體液態氮
利用vacuum真空吸引造成負壓以下降liquid nitrogen 溫度-196達-210

Cooling Solution

To achieve a rapid decrease in temperature, the cooling solution most often used is liquid nitrogen (–196°C). We used slush nitrogen (–210°C), which is obtained by a vacuum pump (Telstar TOP-3; Telstar S.A., Terrassa, Spain) that solidifies part of the liquid nitrogen in few minutes. On return to normal atmospheric pressure, the nitrogen collapses, and the subcooled liquid nitrogen has solid particles in it commonly referred to as “slush” (Fig. 2). The advantage of slush nitrogen lies not only in the temperature difference with respect to liquid nitrogen (∼20°C less) but also in the reduction of the Leiden frost effect, which is the formation of a layer of vapor around the sample when immersed in the cryogenic liquid from room temperature decreasing the cooling rate 14 and 15.

Figure 2. 

Slush nitrogen.

Figure options

蔗糖在冷凍劑中扮演提高滲透壓, 將細胞內水分脫出, 縮短抗凍劑EG, DMSO滲透入細胞內之時間, 減少抗凍劑EG, DMSO毒性

蔗糖在解凍劑中扮演滲透壓緩衝劑, 藉維持細胞外高滲透壓, 避免水分快速滲入細胞內, 減少osmotic shock


http://www.intechopen.com/books/recent-advances-in-cryopreservation/the-maining-of-cryopreservation-for-in-vitro-fertilization-patients


Cryoprotectant agents are essential for the cryopreservation of cells. Basically two groups of cryoprotectants exist: 1) permeating (glycerol, ethylene glycol, dimethyl sulphoxide); and 2) nonpermeating (saccharides, protein, polymers) agents. The essential component of a vitrification solution is the permeating agent. These compounds are hydrophilic non-electrolytes with a strong dehydrating effect. Furthermore, these CPAs are able to depress the “freezing point” of the solution. Regarding the high concentration of cryoprotectant used for vitrification, and in view of the known biological and physiochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of these additives, but it may also be manifested in the form of cryo-injury above and beyond that seen occurring due to classical causes of cell damage (osmotic toxicity and ice formation) during cryopreservation. In spite of this, the permeating CPA should be chosen firstly by their permeating property, and secondly on the basis of their potential toxicity. Because the permeating CPA is responsible for the toxicity (the key limiting factor in cryobiology), different cryoprotectants have been tested for their relative toxicity, and the results indicate that ethylene glycol (EG; MW 62.02) is the least toxic followed by glycerol. Additionally, these highly permeating cryoprotectants are also more likely to diffuse out of the cells rapidly and the cells regained their original volume more quickly upon warming, thus preventing osmotic injury. Therefore, the most common and accepted cryoprotectant for vitrification procedures is ethylene glycol (EG). Today EG is more commonly used in an equimolar mixture with DMSO. Often additives are added to the vitrification solution such as disaccharides. Disaccharides, for example sucrose, do not penetrate the cell membrane, but they help to draw out more water from cells by osmosis, and therefore lessen the exposure time of the cells to the toxic effects of the cryoprotectants. The non-permeating sucrose also acts as an osmotic buffer to reduce the osmotic shock that might otherwise result from the dilution of the cryoprotectant after cryostorage. In addition, permeating agents are able to compound with intracellular water and therefore water is very slowly removed from the cell. Hence the critical intracellular salt concentration is reached at a lower temperature. Removal of the cryoprotectant agent during warming can present a very real problem in terms of trying to reduce toxicity to the cells. Firstly, because of the toxicity of the vitrification solutions, quick dilution of them after warming is necessary; and secondly, during dilution water permeates more rapidly in to the cell than the cryoprotective additive diffuses out. As a consequence of the excess water inflow the cells are threatened by injury from osmotic swelling. In this situation the non-permeating sucrose acts as an osmotic buffer to reduce the osmotic shock. During warming using a high extracellular concentration of sucrose (e.g., 1.0M) counterbalances the high concentration of the cryoprotectant agents in the cell, as it reduces the difference in osmolarity between the intra-and extracellular compartments. The high sucrose concentration cannot totally prevent the cell from swelling, but it can reduce the speed and magnitude of swelling [25, 26, 27].

玻璃化冷凍過程提高冷凍液溫度可能會提高其毒性
玻璃化冷凍過程提高時間>15min可能會提高其毒性
PROH 毒性> DMSO毒性> or = EG毒性
合併使用多種抗凍液&下降其濃度可減少其毒性

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217997/

PLoS One. 2011;6(11):e27604. doi: 10.1371/journal.pone.0027604. Epub 2011 Nov 16.

Comparison and avoidance of toxicity of penetrating cryoprotectants.

Szurek EA1, Eroglu A.

Author information

Abstract

The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ∼23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.

Figure 2

Time- and temperature-dependence of CPA toxicity.

Ovulated mouse oocytes were exposed to a 1.5 M solution of each CPA at 37°C for 0, 15, and 30 minutes and evaluated for their (A) post-exposure survival, (B) fertilization, (C) embryonic development, (D) parthenogenetic activation, and (E) ploidy. Data shown are mean±SEM except for the ploidy rates, which represent percentage of total number of euploid eggs. The total number of oocytes (n) used in each group was also shown. The differences between the control, DMSO and EG groups were not significant while only a few oocytes survived exposure to 1.5 M PROH at 37°C. Therefore, parthenogenetic activation and ploidy were not evaluated in the PROH group. N/A: not applicable.