Survival and development of human embryos was compared following slow cooling versus vitrification involving more than 13,000 vitrified embryos. In addition, the efficacy of an open system, the Cryotop, and a closed vitrification system, the CryoTip™, were compared using human blastocysts. One hundred percent of vitrified human pronuclear stage embryos survived and 52% developed to blastocysts as compared with 89% survival and 41% blastocyst development after slow cooling. Similar survival rates were seen with vitrification of 4-cell embryos (98%) as compared with slow cooling (91%). Furthermore, 90% of vitrified blastocysts survived and resulted in a 53% pregnancy rate following transfer, as compared with 84% survival and 51% pregnancy rates following slow cooling. All corresponding values were significantly different. When the closed and open vitrification systems were compared, no difference was found with regard to supporting blastocyst survival (93 and 97% for CryoTip and Cryotop respectively), pregnancies (51 versus 59% respectively) and deliveries (48 versus 51% respectively). Vitrification is a simple, efficient and cost-effective way to improve cumulative pregnancy rates per cycle. The use of the closed CryoTip system eliminates the potential for embryo contamination during cryopreservation and storage without compromising survival and developmental rates in vitro and in vivo.

https://www.rbmojournal.com/article/S1472-6483(10)61169-8/pdf

Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination

BACKGROUND Successful cryopreservation of oocytes and embryos is essential not only to maximize the safety and efficacy of ovarian stimulation cycles in an IVF treatment, but also to enable fertility preservation. Two cryopreservation methods are routinely used: slow-freezing or vitrification. Slow-freezing allows for freezing to occur at a sufficiently slow rate to permit adequate cellular dehydration while minimizing intracellular ice formation. Vitrification allows the solidification of the cell(s) and of the extracellular milieu into a glass-like state without the formation of ice. OBJECTIVE AND RATIONALE The objective of our study was to provide a systematic review and meta-analysis of clinical outcomes following slow-freezing/thawing versus vitrification/warming of oocytes and embryos and to inform the development of World Health Organization guidance on the most effective cryopreservation method. SEARCH METHODS A Medline search was performed from 1966 to 1 August 2016 using the following search terms: (Oocyte(s) [tiab] OR (Pronuclear[tiab] OR Embryo[tiab] OR Blastocyst[tiab]) AND (vitrification[tiab] OR freezing[tiab] OR freeze[tiab]) AND (pregnancy[tiab] OR birth[tiab] OR clinical[tiab]). Queries were limited to those involving humans. RCTs and cohort studies that were published in full-length were considered eligible. Each reference was reviewed for relevance and only primary evidence and relevant articles from the bibliographies of included articles were considered. References were included if they reported cryosurvival rate, clinical pregnancy rate (CPR), live-birth rate (LBR) or delivery rate for slow-frozen or vitrified human oocytes or embryos. A meta-analysis was performed using a random effects model to calculate relative risk ratios (RR) and 95% CI. OUTCOMES One RCT study comparing slow-freezing versus vitrification of oocytes was included. Vitrification was associated with increased ongoing CPR per cycle (RR = 2.81, 95% CI: 1.05-7.51; P = 0.039; 48 and 30 cycles, respectively, per transfer (RR = 1.81, 95% CI 0.71-4.67; P = 0.214; 47 and 19 transfers) and per warmed/thawed oocyte (RR = 1.14, 95% CI: 1.02-1.28; P = 0.018; 260 and 238 oocytes). One RCT comparing vitrification versus fresh oocytes was analysed. In vitrification and fresh cycles, respectively, no evidence for a difference in ongoing CPR per randomized woman (RR = 1.03, 95% CI: 0.87-1.21; P = 0.744, 300 women in each group), per cycle (RR = 1.01, 95% CI: 0.86-1.18; P = 0.934; 267 versus 259 cycles) and per oocyte utilized (RR = 1.02, 95% CI: 0.82-1.26; P = 0.873; 3286 versus 3185 oocytes) was reported. Findings were consistent with relevant cohort studies. Of the seven RCTs on embryo cryopreservation identified, three met the inclusion criteria (638 warming/thawing cycles at cleavage and blastocyst stage), none of which involved pronuclear-stage embryos. A higher CPR per cycle was noted with embryo vitrification compared with slow-freezing, though this was of borderline statistical significance (RR = 1.89, 95% CI: 1.00-3.59; P = 0.051; three RCTs; I2 = 71.9%). LBR per cycle was reported by one RCT performed with cleavage-stage embryos and was higher for vitrification (RR = 2.28; 95% CI: 1.17-4.44; P =  0.016; 216 cycles; one RCT). A secondary analysis was performed focusing on embryo cryosurvival rate. Pooled data from seven RCTs (3615 embryos) revealed a significant improvement in embryo cryosurvival following vitrification as compared with slow-freezing (RR = 1.59, 95% CI: 1.30-1.93; P < 0.001; I2 = 93%). WIDER IMPLICATIONS Data from available RCTs suggest that vitrification/warming is superior to slow-freezing/thawing with regard to clinical outcomes (low quality of the evidence) and cryosurvival rates (moderate quality of the evidence) for oocytes, cleavage-stage embryos and blastocysts. The results were confirmed by cohort studies. The improvements obtained with the introduction of vitrification have several important clinical implications in ART. Based on this evidence, in particular regarding cryosurvival rates, laboratories that continue to use slow-freezing should consider transitioning to the use of vitrification for cryopreservation.

Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance

Treatment processes for source-separated urine.

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.

https://www.rbmojournal.com/article/S1472-6483(10)61091-7/pdf

Are programmable freezers still needed in the embryo laboratory? Review on vitrification

results: Cryopreservation led to a significant increase in percentage DNA fragmentation, percentage 8OHdG and percentage caspase positive cells (P , 0.001). Percentage DNA fragmentation was positively correlated with percentage 8OHdG before (r ¼ 0.756, P , 0.001) and after cryopreservation (r ¼ 0.528, P ¼ 0.017). The addition of 50 and 100 mM genistein to the cryoprotectant had a significant protective effect on sperm DNA (P , 0.001) although the caspase inhibitor demonstrated no difference to the control. conclusions: Human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspases and apoptosis. The estrogenic compound genistein may be useful in reducing this effect but larger trials are needed to confirm this.

/pdf/cryopreservation-induced-human-sperm-dna-damage-is-4712z8xkjx.pdf

Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis

Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates

Blastocysts were cryopreserved by a new two-step ultra-rapid cooling in super-cooled liquid nitrogen (-205°C). There were 308 mouse blastocysts collected from fertile B6CBF1 mice and 249 human blastocysts collected from 51 couples treated with IVF. The blastocysts were super-cooled by a Vit-Master and cryoloops after treatment in 50 and 100% vitrification solution (VS) for 2 min and 30 s respectively. The 100% VS was composed of 20% ethylene glycol 20% dimethylsulphoxide and 0.5 mol/l sucrose in human tubular fluid medium with 20% human serum albumin. The embryos were warmed after treatment in 0.25 and 0.125 mol/l sucrose for 2 and 3 min respectively. The survival of embryos was observed after re-swell. The survival rate (SR) and hatching rate (HR) of mouse blastocysts in the super-cooled the cryosolution-treated and control groups were not significantly different (SR 87 95.5 and 100%; HR 50 33 and 44.6% respectively; P > 0.05). After 96 super-cooled human blastocysts were warmed 60 survival blastocysts were transferred into 13 patients. The successful SR and pregnancy rate (PR) for the super-cooled blastocyst group were 77.1% (74 out of 96) and 53.8% (seven out of 13). The ultra-rapid vitrification of blastocysts with a successful SR and PR could be used to replace classical slow cooling. (authors)

Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification

https://www.fertstert.org/article/S0015-0282(05)00847-2/pdf

Sperm DNA Fragmentation Negatively Correlates With Velocity and Fertilization Rates but Might not Affect Pregnancy Rates

Purpose: This study determined the incidence of sperm nuclear DNA fragmentation, mitochondrial dysfunction, and chromosomal aneuploidy. The results were correlated with the semen analysis parameters and fertilization rates.

/pdf/dna-fragmentation-mitochondrial-dysfunction-and-chromosomal-y5ov9cnpsn.pdf

Tzu-Chun Cheng

Papers

Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates

Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification

Sperm DNA Fragmentation Negatively Correlates With Velocity and Fertilization Rates but Might not Affect Pregnancy Rates

DNA fragmentation, mitochondrial dysfunction and chromosomal aneuploidy in the spermatozoa of oligoasthenoteratozoospermic males.

Laser-assisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate in women with advanced age