AAA Mini-Research Series – Part 3
Introduction
Cryopreservation has become a cornerstone of assisted reproductive technology (ART), allowing IVF laboratories to “stop biological time” by freezing sperm, oocytes, and embryos for future use (Ayeni et al, 2023). The term vitrification comes from the Latin vitreum (“glass”) and describes ultra-rapid freezing that solidifies cells into a glass-like state without ice crystal formation (Sofi et al., 2022). Since the first successful birth from a frozen embryo in 1983 and the first pregnancy from a frozen oocyte in 1986, interest in cryopreservation techniques has grown tremendously (Sofi et al., 2022). Over the past few decades, innovations in cryopreservation – especially the shift from slow programmable freezing to vitrification – have dramatically improved survival and pregnancy outcomes in IVF. This article reviews global innovations in cryopreservation and vitrification, highlighting how they enhance clinical outcomes for oocytes, embryos, and sperm. We also discuss advances in cryoprotectants, carrier devices, freezing equipment, and quality control systems, and consider how these innovations can be adopted in Nigeria’s IVF market.
From Slow Freezing to Vitrification: A Paradigm Shift
Traditional slow freezing (SF) gradually cools cells in programmable freezers, using lower concentrations of cryoprotectants and controlled ice formation. In contrast, vitrification employs high cryoprotectant concentrations and extremely rapid cooling, avoiding ice crystal formation entirely (Sofi et al., 2022). Vitrification emerged as a game-changer because ice crystals in slow freezing can damage cell structures, whereas vitrification’s glass-like solidification preserves cellular integrity. Clinical evidence strongly favors vitrification for oocytes and embryos – for example, randomized trials found significantly higher clinical pregnancy rates with oocyte vitrification compared to slow freeze (relative risk ~3.9) (Behnke et al., 2019). As a result, vitrification has largely replaced slow freezing worldwide for eggs and embryos due to superior survival and implantation outcomes (Behnke et al., 2019). This advance opened new opportunities in ART, including elective egg banking for fertility preservation and “freeze-all” IVF cycles where all embryos are vitrified for later transfer (Practice Committees of ASRM & SART, 2021). By improving post-thaw viability, vitrification enables safer single embryo transfers (minimizing multiples) and helps avoid ovarian hyperstimulation syndrome by deferring embryo transfer (Nordica, n.d.). In summary, the shift to vitrification is an essential innovation that underpins many modern IVF strategies.
Innovations in Cryoprotectants
Cryoprotectants (CPAs) are chemicals that protect cells from freezing injury. Traditional protocols use a combination of permeating CPAs (like dimethyl sulfoxide DMSO, ethylene glycol EG, or glycerol) and non-permeating CPAs (like sucrose) to dehydrate cells and prevent ice formation. Innovations in CPA formulations focus on maximizing protection while minimizing toxicity. One strategy is combining multiple CPAs at lower individual doses, which reduces toxicity compared to a single high-concentration CPA (Sofi et al., 2022). For example, human oocyte vitrification solutions often use EG + DMSO together with sucrose, each at moderate levels, to balance efficacy and safety. Another advance is the use of sugar molecules like trehalose as alternatives to sucrose. Trehalose has high glass-forming ability and membrane-stabilizing properties. A recent study comparing trehalose-based vs. sucrose-based vitrification media for human blastocysts found that trehalose significantly improved outcomes – the trehalose group had higher post-warming implantation rates (52.8% vs. 43.9%) and more high-quality embryos (Kim et al., 2025). This suggests that optimizing non-permeating CPAs can enhance embryo viability. Additionally, many vitrification media now include macromolecules such as hydroxypropyl cellulose (HPC) or human serum albumin (HSA) to osmotically buffer and protect cells during CPA exposure (Kitazato, 2013). Overall, modern cryoprotectant innovations aim to reduce toxicity, prevent osmotic shock, and improve post-thaw survival, thereby increasing the safety and efficacy of cryopreservation.
Advances in Carrier Devices and Vitrification Tools
A critical innovation in vitrification has been the development of specialized carrier devices that achieve ultrafast cooling by minimizing solution volume. Vitrification devices are broadly classified as “open” or “closed” systems depending on whether the sample contacts liquid nitrogen (LN₂) directly. Open carriers maximize cooling rates but expose samples to LN₂, whereas closed carriers avoid direct contact to improve sterility (at some cost to cooling speed) (Sofi et al., 2022).
Cryotop: Among the open systems, the Cryotop is the most widely used micro-volume device. It consists of a narrow thin plastic strip attached to a handle, on which a tiny droplet (<0.1 μL) containing the embryo or oocyte is loaded and vitrified. By drastically reducing volume, Cryotop achieves extremely high cooling (~23,000°C/min) and warming (~42,100°C/min) rates. This innovation has translated into outstanding clinical results – Cryotop vitrification routinely yields oocyte survival rates >90%, with one study reporting 99.4% survival of warmed oocytes along with healthy live births (Antinori et al., 2007). Cryotop has been credited with the highest number of IVF births worldwide from frozen eggs/embryos (Sofi et al., 2022), and it exemplifies how engineering a thin-film carrier can virtually eliminate ice crystal risk.
CryoLoop: Another pioneering device is the CryoLoop, a small nylon loop (0.7–1.0 mm) on a steel handle. The loop is dipped in a thin film of cryoprotectant solution and the sample is placed on the loop, which is then plunged into LN₂ inside a cryovial (Sofi et al., 2022). The CryoLoop’s design (no insulating straw wall and very low volume) allows cooling rates above 10,000°C/min. Studies demonstrated successful mouse and human blastocyst vitrification with CryoLoop, with human fertilization rates around 74% in one report. This device proved that even a simple loop of nylon could facilitate vitrification, and it has been used for embryos, oocytes, and even sperm.
Closed Systems: To improve biosecurity, various closed vitrification devices have been introduced. For example, the CryoTip is a thin plastic capillary straw that is heat-sealed before immersion in LN₂, preventing direct LN₂ contact. Similarly, High Security Vitrification (HSV) straws and VitriSafe devices encase the sample in a sealed container throughout cooling and storage (Sofi et al., 2022). These closed tools slightly reduce cooling/warming rates but mitigate contamination risk. Innovations have focused on narrowing the carrier walls to regain thermal speed – e.g., the Cryotop-SC (closed version of Cryotop) uses a sealed straw over the Cryotop strip. A trial comparing Cryotop-SC to the open Cryotop found the closed system’s oocyte survival was ~87.3% vs. 91.7% for the open device. Notably, fertilization and pregnancy rates were equivalent in both groups (Kitazato, 2013), indicating that well-designed closed systems can approach the efficacy of open carriers while providing safer, aseptic storage. Another novel carrier is the Kitasato Vitrification System, which incorporates a porous membrane to absorb excess fluid from the sample, further reducing vitrification volume (Sofi et al., 2022). This approach of solution absorbance is an innovation to achieve ultra-rapid cooling without direct LN₂ exposure. In practice, labs choose a device balancing maximum survival with logistical considerations like ease of use and regulatory compliance. The broad range of vitrification carriers – from open Cryotops and open-pulled straws to closed sealed systems – reflects ongoing innovation to optimize cooling rates and biosafety in parallel.
Programmable Freezers and Automation
While vitrification now dominates embryo and oocyte cryopreservation, programmable freezers remain relevant, especially for sperm banking and certain tissues. Programmable freezing machines allow precise control of cooling rates (e.g., 1–2°C per minute) and ice nucleation steps, which was critical for earlier embryo freezing protocols. Modern controlled-rate freezers have improved electronic controls, programmability, and consistency. For instance, current freezer models can store custom cooling programs and interface with lab information systems for monitoring. These ensure reproducible slow-freeze processes for cases where vitrification is not applied (such as some large tissue specimens or research protocols). That said, the clear trend in ART has been toward faster cooling methods for better viability.
An exciting recent innovation is the automation of the vitrification process itself. Traditionally, vitrification is a manual, operator-dependent skill – the embryologist must rapidly transfer embryos through CPA solutions and onto carriers within minutes. To improve standardization, semi-automated vitrification devices have been developed. One example is the Biorocks vitrification system, a compact instrument that automates CPA loading and removal using a hydrogel-based platform (Weng et al., 2025). In a 2024 study, the Biorocks system could process 36 embryos/oocytes per hour with survival outcomes equivalent to manual Cryotop vitrification – mouse oocyte survival was 98% with Biorocks vs 95% with manual method, and human blastocyst re-expansion ~92% vs 90%. The device achieved cooling rates (~31,900°C/min) on par with conventional tools (Antinori et al., 2007). Such automation can increase throughput and reduce variability due to operator skill, which is especially valuable in high-volume labs. Another emerging approach is microfluidic systems that automate CPA equilibration for oocytes, precisely controlling timing and concentration changes (sciencedirect.com, 2025). Programmable microdroplet vitrification and in-straw dilution techniques are also being refined to simplify and shorten warming protocols. In summary, beyond just hardware for slow freezing, the IVF lab is seeing a wave of automation and digital control in cryopreservation – from smarter freezers to robotics-assisted vitrification – all aimed at improving consistency and outcomes.
Quality Control Systems in Cryostorage
Innovations in cryopreservation extend to maintaining sample quality during long-term storage. IVF labs now recognize that rigorous quality control (QC) and monitoring of cryostorage is vital for patient safety. Traditional practice relied on simple stainless-steel LN₂ dewars that require manual refilling. Today, many laboratories employ large-capacity LN₂ tanks (160–1000 L) equipped with computerized monitoring, temperature sensors, and auto-fill systems (Behnke et al., 2019). These systems continuously track LN₂ levels and can automatically replenish LN₂ from a bulk source, drastically reducing the risk of accidental warming due to missed refills. Alarm and remote alert features are standard, so staff are notified immediately of any temperature deviations or low LN₂ levels. Such technology upgrades were prompted by well-publicized tank failures; consequently, best-practice guidelines now emphasize comprehensive risk management for cryostorage (Behnke et al., 2019).
Another aspect of quality control is ensuring aseptic conditions and traceability. Many IVF labs have implemented double witnessing and electronic tracking for cryo samples (barcodes or RFID tags on straws) to prevent mix-ups. Additionally, regular QC checks involve verifying freezer accuracy, LN₂ tank vacuum integrity, and performing test thaws of samples or vial temperature mappings to ensure storage conditions remain optimal (Vitrolife, 2018). Quality management systems (QMS), such as ISO 15189 or specific tissue bank accreditations, provide frameworks for IVF centers to document and audit all cryopreservation procedures. In Nigeria, where formal ART regulations are still developing (Ayeni et al., 2023), IVF clinics can voluntarily adopt such international QC standards to build trust and ensure sustainability. By investing in state-of-the-art storage tanks, alarm systems, and staff training in cryo handling, clinics can safeguard the precious reproductive materials entrusted to them.
Clinical Outcomes and Efficacy of Modern Techniques
The ultimate measure of any cryopreservation innovation is its impact on clinical outcomes – namely, cell survival after thaw, fertilization competence, embryo development, and pregnancy rates. Modern vitrification techniques have achieved remarkable success in this regard. With optimized protocols, post-thaw survival rates for vitrified embryos and oocytes typically range from 80–95%. For example, using the Cryotop method, human oocytes can reach >90% survival after warming (Kitazato, 2013), and one report documented 328 out of 330 vitrified oocytes surviving (99.4%) (Antinori et al., 2007). Embryos fare similarly well: blastocysts vitrified on open devices often re-expand and continue developing at rates comparable to fresh embryos (Sofi et al., 2022). In contrast, slow-frozen embryos and oocytes historically had lower survival (often 50–75%), underscoring how far outcomes have improved. High survival directly translates to higher cumulative pregnancy and live birth rates. Clinical studies have found that vitrified oocytes yield pregnancy rates approaching those of fresh eggs, enabling effective oocyte donation and fertility preservation programs (Behnke et al., 2019). For blastocysts, vitrification has been shown to maintain implantation potential – one analysis noted no significant difference in implantation or birth rates between fresh vs. vitrified blastocysts in IVF, validating the safety of freezing embryos. Sperm cryopreservation is a more mature field; even with slow freezing, sperm survival and functionality post-thaw are usually good, though newer methods (like sperm vitrification on specialized carriers) are being explored to further improve motility and DNA integrity (sciencedirect.com, 2025).
Crucially, the consistency of outcomes has improved with innovation. Advanced carrier devices and CPA protocols yield reliable results with minimal variability, and improved QC means fewer catastrophic losses. This consistency allows IVF clinicians to confidently incorporate cryo-strategies (freeze-all cycles, egg banking, etc.) knowing that frozen-thawed gametes and embryos will perform well. Efficacy metrics such as survival rate, fertilization rate, blastulation rate, and pregnancy per thawed embryo are now commonly tracked in IVF lab KPIs, reflecting how cryopreservation success directly affects clinical success. In summary, innovations in vitrification and cryopreservation have led to excellent survival and reproductive outcomes, effectively making frozen embryos and oocytes nearly as viable as fresh ones in many scenarios. This enhances patient options and optimizes the overall efficiency of IVF treatments.
Adopting Innovations in Nigeria’s IVF Market
Nigeria’s IVF sector is growing rapidly, driven by rising infertility awareness and demand for advanced fertility services. To remain competitive and improve success rates, Nigerian IVF laboratories and clinics can adopt the latest cryopreservation innovations described above. First, transitioning fully from any remaining slow-freeze methods to vitrification for oocytes and embryos is paramount. Many leading clinics in Nigeria already offer vitrification-based egg, embryo, and sperm banking (Nordica, n.d.). Ensuring embryologists are well-trained in vitrification techniques (e.g. via workshops or certification programs) will help standardize outcomes. Collaborating with global suppliers of cryo equipment is also key – for instance, importing quality-assured carrier devices (Cryotop kits, closed straw systems, etc.) and vitrification media from reputable manufacturers (Kitazato, Vitrolife, CooperSurgical, etc.) can bring world-class capabilities to Nigerian labs.
Another consideration is the local infrastructure and regulatory context. Reliable supply of liquid nitrogen and lab facilities to maintain ultra-low temperatures are crucial; clinics should invest in adequate LN₂ storage capacity, back-up tanks, and generators to safeguard against power or supply disruptions. Given that Nigeria currently lacks specific legislation on embryo cryopreservation (Ayeni et al., 2023), IVF providers must self-impose strict ethical and quality standards – for example, clear patient consent for storage duration, protocols for discard or donation of unused embryos, and adherence to global guidelines (ASRM, ESHRE) for cryo practices. Adopting a robust quality management system will enhance consistency; Nigerian centers can work toward certifications or accreditation that benchmark their cryo lab practices against international norms.
From a strategic perspective, embracing cryopreservation innovations will improve clinical outcomes (higher pregnancy and live birth rates), thereby boosting clinic reputations and patient confidence. It also opens avenues for service expansion – fertility preservation services for cancer patients or elective egg freezing for career women are still emerging in Nigeria but have substantial potential (Nordica, n.d.). By offering state-of-the-art vitrification, Nigerian clinics can cater to these needs, contributing to the sustainability and growth of the IVF market. Furthermore, equipment suppliers and biomedical firms have an opportunity to partner with local providers to customize solutions (for example, affordable yet effective LN₂ monitoring systems suitable for the region). In essence, contextualizing global innovations to Nigeria involves not just acquiring technology but also training personnel, educating patients, and establishing frameworks that ensure these advancements are used safely and effectively for the benefit of patients.
Conclusion
Innovations in cryopreservation and vitrification techniques have revolutionized IVF laboratory practice. The evolution from slow freezing to rapid vitrification, improvements in cryoprotectant formulations, introduction of high-performance carrier devices, and advances in automation and quality control have collectively enhanced the viability of frozen-thawed gametes and embryos. These technical strides translate into tangible clinical benefits: higher survival rates, improved pregnancy outcomes, and more flexible, patient-friendly treatment options. For IVF professionals and fertility clinicians, staying abreast of these developments is crucial for maintaining high success rates and expanding fertility services. Equipment suppliers also play a role in delivering the tools and training needed to implement cutting-edge cryotechniques. Ultimately, embracing these innovations – and adapting them to local contexts such as Nigeria’s emerging IVF market – will promote sustainable IVF practice, enabling clinics to safeguard fertility for the future while achieving excellent clinical outcomes today.
Photo Credit: https://www.irvinesci.com/
References
- Antinori, M., Licata, E., Dani, G., Cerusico, F., Versaci, C., & Antinori, S. (2007). Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries. Reproductive Biomedicine Online, 14(1), 72–79pubmed.ncbi.nlm.nih.gov.
- Ayeni, J. V., Omidoyin, T. J., & Bamidele, I. (2023). Cryopreservation of human embryos in an in-vitro fertilisation procedure: The need for a legal framework in Nigeria. F1000Research, 12, 569f1000research.com.
- Behnke, E. J., Schiewe, M. C., et al. (2019). Comprehensive assessment of cryogenic storage risk and quality management concerns: Best practice guidelines for ART labs. Journal of Assisted Reproduction and Genetics, 36(1), 5–14pmc.ncbi.nlm.nih.govpmc.ncbi.nlm.nih.gov.
- Kim, H. R., Kwon, W. H., Choi, E. J., Kim, M. H., Choi, E., Kim, H., … & Kong, I. K. (2025). Comparison of a commercial trehalose-based vs. sucrose-based vitrification solution for human blastocysts: Effects on outcomes. Middle East Fertility Society Journal, 30(11)mefj.springeropen.com.
- Kitazato. (2013). Cryotop-SC results (closed system) [Company news release]. Kitazato IVF. (Reported 87.3% vs 91.7% oocyte survival in closed vs open Cryotop)kitazato-ivf.com.
- Nordica Lagos. (n.d.). Not ready? Preserve your fertility for the future [Web page]. Nordica Fertility Center Lagosnordicalagos.orgnordicalagos.org.
- Practice Committees of ASRM & SART. (2021). Best practices for rapid-cooling vitrification of oocytes and embryos: A committee opinion. Fertility and Sterility, 115(2), 305–310rbmojournal.com.
- Sofi, K. A., Khatun, A., & Yousuf, N. (2022). Recent advancements in vitrification cryodevices for gamete and gonadal tissue. CryoLetters, 43(3), 127–139ingentaconnect.comingentaconnect.com.
- Wang, S., Chen, L., Fang, J., & Sun, H. (2024). A compact, high-throughput semi-automated embryo vitrification system based on hydrogel. Reproductive Biomedicine Online, 48(5), Article 103769pubmed.ncbi.nlm.nih.govpubmed.ncbi.nlm.nih.gov.
- Vitrolife Blog (2018). How to monitor liquid nitrogen storage tanks in IVFasrm.orgvaisala.com;
- Kitazato IVF (2017). Vitrification media: How to guarantee safety in the lab?kitazato-ivf.com (discussing HSA and HPC in media). These complement the scholarly references by highlighting practical quality control measures and media composition considerations in IVF labs.