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Background/Objectives : Open and closed vitrification systems are commonly employed in oocyte cryopreservation; however, there is limited evidence regarding a comparison of their separate impact on oocyte competence. This study uniquely brings to the literature, data on the effect of open versus closed vitrification systems on laboratory and clinical outcomes, and the effect of cooling and warming rates. Methods : A systematic search of the literature was performed using the databases PubMed/MEDLINE and the Cochrane Central Library, limited to articles published in English up to January 2023. A network meta-analysis was conducted comparing each vitrification system versus fresh oocytes. Results : Twenty-three studies were included. When compared to fresh oocytes, both vitrification devices resulted in lower fertilization rates per MII oocyte retrieved. When comparing the two systems in terms of survival rates, no statistically significant difference was observed. However, interestingly open systems resulted in lower cleavage and blastocyst formation rates per 2 pronuclear (2PN) oocyte compared to fresh controls, while at the same time no statistically significant difference was detected when comparing closed devices with fresh oocytes. Conclusions : In conclusion, closed vitrification systems appear to exert a less detrimental impact on the oocytes' competence, which is reflected in the blastocyst formation rates. Proof of superiority of one system versus the other may lead to standardization, helping to ultimately determine optimal practice in oocyte vitrification.

Vitrified, or "frozen", donor eggs can either be fertilized and cultured for fresh transfer (group 1), or fertilized and cryopreserved for transfer in a "frozen embryo transfer" cycle (group 2). This study compared the pregnancy rates between the two groups. Frozen donor egg cycles (N = 1213) were analyzed at the World Egg Bank. The outcome studied was clinical pregnancy rate. Cycles included only single embryo transfers (ET) without preimplantation genetic testing (PGT). A total of 600 cycles met the inclusion criteria. Group 1 included 409 cycles and group 2 had 191 cycles. There was no statistical significance in clinical pregnancy rate between the two groups (38.63% vs 32.46%, p = 0.14). Mean embryo age was higher in group 2 (5.1 vs. 5.4 days, p < 0.01). The compounding effect of vitrification when applied to two distinct stages (oocyte and embryo), has not been studied. When comparing the two groups, we found no difference in pregnancy rate. However, there was a trend towards fewer pregnancies in group 2. A larger study should be done to determine the validity of this result (Ramadan et al. in Fertil Steril, 2020)., (© 2022. The Author(s).)

The purpose of this review is to educate the reader on the role that cryopreservation has played and continues to play in the ever-evolving field of assisted reproductive technologies, specifically in clinical human fertility treatment. We discuss the science behind the cryopreservation methods and investigated some of the major considerations that any clinic or cryobank faces in terms of risks and liabilities, physical challenges that accompany the constantly growing collection of cryopreserved specimens, and what this means on the ethical and legal front. Finally, we take a glimpse in the future to explore what may be on the horizon for the preservation of gametes and reproductive tissues., (Copyright © 2022. Published by Elsevier Inc.)

Examine good tissue practices as relates to in vitro fertilization, biopsying, and vitrificationto compare current knowledge of ova, sperm, and embryos as vectors for disease transmission as it relates to our current knowledge regarding the SARS-CoV-2 virus.Unknown risks relating to the SARS-CoV-2 virus and sperm, ova, and embryos necessitate a reexamining of how human IVF is performed. Over the last decade, improvements in cryosurvival and live birth outcomes have been associated with zona pellucida breaching procedures (e.g., blastocyst collapsing and biopsying). In turn, today embryos are generally no longer protected by an intact zona pellucida when vitrified and in cryostorage. Additionally, high security storage containers have proven to be resilient to potential cross-contamination and reliable for routine human sperm freezing and embryo vitrification.Several options to current IVF practices are presented that can effectively mitigate the risks of cross-contamination and infection due to the current Covid-19 pandemic or other viral exposures. The question remains; is heightened security and change warranted where the risks of disease transmission likely remain negligible?

Purpose: To determine liquid nitrogen evaporation rates of intact liquid nitrogen storage tanks and tanks with their vacuum removed., Methods: Donated storage tank performance (LN2 evaporation) was evaluated before and after induced vacuum failure. Vacuum of each tank was removed by drilling through the vacuum port. Temperature probes were placed 2 in. below the bottom of the styrofoam cap/plug, and tanks were weighed every 3 h. Evaporation rate and time from failure to the critical temperature was determined., Result: Storage tanks with failed vacuum have a much higher evaporation rate than those with intact vacuum; evaporation rates increased dramatically within 3 to 6 h in the smaller tanks, and time to complete depletion varied according to starting LN2 volume. Tanks with storage racks/specimens may have altered evaporation profiles compared to tanks without. Locating temperature probes 2 in. below the styrofoam cap/plug suggests that for most applications, alarms would sound approximately 1 h prior to reaching the critical warming temperature, approximately - 130 °C. External signs of vacuum loss were dramatic: vapor, frost, and audible movement of air., Conclusion: For the first time, we have data on how liquid nitrogen storage tanks behave when their vacuum is removed. These findings are conservative; each lab must consider starting volume, tank size/capacity, function (storage or shipping), age, and pre-existing evaporation behavior in order to develop an emergency response to critical tank failure. Times to complete failure/evaporation and critical warming temperature after vacuum loss are different; these data should be considered when evaluating tank alarm systems.

The recent failure of two liquid nitrogen storage tanks at two separate facilities in the United States has highlighted the need to reexamine our approach to how reproductive laboratories handle the storage of tissue. If we wish to truly understand how we can increase the security and safety of stored reproductive tissue, we really need to understand how often these events (tank failure) occur and what are the ultimate causes of these failures. Are there design flaws in the storage tanks or are these really failures in protocols and following protocols? Unfortunately, because these cases often involve serious litigation, most do not want to or are forbidden to talk about these failures and so we have little data to base recommendations on. Despite the paucity of data, we must come up with protocols and methods to safeguard tissue stored in these tanks. At the same time, we must be careful that we do not introduce potential solutions to these problems that instead of increasing the safety of stored tissue, we exacerbate the problem. Attention should be paid to where tanks are located so that they can be observed throughout the day. Decisions regarding whether tissue will be stored on-site or off-site, whether small- or large-capacity tanks should be used, or whether a patient's specimens should be split among several tanks should not be based on convenience or cost, but on the ability to reduce risks to these precious specimens. Ongoing quality control (QC) must include checking tanks for both visual clues and analyzing evaporation rates of each tank. Finally, each laboratory must have protocols in place that address all the issues involved in maintaining a safe storage system. These protocols must be understood by all those involved in the safeguard of patients' tissue. We may never eliminate all accidents with stored reproductive tissue, but we can reduce the probability of future accidents. In this article, we will discuss how to manage the risks inherent with the storage of reproductive tissue, where to store tissue, how to store them, how to reduce risks through quality control methods, and finally, options available for inventory management., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Several high-profile cases involving in vitro fertilization have recently received considerable media attention and highlight the importance of assuring patient and tissue identification. Within the assisted reproductive technology (ART) laboratory, there are many steps where wrong patient or tissue identity could have drastic results. Erroneous identity can result in tragic consequences for the patient, the laboratory, and for those working in the program as a whole. Such errors can result in enormous psychological and financial costs, as well as a loss in confidence. There are several critical steps that should be taken every single time and for each specific procedure performed in the ART laboratory to ensure the correct identification of patients and their tissue. These steps should be detailed in protocols that include the method of identification, the two unique identifiers that will be used, the sources of these identifiers, and often a system in which more than one person is involved in the identification. Each protocol should ideally include a checklist that is actively used for the implementation of each procedure. The protocol should also indicate what to do if the identification does not match up, including rapid handling and notification of the patient involved in the error. All ART laboratories should instill in their employees an atmosphere of full and open disclosure for cases where mistakes are made., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Objective: To evaluate the validity of collecting day 3 embryo morphology variables into the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS)., Design: Retrospective., Setting: National database-SART CORS., Patient(s): Fresh autologous assisted reproductive technology (ART) cycles from 2006-2007 in which embryos were transferred singly (n=1,020) or in pairs (n=6,508) and embryo morphology was collected., Intervention(s): None., Main Outcome Measure(s): Relationship between live birth, maternal age, and morphology of transferred day 3 embryos as defined by cell number, fragmentation, and blastomere symmetry. Logistic multiple regressions and receiver operating characteristic curve analyses were applied to determine specificity and sensitivity for correctly classifying embryos as either failures or successes., Result(s): Live birth rate was positively associated with increasing cell number up to eight cells (<6 cells: 2.9%; 6 cells: 9.6%; 7 cells: 15.5%; 8 cells: 24.3%; and >8 cells: 16.2%), but was negatively associated with maternal age, increasing fragmentation, and asymmetry scores. An area under the receiver operating curve of 0.753 (95% confidence interval 0.740-0.766) was derived, with a sensitivity of 45.0%, a specificity of 83.2%, and 76.4% of embryos being correctly classified with a cutoff probability of 0.3., Conclusion(s): This analysis provides support for the validity of collecting morphology fields for day 3 embryos into SART CORS. Standardization of morphology collections will assist in controlling for embryo quality in future database analyses., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

A misconception in the field of reproductive medicine is that there is a significant risk of cross-contamination during gamete or embryo cryostorage. This article is a review of the available literature on animal models and human IVF and it suggests otherwise. There is a negligible risk of cross-contamination in IVF working conditions., (Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Standardization of morphologic assessment for an embryo grading system was developed and is being implemented by the Society for Assisted Reproductive Technology (SART). A recent European consensus conference of embryologists from Europe and America is working toward adopting an embryo classification system modeled similarly to that of SART that, if adopted, would produce a de facto international standard to aid cross-border collaboration., (Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the sensitivity of human sperm survival bioassay to using known concentrations of potential toxin of formalin and to elevate the application value of human sperm motility assay as a quality control method in detecting the components used in IVF program., Methods: Fresh semen was obtained from healthy males at andrology laboratory by masturbation. Sperm was processed on a gradient column of isolate medium and PBS medium. In experiment 1, the medium with 0.25%, 0.75% concentration of formalin and control medium were added to the Falcon culture tubes containing HTF medium with or without 0.3% bovine albumin serum and with or without light mineral oil. In experiment 2, in 3 types of culture tubes containing HTF medium with or without 0.3% bovine albumin serum and with or without light mineral oil, the sperm was exposed to each culture tube and cultured for 24 and 48 hrs at room temperature, and the motile sperms were counted under the microscope., Results: The average sperm motility index in the HTF medium with 0.25% formalin at 24 hrs was 0.594 +/- 0.331, significantly higher than in the HTF medium with 0.75% formalin (0.450 +/- 0.284) (P < 0.01). In the medium containing 0.25% and 0.75% formalin with 0.3% bovine albumin serum and light mineral oil, the average sperm survival indexes were 0.683 +/- 0.334 and 0.527 +/- 0.345, respectively, higher than without bovine albumin serum and light mineral oil (0.394 +/- 0.311 and 0.424 +/- 0.311). The average sperm index of 7 ml tissue culture tube made in Denmark was 0.677 +/- 0.335, higher than the other two types of culture tubes made in the USA (0.551 +/- 0.317 and 0.596 +/- 0.327) (P < 0.001). When the sperm cultured in the medium with 0.3% bovine albumin serum and light mineral oil, the average sperm survival indexes were 0.821 +/- 0.259 and 0.645 +/- 0.335, respectively, higher than without bovine albumin serum or light mineral oil (0.571 +/- 0.321 and 0.395 +/- 0.245) (P < 0.01)., Conclusion: The sperm survival bioassay is a sensitivity quality control method to detect the components in the IVF laboratory. The 7 ml tissue culture tube made in Denmark is most suitable for culturing human embryos. Sperm can be protected when cultured in the medium with 0.3% albumin bovine serum and light mineral oil.

Objective: To evaluate the level of variance produced in a multicenter study with the use of a computer-assisted sperm morphology analyzer., Design: A multicenter, prospective, blinded study., Setting: Assisted reproduction research laboratories., Patient(s): Semen samples produced for assisted reproductive procedures., Intervention(s): Hamilton Thorne Research (Beverly, MA) integrated visual optical system semen analyzers were used at five different centers to evaluate the same set of 30 slides that were prepared and numerically coded at Tygerberg Hospital in Tygerberg, South Africa., Main Outcome Measure(s): The percentage of normal sperm., Result(s): Interlaboratory coefficients of variation (CVs) ranged between 16.31% and 23.09%. One of the participating laboratories produced an approximately 14% (-6.5-7.7) limits of agreement analysis, with a CV of 11.36%, for its duplicate readings. The use of a 10% normal sperm morphology cutoff point to determine discordance levels produced rates ranging between 10% and 23.3% for the interlaboratory and intralaboratory readings. This level of discordance equates with < or = 7 of the corresponding readings from two laboratories falling into a different normal sperm morphology group (< or = 10% or >10%)., Conclusion(s): The magnitudes of variation produced by the readings performed in our study reached the same level as for the manual evaluation of sperm morphology. A < 10% CV can be obtained if the correct quality control measures are implemented.

A transgenic animal represents an enormous investment in time and money. Animals can be destroyed through disease, fire, malfuncnons in the control of the environment, negligence, sabotage, or accidental disposal. Researchers can protect valuable transgenic lines from accrdental destruction by "banking" them in liquid nitrogen. Cryopreservation can also reduce animal costs by decreasing the number of live animals investigators must maintain. Often, when one is trying to produce a transgenic animal, some lines will be derived that may not initially appear interesting. These animals can be stored in liquid nitrogen for future recovery and study. The maintenance of just one line of mice, say 25 mice at 15 cents/d, can cost over $1000 (US) in a single year.

Although a method exists for freezing sheep embryos directly into liquid nitrogen without a programmable freezing machine, a more traditional slow cooling method is described here. Either dimethyl sulfoxide (DMSO) or ethylene glycol can be used as a cryoprotectant in this slow cooling method. Pregnancy rates for frozen embryos are similar to those of fresh embryos. For a general discussion of cryopreservation, the reader is referred to Chapter 25.

The following protocol is for freezing rat blastocysts by direct plunging into liquid nitrogen. Although this protocol is designed for freezing the embryos in polypropylene cryotubes, it could readily be adapted to 0.5-mL straws. For a general discussion of cryopreservation, the reader is referred to Chapter 25.

Advances in cryopreservation enable one to freeze embryos without the use of a programmable freezing machine or complex protocols. These methods achieve high rates of survival when mouse embryos are frozen. Understanding the factors that influence the survival of cryopreserved embryos can aid troubleshooting and in adapting freezing strategies from mice to other species. Frozen stocks of transgenics can be maintained indefinitely in liquid nitrogen. Cryopreservation of these valuable animals not only protects them from environmental catastrophes, but also is an economical method of storing lines for future detailed analysis.

Epididymal sperm were collected from C57Bl6/J X DBA2/J (B6D2) males and allowed to capacitate for 2 hr. When cumulus-free oocytes were exposed to sperm for 15 min in either the presence (6.0 mM) or absence of caffeine, fertilization did not occur. However, when cumulus cells were left intact, 23% of oocytes were fertilized in caffeine-free medium and 62% in caffeine-containing medium. When cumulus-free oocytes were incubated with sperm for 30 min, none was fertilized in the absence of caffeine, but 33% were fertilized when 6.0 mM caffeine was present (P less than .02). These effects of caffeine were on the sperm, as sperm exposed to caffeine and then coincubated with oocytes for 15 min in essentially caffeine-free media fertilized a similar percent of oocytes (93%) as when sperm and oocytes were exposed to caffeine during the fertilization period (86%). When sperm were capacitated in caffeine-containing medium, the percentage of ova fertilized was similar to capacitation without caffeine. We conclude that both cumulus cells and caffeine speed up the fertilization process with mouse gametes and that the effect of caffeine is on the sperm, but not due to more rapid capacitation.