Tao et al. Reproductive Biology and Endocrinology (2020) 18:17 https://doi.org/10.1186/s12958-020-00580-5 REVIEW Open Access Human sperm vitrification: the state of the art Yong Tao*, Erika Sanger, Arpornrad Saewu and Marie-Claude Leveille Abstract Sperm cryopreservation has been widely used in assisted reproductive technology (ART) and has resulted in millions of live births. Two principal approaches have been adopted: conventional (slow) freezing and vitrification. As a traditional technique, slow freezing has been successfully employed and widely used at ART clinics whereas the latter, a process to solidify liquid into an amorphous or glassy state, may become a faster alternative method of sperm cryopreservation with significant benefits in regard to simple equipment and applicability to fertility centers. Sperm vitrification has its own limitations. Firstly, small volume of load is usually plunged to liquid nitrogen to achieve high cooling rate, which makes large volume sample cryopreservation less feasible. Secondly, direct contact with liquid nitrogen increases the potential risk of contamination. Recently, new carriers have been developed to facilitate improved control over the volume and speed, and new strategies have been implemented to minimize the contamination risk. In summary, although sperm vitrification has not yet been applied in routine sperm cryopreservation, its potential as a standard procedure is growing. Keywords: Cryopreservation, Vitrification, Contamination, Liquid nitrogen, Spermatozoa, Semen Background births from frozen sperm after 4 decades storage in LN2 Over 8 decades ago, in 1938, Luyet and Hoddap per- had been reported [6]. Cryopreservation of spermatozoa formed the first vitrification of frog sperm in liquid air has been the most valuable and used way to preserve the [1]. Polge et al. [2] at the National Institute for Medical fertility of males, including those undergoing chemother- Research in London, England claimed “Revival of sperm- apy or radiotherapy, those with severe oligospermia or atozoa” after they successfully froze sperm samples from ejaculatory disorder, and those with disorders leading to various species with glycerol [2]. Sperm cryopreservation testicular damage. has been widely used in assisted reproductive technology Currently, there are two principal methods applied to (ART) programs, including infertility treatment and can- the cryopreservation of human sperm, namely, conven- cer patient fertility preservation. Its application can be a tional freezing and vitrification. Conventional freezing, therapeutic solution for patients with male infertility. slow freezing, a traditional technique, has been success- The first attempt using vitrified sperm for human live fully employed for the cryopreservation of human sperm birth was reported in 1953 by two researchers from State many times in the past. In contrast, vitrification is a University of Iowa, using sperm frozen on dry ice [3], novel technique, and has been a quickly growing alterna- and soon after the publication, normal live birth was tive method of rapid freezing. declared. About one decade later, the same group tried Traditional cryopreservation techniques are still widely with liquid nitrogen (LN2) and succeeded [4, 5]. Live used at ART clinics all over the world, but vitrification has become more effective for cryopreserving human * Correspondence: [email protected] spermatozoa after the efforts in past decade [7]. Vitrifi- Ottawa Fertility Center, 100-955 Green Valley Crescent, Ottawa, ON K2C 3V4, cation, by directly plunging the sperm samples into LN2, Canada © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tao et al. Reproductive Biology and Endocrinology (2020) 18:17 Page 2 of 10 is a fast, simple and cost-effective method to cryopreserve into washed and unwashed halves, and each group was human spermatozoa. This method of rapid freezing causes split into two aliquots: one group cryopreserved by con- no damage from intracellular ice crystallization during ventional freezing while the other by vitrification. It was cooling. Live births were achieved with vitrified spermato- demonstrated that conventional freezing resulted in zoa by intracytoplasmic sperm injection (ICSI) [8]andby higher motility and viability, while spermatozoa under- intrauterine insemination [9]. A healthy birth using a vitri- going vitrification were healthier regarding morphology fied sperm sample was recently reported in Spain after a and had fewer defects of sperm head, midpiece and tail Day 5 blastocyst was monitored and transferred [10]. [32]. Very recently, Pabon et al. [33] used 47 human Cryoinjury, also called cryodamage, is the damage of sperm samples to compare the efficiency of vitrification cryopreserved biological materials due to water phase and conventional freezing protocols. They found vitrifi- changes at low temperatures. The mechanisms involve cation was optimal for sperm cryopreservation as vitrifi- osmotic rupture by extra- or intra-cellular ice formation. cation protocol resulted in better motility recovery and Cell viability largely depends on the integrity of plasma higher mitochondrial activity [33]. Similar results were membrane as well as the cellular organelles inside [11]. reported previously by other researchers [22, 24, 25, 27]. The cooling velocities also affect the physicochemical A recent study recruited 20 subfertile men with semen and biophysical reactions, and thus alter the survival. characteristics of severe oligoasthenozoospermia to com- The emergence of proteomics and transcriptomics may pare the effects of two approaches and found that the provide some inspiration to understand cryoinjury vitrification method using only nonpermeable cryopro- mechanisms [12–14]. tectants was an effective alternative to the conventional Another perspective was raised to elucidate the mecha- slow-freeze technique [31]. Using epididymal and tes- nisms of cryoinjury using a fish spermatozoa model. It is ticular spermatozoa, Epis et al. also demonstrated that known that mitochondrial membrane potential changes vitrification resulted in higher mitochondrial membrane occur during cryopreservation, and these changes reflect potential and motility than conventional freezing [34]. the functional normality of cryopreserved spermatozoa. In order to compare the effectiveness of two methods, it Compared to human spermatozoa, fish spermatozoa have is also legitimate to review and summarize the previously lower mitochondrial membrane cryostability. Therefore, published and related articles. Recently, a systematic re- fish spermatozoa can be used as a model to investigate view and meta-analysis was undertaken to compare these cryostability in human spermatozoa damage [15, 16]. methods [35]. The authors reviewed a total of 2428 pub- lished articles and 13 randomized controlled trials includ- Is vitrification superior to conventional freezing? ing 486 vitrified and 486 conventional cryopreserved Sperm vitrification has been proposed as an alternative sperm samples. They concluded that vitrification was su- after the efforts in past years [7]. Human embryo cryo- perior to conventional freezing in post-thaw motility, in- preservation which has been well established may pro- cluding both total motility and progressive motility, vide some clues for sperm cryopreservation. Reed et al. although the efficacy of vitrification varied by vitrification [17] reviewed laboratory and clinical data for all frozen protocol and sample quality [35]. embryo replacement cycles from 2012 to 2015 and con- Collectively, human sperm vitrification has shown in- cluded that vitrification was more effective, as indicated creasing potential although the procedure needs further by higher survival in vitrified embryos compared to optimization as discussed later. slow-cooling cryopreservation. Furthermore, vitrification is also reliable and simple to learn and implement in the Cryoprotectants and cryodevices in sperm vitrification laboratory while clinical pregnancy and implantation Different from embryo vitrification, sperm vitrification with rates outcomes are similar [17], which is supported by highly concentrated, permeable cryoprotectants is not suit- other studies have [18–20]. able for spermatozoa because mammalian spermatozoa ex- One way to address whether vitrification is superior to posed to hypertonic conditions during cryopreservation conventional freezing is to directly compare the two results in osmotic shock, and causes the sperm tail