3D-printed equipment to decouple (powder) X-ray diffraction sample preparation and measurement.

An alternative storage method to separate sample preparation from single-crystal and powder X-ray diffraction measurements at home source diffractometers is described. For single crystals, a setup is presented which allows storage of preselected crystals under cryogenic and ambient temperatures. For powders, a disposable sample holder is introduced. The method is suitable for the storage of air- and moisture-sensitive samples. Equipment made of biodegradable polylactic acid is produced by 3D printing and can be adapted to individual needs. As 3D printers are widely available at research institutions nowadays, models of the presented equipment are provided for the reader to allow easy reproduction.

Basic and applied features in the cryopreservation progress of bovine embryos.

The expansion of the use of in vitro production techniques has revolutionized the bovine embryo market. In the last decade, we have seen the number of in vitro produced (IVP) embryos surpass the number of in vivo-derived (IVD) embryos obtained worldwide. Concomitantly, other biotechnologies were also improved, following the global trend. Embryo cryopreservation has received special attention, as it is one of the tools capable of disseminating in vitro production. Currently, two protocols are available: slow freezing and vitrification. Both have advantages and disadvantages regarding their application and, many aspects need to be considered before their use. In this review, we discuss in vitro production market trends, cellular and molecular features involved in embryo response to cryopreservation, and addressed cryo-storage period and embryonic developmental stage on cryosurvival. In addition, we also presented an overview of some aspects that impact the pregnancy rate following transfer of fresh and cryopreserved IVP embryos.

The effect of extended cryo-storage following vitrification on embryo competence: a systematic review and meta-analysis.

PURPOSE: Few studies explored whether prolonged cryo-storage after vitrification affects embryo competence and perinatal outcomes. This systematic review and meta-analysis aims at highlighting any putative impact of cryo-storage duration on cryo-survival, miscarriage, live birth and major malformations. METHODS: A systematic review was performed using MEDLINE (PubMed), ISI Web of Knowledge, Scopus and Embase databases up to June 2021. Data were combined to obtain a pooled OR, and meta-analysis was conducted using a random effects model. Out of 1,389 screened abstracts, 22 papers were assessed for eligibility, and 5 studies were included (N = 18,047 embryos). Prolonged cryo-storage was defined as > 12 months (N = 3389 embryos). Subgroup analysis was performed for untested vitrified cleavage stage embryos (N = 1739 embryos) and for untested and euploid vitrified blastocysts (N = 13,596 and 2712 embryos, respectively). RESULTS: Survival rate, miscarriage, live birth and major malformation rates were all similar in the two groups. CONCLUSION: These data further support the safety of long-term cryo-storage of human embryos beyond 12 months. This is reassuring for good prognosis patients with surplus embryos, couples seeking a second child from supernumerary embryos and women postponing the transfer for clinical or personal reasons.

Clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer are independent of cryo-storage duration.

RESEARCH QUESTION: The study aimed to retrospectively evaluate the impact of cryo-storage duration on clinical, obstetric and perinatal outcomes after vitrified-warmed euploid blastocyst transfer. DESIGN: This was an observational study including 2688 vitrified-warmed euploid single blastocyst transfers that was conducted at a private IVF centre between May 2013 and March 2020. It included a total of 1884 women (age 38 ± 3 years) undergoing at least one transfer after preimplantation genetic testing for aneuploidies. The euploid blastocysts transferred were clustered into seven groups according to the cryo-storage duration between vitrification and warming: ≤60 days (n = 646; control group), 61-90 days (n = 599), 91-180 days (n = 679), 181-360 days (n = 405), 361-720 days (n = 144), 721-1080 days (n = 118) and >1080 days (n = 97). The primary outcome was the live birth rate (LBR) per transfer. The secondary outcomes were miscarriage rate, obstetric and perinatal issues. The data were adjusted for confounders through logistic or linear regressions. RESULTS: A significantly lower LBR was reported for transfers performed within 91-180 days (n = 291/679, 42.9%; P = 0.017), 181-360 days (n = 169/405, 41.7%; P = 0.016) and 361-720 days (n = 57/144, 39.6%; P = 0.034) versus ≤60 days (n = 319/646, 49.4%). However, this was mainly due to top-quality embryos being transferred first when more euploid blastocysts were available, thereby leaving lower quality ones for subsequent procedures. Indeed, the multivariate odds ratios adjusted for confounders showed similar results across all cryo-storage duration clusters. No difference was reported also for all secondary outcomes. CONCLUSIONS: Cryo-storage duration even beyond 3 years from blastocyst vitrification does not affect clinical, obstetric and perinatal outcomes.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.