■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

Effect of single wall carbon nanotubes on human HEK293 cells.

Background In women, anti-Mullerian hormone (AMH) levels may represent the ovarian follicular pool and could be a useful marker of ovarian reserve. The clinical application of AMH measurement has been proposed in the prediction of quantitative and qualitative aspects in assisted reproductive technologies (ART). In men AMH is secreted in both the serum and seminal fluid. Its measurement may be useful in clinical evaluation of the infertile male. Methods The PubMed database was systematically searched for studies published until the end of January 2009, search criteria relevant to AMH, ovarian reserve, ovarian response to gonadotrophin stimulation, spermatogenesis and azoospermia were used. Results AMH seems to be a better marker in predicting ovarian response to controlled ovarian stimulation than age of the patient, FSH, estradiol and inhibin B. A similar performance for AMH and antral follicular count has been reported. In clinical practice, AMH measurement may be useful in the prediction of poor response and cycle cancellation and also of hyper-response and ovarian hyperstimulation syndrome. In the male, the wide overlap of AMH values between controls and infertile men precludes this hormone from being a useful marker of spermatogenesis. Conclusions As AMH may permit the identification of both the extremes of ovarian stimulation, a possible role for its measurement may be in the individualization of treatment strategies in order to reduce the clinical risk of ART along with optimized treatment burden. It is fundamental to clarify the cost/benefit of its use in ovarian reserve testing. Regarding the role of AMH in the evaluation of infertile men, AMH as single marker of spermatogenesis does not seem to reach a satisfactory clinical utility.

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Anti-Müllerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART)

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

BACKGROUND: Anti-Miillerian hormone (AMH) is reported to be a reliable marker of the ovarian response to controlled ovarian stimulation (COS). The objective of this study is to determine whether the serum AMH level can predict ovarian hyperstimulation syndrome (OHSS) prior to selection of COS protocols. METHODS: A cohort of 262 IVF cycles was investigated prospectively, in order to evaluate the predictive value for OHSS by means of certain risk factors, including age, body mass index (BMI), serum estradiol (E 2 ) level, number of retrieved oocytes and basal serum AMH level. RESULTS: The basal serum AMH level predicted OHSS better than age and BMI with a sensitivity of 90.5% and specificity of 81.3%. Both the basal serum AMH level (odds ratio: 1.7856, P = 0.0003) and serum E 2 level on the day of HCG administration (odds ratio: 1.0005, P = 0.0455) proved to be significant predictors of OHSS by logistic regression analysis. However, age (odds ratio: 0.9346, P = 0.049) was the only significant factor for prediction of clinical pregnancy. CONCLUSIONS: The basal serum AMH level could be utilized effectively to predict OHSS and thus to direct the selection of mild COS protocols.

Serum anti-müllerian hormone and estradiol levels as predictors of ovarian hyperstimulation syndrome in assisted reproduction technology cycles

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

BACKGROUND: Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. METHODS: Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). RESULTS: After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). CONCLUSIONS: The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility

Chung-Hsien Liu

Papers

Serum anti-müllerian hormone and estradiol levels as predictors of ovarian hyperstimulation syndrome in assisted reproduction technology cycles

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

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility