Survival,re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems

Purpose

This study evaluated and compared survival, re-expansion, and percentage of live cells of individual Days 5 and 6 human blastocysts that were vitrified and warmed with the Vit Kit Freeze/Thaw (Irvine Scientific, CA), or with two protocols using the Global Fast Freeze/Thaw Kits (LifeGlobal, Canada).

Methods

Frozen/thawed Day 2–3 or discarded embryos were cultured to blastocyst (culture day 5–6). Group 1 blastocysts were vitrified with the Vit Kit (n?=?29) and High Security Vitrification (HSV) devices. Group 2 (n?=?47) and Group 3 (n?=?48) blastocysts were cryopreserved with the Global Fast Freeze Kit and 0.25 ml straws, using a direct plunge or a ?100 °C holding step, respectively. Group 4 (Controls, n?=?30) were not vitrified. Blastocysts were subsequently cultured for 24 h, assessed for survival and expansion, and then stained individually with propidium iodide and Hoechst. Live and total cell number was assessed with ImageJ (NIH), and the percentage of live cells calculated for each blastocyst.

Results

The percentage of live cells was not different between vitrified and control (non-vitrified) blastocysts, thus vitrification did not affect cell survival. Survival (following thawing and after 24 h culture), re-expansion, and percentage of live cells were not different for blastocysts vitrified and warmed between the two vitrification/warming kits, or between the two protocols for the Global Fast Freeze/Thaw Kits.

Conclusions

Blastocyst vitrification can be achieved with equal success using simplified protocols and cheaper and easy to load freezing straws, providing simultaneously increased safety, and efficiency with lower cost, when compared with vitrification using specialized embryo vitrification devices.

     

Slow and ultrarapid cryopreservation of biopsied mouse blastocysts and its effect on DNA integrity index

Purpose  

To evaluate the effect of slow and ultra-rapid freezing on biopsied blastocysts’ DNA integrity.     

Is vitrification standard method of cryopreservation

Cryopreservation of human oocytes and embryos or blastocyts is an important choice in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing other attempts’ costs. It provides an opportunity for patients to have more than one attempt following an ovarian stimulation cycle, thereby decreasing the exposure of patients to exogenous gonadotrophins. Vitrification is a cryopreservation technique that leads to a glass-like solidification. Oocyte, zygote, embryo and blastocyst freezing by vitrification method for cryopreservation have been used for many years beside sperms preservation. Moreover, the use of vitrification technology for ovarian tissue cryopreservation to freeze eggs offers such an elderly women who sometime find more difficulty in conceiving or in maintaining pregnancy till full term because of old age compared to relatively younger women who might get better chances to get a healthy pregnancy. Furthermore, vitrification helps cancer patients who are looking to preserve their fertility later on after completing their treatment.     

Cryopreservation of human oocytes,zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification) methods

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.     

玻璃化法冻融小鼠桑葚胚期和囊胚期胚胎的效果观察

目的 比较玻璃化法冻融小鼠桑葚胚期、囊胚早期和囊胚期胚胎后,胚胎存活及继续发育的能力。方法应用6 mol/L乙二醇和1 mol/L蔗糖的玻璃化冷冻液,冻融小鼠142个桑葚胚期、135个囊胚早期和148个囊胚期胚胎,观察冻融后胚胎存活及继续发育的情况。结果 小鼠桑葚胚期、囊胚早期和囊胚期胚胎的存活率分别为88.0％、73.3％和60.1％,囊胚孵出率分别为73.9%、61.5％和49.3％。桑葚胚期的胚胎存活率及囊胚孵出率均高于囊胚早期,囊胚早期高于囊胚期,各期胚胎存活率、囊胚孵出率比较,差异均有显著性(P<0.05)。结论 玻璃化法冻融桑葚胚期的效果好于囊胚早期及囊胚期,桑葚胚期是卵裂期后胚胎玻璃化冻融的最佳阶段。     

Slow freezing should not be totally substituted by vitrification when applied to day 3 embryo cryopreservation: an analysis of 5613 frozen cycles

Purpose

This study aimed to compare slow freezing (SF) and vitrification (VT) techniques for day 3 embryo cryopreservation in infertile couples.

Methods

This retrospective cohort study enrolled 5613 infertile patients, with 7862 frozen-thawed day 3 embryos and 3845 vitrified-warmed day 3 embryos, from 2010 to 2014, at a single center. The rates of embryo survival, pregnancy, implantation, miscarriage, live birth, and live birth weight were compared between the two groups.

Results

A total of 5613 cycles with 5520 transfers were analyzed. Using SF, the rates of overall embryo survival and fully intact blastomeres were lower than those in VT (91.5 vs. 97.4 % and 68.7 vs. 92.3 %, respectively). The rate of good quality embryos after thawing/warming was lower in SF than in VT. In single frozen embryo transfer cycles (FETs), the pregnancy and implantation rates were similar between the two groups (35.0 vs. 40.8 % and 34.6 vs. 35.9 %, respectively). In double FETs, the pregnancy rate per cycle was also similar between the groups (58.8 vs. 58.4 %). The implantation rate per embryo transfer was significantly higher with SF than with VT (38.8 vs. 34.6 %). With adjustment for maternal age and the number of good quality embryos, differences in implantation rate remained significant (adjusted P value, SF vs. VT P < 0.05). No independent effect was found for the method of cryopreservation on the pregnancy rate. No significant differences in the rates of miscarriage, live birth, and live birth weight were observed between the two techniques.

Conclusions

Despite the significantly low embryo survival rate, fully intact blastomere rate, and good quality embryo rate in SF, the pregnancy and implantation rates were not adversely affected in single and double FETs. SF yielded an equivalent miscarriage rate, live birth rate, and live birth weight compared with VT. The SF protocol to cryopreserve day 3 embryos still should be considered.     

The use of a metal container for vitrification of mouse ovaries, as a clinical grade model for human ovarian tissue cryopreservation, after different times and temperatures of transport

Purpose

Cryopreservation of ovarian tissue is paramount for fertility preservation, with important clinical applications, especially for women suffering from an oncological condition. Several cryopreservation methodologies have been tried in search of better outcomes, especially in terms of primor-dial and primary follicles integrity post-cryopreservation. Vitrification has successfully been applied to ovarian tissue using different carriers for tissue exposure to the liquid nitrogen (LN2).

Methods

We developed an enclosed metal vessel, which has the advantage of a faster heat transfer, when in contact with LN2 avoiding at the same time, the direct contact with tissue. Additionally, we assessed the effect of different times and temperatures of transport between the collection of mouse ovaries and the beginning of cryopreservation, on follicular morphology after vitrification.

Results

Our results suggest that 37 °C and R.T. help to maintain normal primordial and primary follicle morphology for up to 4 hrs after collection and beginning of vitrification in a metal container.

Conclusion

These data show that the metal container is an appropriate carrier for mouse ovary vitrification. The rate of morphologically normal primordial follicles up to 4 hrs.     

A new safe, simple and successful vitrification method for bovine and human blastocysts

This study examined a new method for vitrification of blastocysts that is safe, simple and easy to learn and use. Current vitrification techniques have shortcomings that include the use of dimethyl sulphoxide, one of the more toxic cryoprotectants, and minute containers that are difficult to handle and are usually open to contamination. Cell handling and loading times are very short, which allows no room for user-associated errors and increases the difficulty of the procedure. This study describes a method of vitrification without these shortcomings. Human and bovine blastocysts were exposed to a series of three cryoprotectant solutions and loaded into a 0.25 ml sterile straw, heat sealed at both ends and vitrified. This technique allowed sufficient time for cryoprotectant exposure, loading, sealing and vitrification. Research blastocysts were thawed, cultured for 24 h, and stained for cell viability. The majority survived and on average had few lysed cells. In clinical studies from three different centres, 81.4% of vitrified blastocysts were intact after thawing. Out of 43 transfers with 76 blastocysts replaced, 44.7% implanted, 43.4% yielded a fetal heart beat, and a total of 32 babies have been delivered or are ongoing. The overall clinical pregnancy per transfer rate was 60.4%. The high survival rates and clinical pregnancy rates obtained with this new, safe and easy-to-use vitrification procedure are encouraging.     

The effect of vitrification on ultrastructure of human in vitro matured germinal vesicle oocytes

Objective

To describe the possible effects of cryotop vitrification on maturation rate and ultrastructural morphology of human in vitro matured germinal vesicle (GV) oocytes.

Study design

A total of 301surplus immature GV oocytes obtained from infertile patients were allocated into two groups: (i) GV oocytes (n = 150) matured in vitro (fIVM), and (ii) GV oocytes (n = 151) that were first vitrified, then matured in vitro (vIVM). Supernumerary fresh in vivo matured oocytes (n = 10) were used as controls. The maturation media was Ham's F10 supplemented with FSH + LH and human follicular fluid. After 36 h of incubation, the oocytes were investigated for nuclear maturation and ultrastructural changes using transmission electron microscopy (TEM).

Results

Oocyte maturation rates were reduced (P < 0.001) in vIVM (45.92%) in comparison with fIVM oocytes (75.33%). The rate of degeneration was also significantly higher in vIVM than in the fIVM group (44.4% vs. 6.0%). Large and numerous mitochondria and minute vesicles of smooth endoplasmic reticulum (SER) complexes (MV complexes) were observed in both fIVM and vIVM groups. In addition, TEM revealed a drastic reduction in amount of cortical granules (CGs) at the cortex of vitrified-warmed GV oocytes, as well as appearance of vacuoles and small mitochondria-SER aggregates in the ooplasm.

Conclusion

The vitrification procedure is associated with ultrastructural alterations in specific oocyte microdomains, presumably related to the reduced competence of cryopreserved oocytes for maturation. This information emphasizes the need for further work on advancing the cryotechnology of human oocytes.     

Oocyte cryopreservation: searching for novel improvement strategies

Purpose

To highlight emerging techniques aimed at improving oocyte cryopreservation.

Methods

Review of available and relevant literature through Pubmed and Medline searches.

Results

Oocyte cryopreservation is an increasingly common procedure utilized for assisted reproduction and may benefit several patient populations. Therefore, improving efficiency is paramount in realizing the tremendous promise of this approach. However, in addition to numerous studies looking to improve oocyte cryopreservation efficacy via examination of variables involved with protocol methodology, such as type/concentration of cryoprotectant (CPA), type of storage device, or cooling/warming rates, there are more novel approaches for improvement. These alternate approaches include utilizing different the stages of oocytes, examining alteration of basal media and buffer composition, optimizing CPA exchange protocols and device loading through use of automated technology, as well as examination/manipulation of oocyte cellular composition to improve cryotolerance. Finally, elucidating more accurate or insightful indicators of “success” is crucial for continued improvement of oocyte cryopreservation.

Conclusion

Oocyte cryopreservation has improved dramatically in recent years and is receiving widespread clinical use. Novel approaches to further improve success, as well as improved methods to assess this success will aid in continued improvement.     

Slow freezing and vitrification of mouse morula and early blastocysts

Purpose

To assess the relative success of morula and early blastocyst slow freezing and vitrification in regards to survival and implantation rates utilising protocols which could be clinically implemented as a viable alternative to expanded blastocyst stage freezing.

Methods

Mouse morula and early blastocysts were either slow frozen/thawed or vitrified/warmed. Their subsequent survival, blastocyst development and blastocyst cell number and allocation to either the inner cell mass, trophectoderm or epiblast was assessed. In addition blastocysts were also transferred to pseudopregnant recipients and implantation and fetal development was determined.

Results

Vitrification of both morula and early blastocysts resulted in significantly higher rates of survival and blastocyst development compared to slow freezing. In addition slow frozen early blastocysts had significantly reduced blastocyst cell number compared to control however vitrified morula and early blasocyts and slow frozen morula had equivocal blastocyst cell numbers. Transfer of blastocysts from both methods of cryopreservation resulted in similar implantation rates however the placentas created from slow frozen early blastocysts were significantly lighter than control (95.5 g ± 5.4 vs. 122.0 g ± 4.2 respectively).

Conclusions

Vitrification resulted in significantly higher rates of morula and early blastocyst survival and blastocyst development compared to slow freezing. In addition this study has validated the use of a closed DMSO free vitrification protocol which could then be investigated for use in the clinical setting as an alternative to expanded blastocyst freezing.     

成熟卵母细胞玻璃化冷冻时机及解冻方法对辅助生殖结局的影响

目的 探讨玻璃化冷冻成熟卵母细胞过程中不同冷冻时机及解冻方法对辅助生殖结局的影响.方法 回顾性分析郑州大学第一附属医院生殖医学中心2007年5月-2009年5月实施辅助生殖周期中因不同原因接受成熟卵母细胞冷冻及解冻的不孕症患者30例的临床资料,其中女方双侧输卵管梗阻21例,男方无精症9例.将30例患者按照成熟卵母细胞的冷冻时机和解冻方法不同分为3组:A组共5例,取卵后4～5 h冷冻且采用常规方法解冻;B组共9例,取卵后2 h内冷冻且采用常规方法解冻;C组共16例,取卵后2 h内冷冻且采用改良法解冻.所有卵母细胞均采用玻璃化冷冻.冻存2～12个月解冻,存活的卵母细胞行卵母细胞胞质内单精子注射(ICSI)后进行胚胎移植.观察并比较3组患者的辅助生殖结局及孕期随访情况.结果 (1)B组和C组患者的卵母细胞存活率[(65±33)%、(72±23)%]、移植周期率(9/9、16/16)均明显高于A组[(16±17)%、1/5],差异均有统计学意义(P=0.001、0.021);B组与C组分别比较,差异均无统计学意义(P>0.05).C组的平均胚胎种植率[(33±38)%]、临床妊娠率(9/16)均明显高于B组[(4±11)%、1/9],差异均有统计学意义(P=0.033、0.040).(2)C组内采用自身胚胎移植、赠卵胚胎移植及供精胚胎移植者的平均年龄[分别为(28.6±2.1)、(28.0±4.6)、(28.1±3.4)岁]、卵母细胞存活率[分别为(73±25)%、(88±10)%、(66±25)%]、受精率[分别为(84.6±0.9)%、(79.3±2.0)%、(82.8±15.0)%]、胚胎种植率[分别为(20.0±44.7)%、(33.0±0.1)%、(41.6±41.7)%]及临床妊娠率(分别为1/5、3/3、5/8)之间比较,差异均无统计学意义(P>0.05).(3)A组1例患者行胚胎移植,但未妊娠;B组1例临床妊娠,2个月后胚胎停止发育;C组9例临床妊娠,其中1例妊娠4个月流产,8例已顺利分娩5男婴、4女婴,新生儿染色体及发育均正常,每解冻卵母细胞活产率为5.9%(8/135),平均孕周为(39.4±0.9)周,平均新生儿出生体质量为(3574±569)g.结论 取卵后2 h内玻璃化冷冻及改良法解冻成熟卵母细胞,可以提高胚胎质量及改善临床妊娠结局.     

A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos

Purpose

To compare two different vitrification methods to slow freezing method for cryopreservation of human cleavage stage embryos. Design: Prospective randomised trial. Setting: University assisted reproduction centre. Patient(s): 568 patients (mean age 33.4 ± 5.2) from April 2009 to April 2011.

Methods

1798 supernumerary good-quality cleavage stage embryos in 645 IVF cycles intended to be cryopreserved were randomly allocated to three groups: slow freezing, vitrification with the Irvine® method, vitrification with the Vitrolife® method. Main Outcome Measure(s): Embryo survival and cleavage rates, implantation rate.

Results

A total of 1055 embryos were warmed, 836 (79.2 %) survived and 676 were finally transferred (64.1 %). Post-warming embryos survival rate was significantly higher after vitrification (Irvine: 89.4 %; Vitrolife: 87.6 %) than after slow freezing (63.8 %) (p < 0.001). No differences in survival rates were observed between the two vitrification methods, but a significant higher cleavage rate was observed using Irvine compared to Vitrolife method (p < 0.05). Implantation rate (IR) per embryo replaced and per embryo warmed were respectively 15.8 % (41/259) and 12.4 % (41/330) for Irvine, 17.0 % (40/235) and 12.1 % (40/330) for Vitrolife, 21.4 % (39/182) and 9.9 % (39/395) for slow-freezing (NS).

Conclusions

Both vitrification methods (Irvine and Vitrolife) are more efficient than slow freezing for cryopreservation of human cleavage stage embryos in terms of post-warming survival rate. No significant difference in the implantation rate was observed between the three cryopreservation methods.     

In vitro and in vivo viability of human blastocysts collapsed by laser pulse or osmotic shock prior to vitrification

Purpose  

This study was designed to investigate whether artificial shrinkage, induced by a laser pulse or hyperosmotic sucrose solutions, improves in vitro survival and/or implantation of vitrified-warmed human expanded blastocysts.     

Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer

Trophectoderm biopsy with comprehensive chromosome screening (CCS) has been shown to increase implantation and pregnancy rates. Some patients desire CCS on previously cryopreserved blastocysts, resulting in blastocysts that are thawed/warmed, biopsied, vitrified and then warmed again. The effect of two cryopreservation procedures and two thawing/warming procedures on outcomes has not been effectively studied. Cycles were divided into two groups: group 1 patients underwent a cryopreserved embryo transfer with euploid blastocysts that were vitrified and warmed once; group 2 patients had a cryopreserved embryo transfer of a euploid blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified and warmed. Groups 1 and 2 included 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years, respectively (not significantly different). Blastocyst survival in group 1 (114/116, 98.3%) and survival of second warming in group 2 (21/24, 87.5%) was significantly different (P = 0.0354). There was no difference between biochemical (68.2% and 62.5%) and clinical (61.2% and 56.3%) pregnancy rates, implantation rate (58.4% and 52.4%) and live birth/ongoing pregnancy rate (54.0% and 47.6%) between groups 1 and 2, respectively. Although it is unconventional to thaw/warm, biopsy, revitrify and rewarm blastocysts for cryopreserved embryo transfer, the results indicate that outcomes are not compromised.Trophectoderm biopsy and screening the embryos for chromosomal abnormalities has been reported to increase implantation and pregnancy rates. There is a category of patients requesting chromosomal screening on previously cryopreserved blastocysts. This scenario requires blastocysts to be thawed/warmed, biopsied, cryopreserved, and thawed/warmed again. The effect of double cryopreservation procedures and double thawing/warming procedures on pregnancy is unknown. Patients were divided into two groups, group 1 underwent a cryopreserved embryo transfer with a chromosomally normal blastocyst that was vitrified and warmed once and group 2 included patients that had a cryopreserved embryo transfer of a chromosomally normal blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified, and rewarmed. A total of 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years were included in groups 1 and 2, respectively. The survival rate for group 1 (114 of 116, 98.3%) compared with the second warming for group 2 (21 of 24, 87.5%) was significantly higher. There was no difference between biochemical (68.2% and 62.5%), and clinical pregnancies (61.2% and 56.3%), implantation (58.4% and 52.4%), and live birth/ongoing rates (54.0% and 47.6%) between groups 1 and 2. Although it is unconventional to twice cryopreserve and twice thaw/warm a blastocyst, our results indicate that outcomes are not compromised.     

Vitrification of human oocytes and different development stages of embryos: An overview

Cryopreservation of oocytes and embryos leads to increased cumulative pregnancy rates along with restored costs of artificial reproductive technology (ART), minimizing the risks of ovarian hyper stimulation syndrome (OHSS) through reducing number of stimulated cycles, reducing the risks of multiple pregnancy, allowing patients undergoing chemotherapy and radiotherapy to preserve their fertility and giving hope to those with genetic diseases as turner syndrome and premature ovarian failure or both.Vitrification of oocytes and embryos has reduced the idea of embryo selection because of the high viability rate and it is an area of active research justifying optimism for improved results in cryopreservation.     

The human postmenopausal ovary as a tool for evaluation of cryopreservation protocols towards whole ovary cryopreservation

Purpose  

Cryopreservation of a complete ovary may be a future method for fertility preservation in cancer patients. Difficulties exist in cryopreservation of the relatively large ovarian tissue mass. This study evaluates whether a human postmenopausal ovary can be used, as a complement to animal models, in studies of this research field.     

Comparison of two different media for vitrification and rewarming of human zygotes: Prospective randomized study

Vitrification has been successfully used for cryopreservation of human oocytes, zygotes and embryos. There are various media which contain different combinations and concentrations of cryoprotectants for this technique. In this prospective randomized study, two different vitrification and warming media were compared in human zygotes’ cryopreservation.     

Mechanical zona pellucida removal of vitrified-warmed human blastocysts does not affect the clinical outcome

Research QuestionDoes complete mechanical removal of the zona pellucida modify the outcome of transfer of vitrified–warmed human blastocysts?DesignIn a prospective randomized controlled study, 419 couples were allocated to either zona pellucida-free (n = 209) or zona intact (n = 210) vitrified–warmed embryo transfer. Main outcome measures included clinical pregnancy, implantation and ongoing pregnancy rates.ResultsTransfer of zona pellucida-free blastocysts resulted in clinical pregnancy, implantation and ongoing pregnancy rates (35,9%, 33,9% and 32,1%, respectively), similar to those achieved with zona intact control embryos (39%, 36,4% and 33,1%, respectively).ConclusionTotal mechanical removal of the zona pellucida did not affect the tested parameters of clinical outcomes.