Abstract Study question To date, no publications exist describing Blockchain in IVF (Hickman, 2020). Can Blockchain technology be used for traceability and accountability in IVF laboratory procedures? Summary answer Blockchain is an easy-to-implement technology for incorruptible traceability of a “Virus-Free” vitrification/warming procedure. What is known already In healthcare, Blockchain can become a tool to address challenges regarding sensitive data-sharing and traceability of medical and laboratory procedures. During the Covid-19 pandemic, many authors warned about the role of LN2 as a potential vector for virus contamination, and vitrification and warming were identified as critical procedures for risk of contamination for environments, surfaces, operators and cells. In this study we describe the first application of Blockchain in IVF for incorruptible traceability of a “Virus-Free” vitrification/warming procedure based on the combined use of UVC-Sterilized Liquid Nitrogen (LN2) and CE Medical Devices (CE-MD). Study design, size, duration Report on 2346 Ethereum Blockchain data transactions for IVF laboratory procedures mined from 01/10/2019 to 31/12/2021. The procedures were oocyte/embryo vitrification, warming or handling in LN2 after cryopreservation. For each vitrification, warming and handling a UVC-sterilized batch of LN2 was associated with the code assigned to the vitrification/warming procedure and with the lot number of the single-use sterile vitrification box (N-Sleeve). The clinical results obtained from warmed oocytes/embryos were observed as completion of this process. Participants/materials, setting, methods A Blockchain trusted “Virus-Free” vitrification/warming programme was set up using a specifically designed CE-MD N-Bath-System (Nterilizer-Italy). Each procedure was traced by the CE-MD’s software and a dedicated web application. Finally, data were made incorruptible by Ethereum Blockchain transactions. Before oocyte/embryo warming, vitrification carrier washing with UVC-sterilized LN2 was performed in accordance with Parmegiani et al (2012) and recent international anti-Covid guidelines. Main results and the role of chance Of the 2346 Blockchain transactions 1268 regarded vitrification and cryopreserved specimen handling procedures; 1078 transactions were frozen cell warmings (308 oocytes and 770 embryos) performed on 799 patients. To date, 445 pregnancies have been obtained (pregnancy rate: 41% per cycle; 56% per patient) and 219 babies have been born. Limitations, reasons for caution Ethereum is a decentralized, open-source blockchain with smart contract functionality. Ether is the native cryptocurrency which is highly susceptible to cost changes. Other public or private Blockchains may be used in future in healthcare with more stable transaction costs. Wider implications of the findings This is the first evidence of the application of Blockchain in IVF and many others will probably follow. Blockchain immutable records of LN2 sterilization combined with procedure codes and disposable lots represent incorruptible traces for “Virus-Free” vitrification/warming. During this pandemic 219 babies were born from cryopreservation procedures powered by Blockchain. Trial registration number Not Applicable

To investigate the clinical efficacy of a “Universal Warming” protocol, based on subsequent steps with 1 M and 0.5 M concentration of extracellular cryoprotectant (ECCP), on shipped oocytes. Oocytes are vitrified using different brands of ready-to-use kits which recommend that the use of their own warming kit and combining different vitrification/warming kits may have legal consequences for assisted reproductive (AR) centers, until this practice has been validated with clinical studies. Retrospective multi-center transnational observational study. Number of oocytes warmed 1.898. Vitrification performed with vitrification kit (Kitazato, Japan); warming carried out randomly with two different kits: Kitazato warming kit and Vit Kit®-Thaw (FujiFilm Irvine, USA). Warmed oocytes were assigned to 2 groups: KK (Kitazato/Kitazato) 939, and KI (Kitazato/Irvine) 959. Primary endpoint: survival rate. Secondary endpoints: fertilization rate; blastulation rate; implantation rate; live birth rate. Survival was comparable between the groups: 84.6% (795/939) in group KK vs 82.1% (787/959) in group KI. Fertilization rate was lower (P = 0.027) in group KK (75.7%—602/795) than in group KI (80.4%—633/787). Blastulation and implantation and live birth rates were all statistically comparable between the study groups: blastulation rate was 58.5% (352/602) vs 57.8% (366/633); implantation rate was 41.5% (80/193) vs 45.9% (84/183); live birth rate was 52.5% (62/118) in KK and 45.0% (54/120) in KI. The use of this “Universal Warming” protocol simplifies vitrified oocyte exchange between AR centers in different countries, and overcomes potential regulatory/commercial/availability differences affecting clinical practice.

In previous pilot studies, we paved the way for the clinical use of a single "universal warming protocol" based on subsequent steps with 1 and 0.5 M of extracellular cryoprotectant (ECCP), irrespective of the cryopreservation method used for freezing. Nowadays, embryos are routinely frozen using different commercial brands of ready-to-use vitrification solutions which differ only slightly in their composition. This is the first large-scale clinical study to demonstrate that vitrification and warming solutions of different brands can be successfully combined on vitrified embryos. Retrospective longitudinal cohort study. 847 embryos frozen at cleavage stage obtained from patients' own oocytes. Duration: 01/01/2013 - 31/12/2015. Each patient's embryos were vitrified and warmed in various combinations by using three different kits: i) Vitrification Kit (Kitazato, Japan), ii) Sage Vitrification Kit (Origio, Denmark), iii) "home-made" in house in our laboratory. Vitrification/warming kits from different brands are routinely used in our center and warming procedures are randomly performed with any available kit on a "first-in-first-out" basis, irrespective of the brand used for vitrification. Group names: KK, KS, SK, SS, SH, HK, HS, HH. • KK - vitrification Kitazato / warming Kitazato • KS - vitrification Kitazato / warming Sage, • SK - vitrification Sage / warming Kitazato, • SS - vitrification Sage / warming Sage, • SH - vitrification Sage / warming "in house kit", • HK - vitrification "in house kit" / warming Kitazato, • group HS - vitrification "in house kit" / warming "Sage", • group HH -vitrification "in house kit" / warming "in house kit". No embryos were vitrified with Kitazato and warmed with "in house" kit. Primary endpoint: survival rate (number of embryos surviving per number of embryo warmed). Secondary endpoint: implantation rate (number of embryos implanted per number of embryos transferred). Female mean age and survival rate were statistically comparable between the study groups. Cryo-survival rate was: group KK 96.4% (54/56), group KS 100.0% (13/13), group SK 98.8% (80/81), group SS 97.2% (174/179), group SH 97.6% (40/41), group HK 95.2% (20/21), group HS 99.5% (187/188), group HH 97.4% (261/268). Implantation was generally comparable in all study groups - exceptions were KS 46.2% (6/13) vs. SK 17.5% (14/80 - P=0.049), SS 14.7% (24/163 - P=0.012), HS 14.5% (26/179 - P=0.010), HH 17.3% (45/260 - P=0.025); and SH 30.0% (12/40) vs. SS (P=0.042), HS (P=0.035). This study confirms that it is possible to combine different kits for vitrification/warming. Other large-scale multi-center studies are needed to confirm the implantation potential of these warmed embryos. The "universal warming protocol" (ECCP 1 - 0.5 M) permits warming of vitrified embryos, irrespective of brand, cryoprotectants and basic medium in the vitrification kit. The possibility to combine different vitrification/warming kits may favour embryo exchange between IVF centres.

To study the efficacy and efficiency of a “universal warming protocol” for vitrified human embryos, based on subsequent steps with 1 and 0.5 M concentration of extracellular cryoprotectant (ECCP).

Human embryos are vitrified and warmed with commercial kits. Although vitrification/warming solutions have only slight differences in their composition, it remains to be demonstrated that a warming solution of one brand can be used to thaw embryos vitrified with another. In a previous study, we warmed cryopreserved oocytes using a single protocol based on sequential steps with 1 and 0.5 M extracellular cryoprotectant (ECCP), irrespective of the freezing method used. The aim this study is to verify the efficiency of this "universal protocol" for vitrified embryos. Prospective randomized study on 163 embryos obtained from 99 infertile patients undergoing fertility treatments via ICSI. Inclusion criteria: female age at freezing ≤ 42, embryos not tested for preimplantation genetic screening. Each patient's embryos were first randomized for vitrification with two different kits: Vitrification Kit (Kitazato, Japan) and Sage Vitrification Kit (Origio, Denmark). At warming the embryos were randomly warmed either with Kitazato or Sage warming kit, specifically: • group A - 56 embryos vitrified with Kitazato and warmed with Kitazato, • group B - 55 embryos vitrified with Kitazato and warmed with Sage, • group C - 25 embryos vitrified with Sage and warmed with Kitazato, • group D - 27 embryos vitrified with Sage and warmed with Sage. Randomization performed with a specific software tool (http://www.randomizer.org). The study was closed as soon as the last group reached the minimum number of 25 embryos. Primary endpoint: embryo survival rate (number of embryos surviving per number of embryos warmed). Secondary endpoint: implantation rate (number of embryos implanted per number of embryos transferred). Trial registration number ISRCTN12342851. Kitazato kits contain Threalose as ECCP and are supplemented with Hydroxypropyl Cellulose (HPC). Sage kits contain Sucrose as ECCP and are supplemented with Human serum albumin (HAS). Both involve sequential steps with 1 and 0.5 M ECCP. Vitrification performed with Cryotop SC (Kitazato, Japan). Between-group differences assessed with χ2-method or with Fisher's exact test. Difference significant with P <0.05 Female mean age, survival and implantation rates were statistically comparable between the study groups. Survival rate: group A 100 % (56/56), group B 98.2% (54/55), group C 96.2% (25/26), and group D 96.3 % (26/27). Implantation rate: group A 21.4 % (12/56), group B 20.4% (11/54), 20% (5/25), and group D 19% (5/26) . Worldwide, billions of embryos have been frozen by using different vitrification kits. This study reveals that it is possible to combine Kitazato and Sage kits for vitrification and warming. The "universal warming protocol" based on 1 and 0.5 M of ECCP allows efficient thawing of vitrified embryos, irrespective of brand, cryoprotectants and basic medium contained in the freezing kit. This finding optimizes costs, simplifies lab routines and favours embryo exchange between IVF centers.

The present study reports, as far as is known for the first time, the safety of UV sterilization of liquid nitrogen and hermetical cryostorage of human oocytes by comparing the efficiency of fresh and vitrified sibling oocytes of infertile patients. A prospective randomized study on sibling oocytes of 31 patients was carried out. Metaphase-II oocytes were randomized for intracytoplasmic sperm injection and the supernumerary sibling oocytes were vitrified using a novel Cryotop aseptic procedure (UV liquid nitrogen sterilization and hermetical cryostorage). After unsuccessful attempts with fresh oocytes, vitrified sibling oocytes were injected. Mean outcome measures observed were fertilization, cleavage and top-quality embryo rates. No significant differences were observed between the fresh and vitrified–warmed sibling oocytes: oocyte fertilization was 88.3% versus 84.9%; cleavage 72.6% versus 71.0%; top-quality embryos 33.8% versus 26.3% and mean number of transferred embryos 2.6±0.1 versus 2.5±0.1, respectively. Clinical pregnancy rate per cycle with vitrified–warmed oocytes was 35.5% (implantation rate 17.1%) and seven healthy babies were born. This study demonstrated that UV liquid nitrogen sterilization and hermetical cryostorage does not adversely affect the developmental competence of vitrified oocytes, allowing safe aseptic open vitrification applicable under strict directives on tissue manipulation. Human reproductive cell vitrification can be performed with specific open carriers, which show high efficiency especially with oocytes. In vitrification with open carriers, the risk of contamination of the culture medium at warming through contact between the cells and/or the outer surface of the carrier cannot be excluded if the LN2 used for vitrification/storage is accidentally contaminated. With these carriers, an aseptic vitrification can be performed by sterilizing LN2 for the vitrification procedure and subsequently cryostoring the carriers inside hermetical containers. With the present study, we report, as far as is known for the first time, the safety of ultraviolet LN2 sterilization and hermetical cryostorage of human oocytes by comparing the efficiency of fresh versus vitrified sibling oocytes of infertile patients. A prospective randomized study on intracytoplasmic sperm injection cycles with fresh or vitrified–warmed sibling oocytes of 31 infertile patients was carried out. Mature metaphase-II oocytes were randomized for ICSI; the supernumerary sibling oocytes were vitrified using a novel Cryotop aseptic procedure (sterilization and hermetical cryostorage). No significant differences in the main outcome measures were observed between the fresh and vitrified–warmed sibling oocytes. The clinical pregnancy rate per cycle with vitrified–warmed oocytes was 35.5% (implantation rate 17.1%); resulting in the birth of seven healthy babyes. This study demonstrated that ultraviolet LN2 sterilization and hermetical cryostorage does not adversely affect the developmental competence of vitrified oocytes, allowing a safe aseptic open vitrification applicable under strict directives on tissue manipulation.