Cryopreservation and IVF in the time of Covid-19: what is the best good tissue practice (GTP)?

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?

Day 7 blastocyst euploidy supports routine implementation for cycles using preimplantation genetic testing.

OBJECTIVE: To determine if Day 7 blastocysts merit biopsy, vitrification and transfer consideration by contrasting their aneuploidy and implantation rates to Day 5 and 6 blastocysts. METHODS: A total of 1,925 blastocysts were biopsied from 402 PGT-A cycles over a 12 to 16 month interval. All embryos were cultured under tri-gas, humidified conditions (37ºC) for up to 7 days (168 hours post-insemination). Biopsied blastocysts were vitrified and trophectoderm samples analyzed using NextGen sequencing. Single euploid embryo transfers were performed (n=254) using either a Day 5 (n=145), Day 6 (n=92) or a Day 7 blastocyst (n=16) post-warming. Euploidy rates and pregnancy outcomes were subsequently assessed and differences determined by day of development and blastocyst quality grade. RESULTS: No differences were observed in implantation, pregnancy loss or ongoing pregnancy rates between Day 5 and Day 6 blastocysts. Development to Day 7 accounted for 6.6% of all blastocysts. Euploidy rates were higher in Day 5 blastocysts (53.5%; p<0.05) compared to Day 6 (40.4%) and Day 7 (35.9%). High implantation potential (56.3% to 79.3%) of vitrified-warmed euploid blastocyst occurred independent to the day of development. However, miscarriage/loss rates increased (22.2% vs. 2%; p<0.05) with Day 7 blastocysts, resulting in lower (p<0.05) live birth rates (43.8% vs. 67.4-77.2%). CONCLUSION: Culturing blastocysts to Day 7 has proven beneficial by achieving viable euploid embryos that would have otherwise been discarded. An extra Day of embryo growth allows select patients additional opportunities for in vitro development and possible healthy term live births.

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an "open" system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable µS-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-µm ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (µS-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified µS-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the µS-VTF procedure has not affected its successful application, as high survival rates (> 95%) and pregnancy rates continue today.

Single center validation of routine blastocyst biopsy implementation.

PURPOSE: The study aims to contrast the efficacy of trophectoderm biopsy preimplantation genetic screening (PGS)/vitrification (VTF)-all cycles to past treatment protocols. Specifically, do these applied technologies increase live birth rates on a per cycle/first transfer basis? MATERIALS AND METHODS: An observational, retrospective cohort study of first transfer outcomes was performed in two groups. Group 1 (PGS) included PGS/VTF-all cycles, and group 2 (no PGS) included the first transfer from non-PGS fresh cycles or VTF-ALL cycles. In group 1, all blastocysts were biopsied on days 5/6, vitrified and array CGH performed. Group 2 patients had embryo transfers on day 3 or day 5. All blastocysts were vitrified and warmed according to µS-VTF protocols. Clinical pregnancies and implantation were confirmed by ultrasound and live birth information attained. Results were stratified by age with donor cycles excluded, and to eliminate bias, the same groups were then validated on a per cycle basis. Chi-squared used to determine significance. RESULTS: Analyzing 287 embryo transfers and 1,000+ PGS-tested blastocysts, an overall 97 % increase in live births favored group 1 (PGS). When utilizing PGS/VTF-ALL cycles, patients under 43 years old exhibited higher implantation, clinical pregnancy, and ongoing/live birth rates. Re-analyzing the data to include all cycles initiated revealed higher live birth rates in group 1 age groups ≤34 and 38-40 years old. CONCLUSION: Validating PGS on a per cycle basis eliminated data bias by including patients without blastocysts to biopsy or euploid embryos. Clearly, PGS uses blastocysts more efficiently to achieve success, while many women over 40 may benefit most by understanding why some failures occur. SUPPORT: None.

Validation of microSecure vitrification (µS-VTF) for the effective cryopreservation of human embryos and oocytes.

A novel, aseptic closed system vitrification (VTF) technique for the cryopreservation of embryos and oocytes has been developed and clinically validated in this study. It combines the practicality of embryo-containing sterile flexipettes stored safely and securely with 0.3 ml CBS™ embryo straws possessing weld seals. The cooling and warming rates of this double container system were determined using a data logger. Upon direct plunging into LN(2), the flexipettes cool at an average rate of 1391°C/min, while warming occurs at an average rate of 6233°C/min in a 37°C 0.5 M sucrose bath. Direct deposition of the flexipette into a warming bath insured a rapid transition between -100 and -60°C to minimize potentially harmful recrystalization associated with devitrification. In conclusion, the µS-VTF system has exhibited higher (p<0.05) intact survival, implantation and live birth rates than conventional slow freezing methods. The effective embryo transfer of vitrified blastocysts proved similar to or better than fresh embryo transfer outcomes. The sustained clinical use of µS-VTF has justified a change in our infertility practice. Capsule: The microSecure vitrification (µS-VTF) procedure is a low-cost, non-commercial, aseptic, closed system that offers technical simplicity and repeatability, while effectively attaining an estimated 4:1 warming-to-cooling rate ratio, which supports excellent embryo survival and sustained viability.

Potential risk of monochorionic dizygotic twin blastocyst formation associated with early laser zona dissection of group cultured embryos.

OBJECTIVE: To document the risk of in vitro monochorionic dizygotic twin formation in the implementation of a program of blastocyst biopsy with preimplantation genetic screening (PGS). DESIGN: Case report. SETTING: Private infertility laboratory. PATIENT(S): Prospective PGS patients with intracytoplasmic sperm injection-derived, group-cultured blastocysts over a 3-year period. INTERVENTION(S): Group culture in Global medium (Life Global) to optimize blastocyst formation of zygotes produced for blastocyst biopsy for PGS (n ≤ 8 embryos/25 µL droplet), and laser zona dissection (LZD) of all day-3 cleaved embryos to promote pre-expansion trophectodermal extrusion at the blastocyst stage (i.e., premature hatching). MAIN OUTCOME MEASURE(S): Blastocyst formation and quality grading on days 5 and 6 of in vitro culture for the vitrified embryo transfer of single or dual euploid blastocysts. RESULT(S): Over 3,000 blastocysts were produced in vitro. On two separate occasions, complete trophectodermal amalgamation was observed between two hatching blastocysts. Vitrified single-euploid blastocyst transfers efficiently implanted and established clinical pregnancies similar to dual-euploid blastocyst transfers, without the risk of twin formation. CONCLUSION(S): The amazing occurrence of monochorionic dizygotic twin formation has now been documented in vitro, supporting the theory that assisted reproductive technology may facilitate this rare perinatal condition. Furthermore, we have provided clinical evidence that the transfer of a single-euploid blastocyst can optimize a patient's pregnancy success while reducing potentially undesirable conditions associated with monochorionic twin pregnancies.