Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.
Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients’ future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.
The development and implementation of an efficient cryopreservation program for human oocytes has been a significant breakthrough in reproductive medicine. According to recent evidence, vitrification is the most effective strategy to cryopreserve metaphase II (MII) oocytes, as it results in statistically higher survival rates compared to slow freezing, independently of the patient population (infertile patients or oocyte donation program)1,2,3. The remarkable achievements of oocyte vitrification led the Practice Committees of the American Society for Reproductive Medicine (ASRM) and the Society for Assisted Reproductive Technology (SART) to pronounce this technique to be the most effective for elective fertility preservation in postpubertal women, for both medical and non-medical indications4,5,6. Medical indications for fertility preservation include (i) cancer and autoimmune diseases that require therapies7 such as radiotherapy, cytotoxic chemotherapy, and endocrine therapy (whose detrimental effect on the ovarian reserve is associated with maternal age as well as type and dose of the treatment); (ii) ovarian diseases requiring repeated or radical surgery (such as endometriosis)8; and (iii) genetic conditions (e.g., X-fragile) or premature ovarian failure. In addition, fertility preservation has become a valuable option for all women who have not accomplished their parental objective for non-medical reasons (also known as social freezing).
Regardless of the indication for fertility preservation and according to the major international guidelines on fertility preservation, all patients willing to vitrify their oocytes should receive appropriate counseling to be informed about their realistic chance of success, the costs, risks, and limitations of the procedure9,10,11,12,13. Most importantly, it should be clear that vitrifying a cohort of MII oocytes does not ensure a pregnancy, but that it offers a higher chance of success for future in vitro fertilization (IVF) treatment, if necessary14. In this regard, the woman's age at the time of oocyte vitrification is certainly the most important limiting factor15 as advanced maternal age (AMA; >35 years) is the main cause of female infertility16. Besides a progressive reduction in the ovarian reserve, AMA is associated with an impairment of oocyte competence due to defective physiological pathways such as metabolism, epigenetic regulation, cell cycle checkpoints, and meiotic segregation17. Therefore, the reasonable number of eggs to vitrify mainly depends on maternal age. In women younger than 36 years, at least 8-10 MII oocytes18 are required to maximize the chance of success. In general, the higher the number of vitrified oocytes, the higher is the likelihood of success. Therefore, tailoring ovarian stimulation according to ovarian reserve markers such as anti-Müllerian hormone (AMH) levels or antral follicle count (AFC) is crucial to fully exploit the ovarian reserve in the shortest possible time.
The safety of the whole procedure is the other key issue when enrolling patients for fertility preservation. Clinicians should employ the best strategies to minimize the risks and prevent (i)ovarian hyperstimulation syndrome (OHSS) by using safe approaches such as the gonadotrophin-releasing hormone (GnRH) antagonist protocol followed by a GnRH agonist trigger19 and (ii) the remote, yet possible, risks of peritoneal bleeding, injury to the pelvic structures (ureter, bowel, appendix, nerves), or pelvic infection during oocyte retrieval. Lastly, (iii) traditional regimens for stimulation are associated with supraphysiologic serum estradiol and therefore, are not recommended in estrogen-sensitive diseases such as breast cancer. Protocols involving aromatase inhibitors (such as letrozole or tamoxifen) are more suitable in these cases20,21. In the laboratory setting, the most widespread protocol for oocyte vitrification is still the one first described by Kuwayama and colleagues2,23, which consists of a stepwise procedure involving the gradual addition of cryoprotectants (CPAs). In the first phase (equilibrium/dehydration), oocytes are exposed in a CPA solution containing 7.5% v/v ethylene glycol and 7.5% v/v dimethyl sulfoxide (DMSO), while in the second phase, oocytes are moved to a vitrification solution with 15% v/v ethylene glycol and 15% v/v DMSO, plus 0.5 mol/L sucrose. After a short incubation in the medium of vitrification, the oocytes can be placed in specifically designed, open cryodevices and finally plunged in liquid nitrogen at -196 °C to be stored until use.
Here, the whole clinical and laboratory work-up of a fertility preservation treatment has been described by (i) outlining recommendations for objective and evidence-based counseling, (ii) focusing on the cost-effectiveness of the procedure, and (iii) describing the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures will be detailed along with approaches to maximize their efficacy, efficiency, and safety. As other protocols or adaptations of this protocol exist in the literature, the representative results and the discussion sections of this manuscript only apply to this procedure.
1. Work-up and clinical counseling
NOTE: In case of patients requiring fertility preservation for oncologic reasons, ensure that there is no waiting list for scheduling consultation, and the appointment is provided as soon as possible.
2. Controlled ovarian stimulation protocols for fertility preservation
NOTE: When the time available before starting the cancer treatment is limited, the random-start protocol (i.e., starting ovarian stimulation at any time during the menstrual cycle) is recommended for the ovarian stimulation in oncologic patients who are candidates for fertility preservation. In a fertility preservation program for non-urgent medical reasons or social reasons, conventional stimulation starting in the early follicular phase is preferable, and ovarian stimulation is started based on the menstrual cycle. Controlled ovarian stimulation (COS) approaches should be performed according to the recent European Society of Human Reproduction and Embryology (ESHRE) guidelines24.
3. Oocyte retrieval
4. IVF laboratory
5. Oocyte denudation
6. Oocyte vitrification
NOTE: Perform oocyte vitrification preferably within 38 h of oocyte retrieval and immediately after denudation. The vitrification procedure described here has to be accomplished at room temperature (RT) and by using a stripper pipette with an inner diameter of 170 µm so as not to damage oocytes during manipulation.
7. Oocyte warming
Overview of the fertility preservation program at the center
Over a 12-year period (2008-2020), 285 women underwent at least one oocyte retrieval entailing the vitrification of the whole cohort of mature eggs collected. Most of these women (n=250) underwent a single retrieval, and 35 underwent multiple retrievals. The reasons for undergoing oocyte retrieval for egg vitrification are summarized into 4 categories: medical (except for cancer), cancer, non-medical, and others. Among the 250 women undergoing a single oocyte retrieval for egg vitrification, 8% had medical reasons other than cancer (10 endometriosis, 3 myoma, 4 ovarian cysts, 1 hydrosalpinx), 16% had cancer (31 breast cancer, 3 ovarian cancer, 2 colorectal cancer, 2 Hodgkin's lymphoma, 1 vulvar cancer e 1 cervical cancer), 53% had non-medical reasons, and 23% had other reasons (43 absence of sperm retrieved, 10 risk for OHSS, 4 infections, and 1 fever). This distribution was different among patients undergoing multiple oocyte retrievals for egg vitrification. Specifically, 9% had medical reasons other than cancer (1 endometriosis, 2 reduced ovarian reserve), 6% had cancer (2 breast cancer), 80% had non-medical reasons, and 3% had other reasons (1 absence of sperm retrieved). None of the patients undergoing multiple oocyte retrievals for egg vitrification warmed those eggs, while 78 of the 250 women undergoing a single egg vitrification cycle returned to use those oocytes (Figure 3).
Table 1 summarizes the data of the 250 women undergoing a single oocyte vitrification cycle clustered according to the related reasons. The patients with a medical reason for egg vitrification and the patients undergoing fertility preservation because of cancer were younger (mean maternal age < 35 years) and showed a better ovarian reserve (higher AFCs) than the patients with non-medical or other reasons. However, the mean maturation rates (number of MII oocytes/number of COCs retrieved) were slightly lower (72-73% versus 77-79%), so that the number of oocytes vitrified on average was similar in the 4 groups (9-10 oocytes). Importantly, 9 out of 40 oncologic patients (22.5%) underwent a random-start ovarian stimulation protocol because they had limited time before starting chemo- or radiotherapy. It is interesting that approximately half of the patients with medical reasons other than cancer (53%) and the majority of patients with other reasons for egg vitrification (76%) actually returned for warming. Conversely, very few patients who underwent fertility preservation for cancer (17.5%) or non-medical reasons (13%) used their vitrified oocytes for IVF. Also remarkable is the time elapsed between vitrification and warming among the patients who returned: on average, 283 days in patients with medical reasons other than cancer, 132 days in patients with other reasons, 1264 days in oncologic patients, and 1547 days in patients with non-medical reasons. Regardless of all these relevant differences, the survival rate was similar (83-88%; on an average, 8-11 oocytes were warmed, and 7-9 oocytes survived) between the patients in the 4 groups, thereby confirming the efficacy and safety of oocyte vitrification and warming protocols. Moreover, the survival rate is independent of vitrification and the warming operator's experience (Figure 4A,B). Table 1 shows the fertilization rates in the 4 groups, which are ~70%, except for the patients with non-medical reasons for oocyte vitrification (~80%). However, these data are not comparable owing to a small sample size and the bias of the sperm factor on the fertilization outcome27.
Figure 1: Medium droplet configuration for oocyte cryopreservation. To gradually perform equilibration, oocytes are first placed in a (A) drop of BS and mixed with (B) a drop of ES. After 3 min of incubation, (C) a third drop of ES solution is mixed, and oocytes are incubated for 6-9 min. Abbreviations: HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BS = HEPES-buffered medium; ES = equilibration solution. Please click here to view a larger version of this figure.
Figure 2: Oocyte loading on cryopreservation device. The oocytes are placed on the cryodevice in (A) a single small drop of VS. (B) The stripper pipette is shifted away from the oocytes, and (C) the excess of VS is re-aspirated to leave just a thin layer around each oocyte. Abbreviation: VS = vitrification solution. Please click here to view a larger version of this figure.
Figure 3: Oocyte vitrification cycles performed at the GENERA center for reproductive medicine of Rome (years 2008-2020). Over the 12-year period, 250 patients underwent a single oocyte retrieval for oocyte vitrification, while 35 underwent multiple oocyte retrieval cycles. The inherent reasons for oocyte vitrification are shown in the figure. The patients returning for warming (n=78) belong only to the group of women who underwent a single oocyte vitrification cycle. Please click here to view a larger version of this figure.
Figure 4: Mean survival rate per cohort of warmed oocytes. (A) Vitrification and (B) warming operators' experience.Each patient is included only for the first warming cycle. Statistically significant differences were assessed using Mann-Whitney U-test. Please click here to view a larger version of this figure.
Figure 5: Oocyte morphology at the beginning and the end of the equilibration procedure. To determine the outcome of the equilibration procedure, it may be useful to annotate (A) oocyte morphology before starting the procedure. (B) A sharp shrinkage of the oocyte is observed after first exposure to the cryoprotectant solution. The equilibration procedure can be considered complete when (C) the oocyte has recovered its initial volume. Scale bars = 25 µm. Please click here to view a larger version of this figure.
Reason for oocyte vitrification | Medical other than cancer (n=19) | Cancer (n=40) | Non-medical (n=133) | Other (n=58) |
Maternal age at oocyte retrieval, mean±SD | 33.6±6.4 years | 34.6±5.9 years | 37.0±3.4 years | 38.2±4.3 years |
Basal FSH, mean±SD | 7.5±4.2 IU/L | 7.6±1.7 IU/L | 7.8±4.1 IU/L | 8.8±5.2 IU/L |
AMH, mean±SD | 1.8±1.7 ng/mL | 1.9±0.2 ng/mL | 2.2±2.4 ng/mL | 3.0±2.5 ng/mL |
AFC, mean±SD | 12.7±5.0 | 13.3±7.0 | 11.6±6.1 | 10.7±6.7 |
BMI, mean±SD | 20.1±1.5 | 21.8±1.6 | 20.2±3.1 | 22.9±3.6 |
Duration of stimulation, mean±SD | 10.0±1.5 | 9.6±1.7 | 9.9±2.1 | 10.3±2.1 |
9/40, 22.5% | ||||
Random start | ||||
COCs, total, mean±SD | 239 | 551 | 1493 | 592 |
12.6±5.3 | 13.8±9.1 | 11.2±7.1 | 10.2±8.5 | |
MII oocytes, total, mean±SD | 167 | 400 | 1161 | 462 |
8.8±3.3 | 10.0±7.2 | 8.7±5.7 | 8.0±6.7 | |
Maturation rate, mean±SD | 72.4±13.6% | 72.1±19.6% | 79.5±17.5% | 77.4±22.2% |
Patients returning for warming, total % of patients who underwent vitrification | 10/19, 52.6% | 7/40, 17.5% | 17/133, 12.8% | 44/58, 75.9% |
Days between vitrification and first warming, mean±SD | 283±225 | 1264±764 | 1547±709 | 132±203 |
Warmed MII oocytes, total mean±SD | 82 | 66 | 191 | 409 |
8.2±2.8 | 9.4±4.8 | 11.2±5.8 | 9.3±7.2 | |
Survived MII oocytes, total mean±SD | 73 | 55 | 160 | 345 |
7.3±3.3 | 7.9±4.0 | 9.4±4.8 | 7.8±6.9 | |
Survival rate, mean±SD | 87.6±18.1% | 83.2±1.7% | 85.4%±14.5% | 84.7±21.9% |
Sperm Factor | ||||
N, n, % | 5/10, 50% | 4/7, 57% | 13/17, 76% | 13/44, 29% |
MMF, n, % | 3/10, 30% | 3/7, 43% | 3/17, 18% | 17/44, 39% |
OAT, n, % | 2/10, 20% | – | 1/17, 6% | 11/44, 25% |
NOA, n, % | – | – | – | 3/44, 7% |
Fertilization rate, mean±SD | 70.0±14.4% | 70.0±17.0% | 78.5±20.6% | 71.0±24.3% |
Table 1: Patients undergoing a single cycle of oocyte vitrification. Abbreviations: FSH = follicle stimulating hormone; AMH = anti-Müllerian hormone; BMI = body mass index; COC = cumulus oocyte complexes; MII = metaphase-II; N = normozoospermic; MMF = moderate male factor (1-2 sperm defects); OAT = oligoasthenoteratozoospermic; NOA = non-obstructive azoospermia (sperm were collected through testicular sperm extraction).
Clinical considerations
Although emerging strategies, such as ovarian tissue cryopreservation and in vitro maturation, have been explored, oocyte vitrification after COS is the gold standard technique for fertility preservation. In this scenario, the number of oocytes retrieved and cryopreserved should be maximized in the shortest possible time as most cancer patients might benefit solely from one ovarian cycle before they have to commence their cancer treatment(s). Thus, a proper ovarian stimulation protocol is crucial to fully exploit the ovarian reserve and increase the cumulative chance of a future live birth28, an outcome strongly dependent on maternal age at oocyte retrieval. In this regard, an ideal age for oocyte vitrification for fertility preservation purpose has not been proposed yet29, and a consensus is required between either 35 years18 or 37 years30,31. However, fertility preservation should be accessible for all patients, including women older than 37 years, provided that they are given proper counselling on the success rates based on their age.
The protocol and starting dose of medications should be outlined according to the gynecologist's judgment and most importantly, based on the time available32,33. Starting conventional stimulation in the early follicular phase is recommended when time is not an issue. However, if required, the random-start approach is feasible to minimize delays in commencing urgent cancer/medical treatments34,35. Follicular development has been reported to be an extremely dynamic process as multiple waves of follicle recruitment have been described throughout a single ovarian cycle. Although the biological mechanisms behind this phenomenon still need to be unveiled, competent oocytes can be retrieved and cryopreserved independently of the phase of the menstrual cycle in which COS is started36.
In this center, ovarian stimulation is typically performed using the GnRH antagonist protocol and 150-300 IU/day of recombinant-FSH or human menopausal gonadotropin. In specific populations of patients older than 35 years, with LH deficiency, or showing suboptimal response after standard COS, LH might be added during COS to increase the recruitment and growth of follicles through a synergic action with insulin-like growth factor 116,37,38. Moreover, the GnRH antagonist protocol followed by a GnRH agonist trigger has been reported to be a short, safe, and highly convenient stimulation protocol to maximize ovarian response and minimize the risk for OHSS39. In patients with estrogen-sensitive diseases such as breast cancer, gonadotropins associated with aromatase inhibitors, such as letrozole, are administered20.
In fact, a recent review showed that letrozole involves similar ovarian response as conventional stimulation with no complications in terms of congenital defects, malignancy recurrence, and increased mortality40. Similarly, tamoxifen might be used during COS in case of estrogen-sensitive tumors, which, like letrozole, showed no impact on oocyte competence41,42. However, this needs to be confirmed in larger studies. In this center, letrozole is administered from day 1 of stimulation up to day 7 after oocyte retrieval in case of estradiol-sensitive tumors. The risk for OHSS, which is the most important complication of COS that would also further delay anticancer treatments, should be considered, particularly in young patients with high AFCs. In these cases, triggering ovulation with a GnRH agonist instead of human chorionic gonadotropin (hCG) has proven to be beneficial. Other issues that must be prevented are (i) thrombo-embolism, which requires the administration of low-molecular weight heparin during COS, and (ii) a reduced ovarian response after COS43. To compensate for the latter, two consecutive stimulations in a single ovarian cycle might be performed. This novel unconventional COS protocol, known as DuoStim, entails follicular and luteal phase stimulations and two oocyte retrievals in a short timeframe (~15 days) and should be investigated for fertility preservation purpose in the future44,45,46.
Other possible strategies for fertility preservation in women are: (i) ovarian tissue cryopreservation, which is the only option available for prepubertal females. This possibility is promising for restoring reproductive and endocrine activity and avoiding delay in starting cancer treatment as no hormonal stimulation is needed. However, it is still experimental and requires laparoscopic surgery with later transplantation, and there is the risk for orthotopic cancer retransmission. (ii) Embryo cryopreservation, which involves a higher survival rate after warming, but delays cancer treatment and requires a male partner or donor to be involved, thereby also limiting a woman's future reproductive autonomy47. Moreover, it might be subject to legal limitations in some countries. Considering these limitations, oocyte vitrification is considered the most established and ethically acceptable approach. Ideally, all major hospitals, where women affected from cancer in their reproductive age are treated, should provide programs for fertility preservation, and the whole process should be free of charge for these patients to ensure equal access to this program. Still, oocyte cryopreservation must be performed solely in centers with enough expertise not to affect oocyte competence in the process48.
Critical steps in the vitrification protocol and troubleshooting
Vitrification is a pseudo second-order phase transition (IUPAC Compendium of Chemical Terminology) that induces a glass-like solidification inside the cells preventing ice crystal nucleation and growth, the main causative factor of cell injury. To achieve proper vitrification, a combination of at least two CPAs is required (typically ethylene glycol and DMSO as permeating agents and sucrose as the non-permeating agent) along with an extremely high cooling rate (>20,000 °C/min) achieved either by minimizing loading volumes or by direct exposure of the samples to liquid nitrogen (open devices). As oocytes are the largest cells of the body, they contain the largest amount of water. Therefore, they are more sensitive to freezing injuries than embryos. During oocyte cryopreservation, damage to intracellular organelles (e.g., the cytoskeleton or meiotic spindle), alteration of membrane permeability, zona pellucida hardening, oocyte activation, alteration of the biochemical pathways, and possibly cell death can occur49. For this reason, a delicate balance between multiple factors must be ensured for successful preservation of oocyte viability and developmental competence. To achieve consistency in the survival rate after vitrification and reach the benchmark levels, all the crucial steps of the procedure must be strictly controlled.
CPAs, cell osmolality, and cooling rates
To increase the probability of vitrification, the viscosity of the medium (and therefore CPA concentration) must be maximized. However, the toxicity of the CPAs should always be kept under control50. To this end, it is crucial to minimize both the time of cell exposure to the CPAs and the loading volumes, and always work at room temperature (25-27 °C)51. Other protocols involve different combinations of CPAs and cryodevices and are carried out at 37 °C; however, they have not been described here. Thus, the notes, representative results, and discussion sections of this manuscript only apply to the vitrification protocol detailed here.
During cryopreservation, the oocytes are exposed to CPA solutions of increased osmolyte concentration to promote cell dehydration and cytoplasmic shrinkage. During warming, they are exposed to solutions with decreased osmolyte concentration to restore the cytoplasmic volume. During vitrification, the oocytes are dehydrated, and unintentional fluctuations in cell volume can cause severe osmotic shock, thereby compromising survival and developmental potential after warming52. Finding the optimal cooling rate is a key aspect for preserving cell viability as it affects the osmolality53 and cell developmental potential54. For instance, when a cell is exposed to cooling rates slower than the optimal rates, it may be extensively exposed to hypertonic conditions leading to cell death.
To achieve the optimal cooling rate, the loading volumes should be minimized, RT should be controlled so as not to affect the speed of the equilibration process, and samples should be directly exposed to liquid nitrogen. One way to assess when equilibration has been accomplished is to annotate the morphology of the oocyte (in particular, the width of the perivitelline space and the thickness of the zona pellucida) before the beginning of the procedure (Figure 5A). After an initial phase of cell volume reduction (Figure 5B), the oocyte is expected to recover its initial volume (Figure 5C). Although correct pH is maintained during oocyte handling under air atmosphere because of the zwitterions buffering the vitrification-warming solutions, medium osmolality is instead particularly dependent on temperature53. Therefore, the method of dish preparation is critical to reduce any possible detrimental effect55.
Oil overlay is certainly pivotal to prevent evaporation and avoid any increase in osmolality in the IVF media56. However, it cannot be used while performing the vitrification and warming procedures. Therefore, some tips are important for operators: (i) prepare the droplets as quickly as possible and immediately before use; (ii) consider larger volumes of droplets to minimize shifts in osmolarity (<30 µL is not recommended); (iii) when environmental conditions cannot be standardized, a 6-well plate can be used, and sterile water or PBS can be added to the adjacent reservoir to limit evaporation; (iv) pay attention to the date of the first opening of the vial of medium as osmolality can change if the bottle is repeatedly opened.
Oocyte warming and oocyte devitrification
The most crucial step that may affect the consistency in the survival rate is definitely the warming process57. During this step, CPAs are gradually removed from the oocyte and diluted to prevent any potential cytotoxic effect. Devitrification, namely the formation of ice nuclei or ice crystals during the warming of a vitrified solution or accidentally during audit, transport, or storage in vapor, is one of the main risks of vitrification58,59,60. Therefore, to prevent devitrification and injury during warming, the difference in temperature between the first and last steps of the process must be maximized. As shown by Seki and Mazur57 in murine oocytes subject to vitrification and warming at different rates, the faster the warming, the higher the survival. Volume and temperature of the TS are the main factors to control: TS should be properly warmed to 37 °C (at least 1 h before the procedure), and the liquid nitrogen rack should be filled to the edge of its capacity and placed as close as possible to the stereomicroscope. The operator should be as fast as possible while transferring the cryopreservation carrier from the liquid nitrogen to the TS. Because of the high efficiency of vitrification, a universal warming protocol can be used irrespective of the freezing protocol involved, making the management of all the warming cycles easier even when oocytes are imported from a different IVF center61.
Open devices and risk of contamination
The employment of open systems and direct exposure of the samples to liquid nitrogen are required to achieve the extremely high cooling and warming rates that support the effectiveness of this protocol. Although vitrification is a procedure that poses low risk for cross-contamination, because it involves very small volumes and is carried out after several sample washings that dilute any putative viral load, it is essential to adopt all precautionary measures to increase its safety. Based on current evidence, closed systems do not offer a competitive alternative to open systems at least for oocyte vitrification62,63. To maintain the effectiveness of open vitrification systems while minimizing the risks associated with direct contact with liquid nitrogen, the latter might be sterilized by ultraviolet irradiation64,65. Alternatively, vapor storage tanks might be used, which are known to pose a lower risk of contamination than conventional ones, but have proven to be effective in preserving oocyte viability66,67.
Importance of constant monitoring of key performance indicators for oocyte vitrification programs
In an IVF laboratory, the monitoring of key performance indicators (KPIs) is essential for monitoring and constantly improving results68. In general, when defining KPIs for monitoring processes and procedures, three main areas should be considered: structure, process, and outcome. Structural KPIs measure the quality of the IVF laboratory by outlining the characteristics of physical and human resources. An example of a structural KPI related to the facility in a cryopreservation program might be the number of cryotanks relative to the total number of ART procedures requiring cryopreservation conducted in a given time period or the number of cryo-tanks relative to the total square meters of the cryoroom. However,human resources and in particular, operator skills are of utmost importance when dealing with a delicate procedure such as vitrification. In fact, although extremely effective, the vitrification technique is a challenging procedure involving several procedural phases with stringent timings that must be strictly controlled: a very small amount of a significantly viscous medium should be managed over a very short period.
No freezing machine with specific cooling parameters setting is involved in the procedure; therefore, standardization of protocol details and training are essential. The manual process has strict skill requirements that must be fulfilled to obtain consistent and highly reproducible cell survival rates. Specific training should be provide to each novice operator to make them proficient in controlling all the critical points of the procedure, in particular, the exposure time of the samples to CPAs and the handling of the cells in a highly viscous medium. However, according to Dessolle and coauthors,69 the learning curve for the vitrification procedure should not be so long even for junior embryologists as the achievement of proficiency is mainly limited by manipulation challenges. Once expertise in vitrification has been achieved, the performance of each individual operator must be accurately and regularly verified through the use of KPIs to regularly assess the maintenance of competency values established by consensus papers70. Moreover, operator confidence/competence must be comparable so as to not affect the outcomes.
Process KPIs measure how well the IVF laboratory works. Vitrification and warming protocols and procedures should be conducted in a timely manner, and fluctuations in culture conditions should be minimized by paying particular attention to maintaining adequate osmolarity and temperature for the preservation of not only oocyte developmental competence, but also of operator safety while handling liquid nitrogen. Examples of process KPIs arethe percentage of laboratory staff injuries while handling liquid nitrogen per number of IVF procedures per year, the percentage of gametes/embryos lost/damaged during vitrification/warming procedures, and audits per number of procedures per year.
Finally, outcome KPIs measure the effectiveness of the IVF laboratory and generally refer to postwarming oocyte survival defined as the proportion of morphologically intact oocytes at the time of ICSI (in case of oocyte vitrification, the competency value should be >50% and the benchmark value is 75%)70. Additionally, the rates of fertilization (<10% lower than the fresh oocytes inseminated at the center from a comparable patient population), embryo development (the same as a comparable patient population using fresh oocytes), and implantation (<10-30% lower than a comparable population of fresh embryos at the center) are applicable as outcome KPIs for vitrified oocytes70. However, clinical outcomes are more subject to couple-specific characteristics than to faulty clinical procedures71,72.
The authors have nothing to disclose.
None
Collection | |||
Equipment | |||
Hot plate | IVF TECH | ||
Lab Markers | Sigma Aldrich | ||
Laminar Flow Hood | IVF TECH | Grade A air flow | |
Stereomicroscope | Leica | Leica M80 | |
Thermometer | |||
Test tube Warmer | |||
Tri-gas incubator | Panasonic | MCO-5M-PE | 02/CO2 |
Vacuum Pump | Cook | K-MAR-5200 | |
Consumables | |||
CSCM (Continuos single culture complete) medium | Fujifilm Irvine Scientific | 90165 | IVF culture medium supplemented with HSA |
Mineral Oil for embryo culture | Fujifilm Irvine Scientific | 9305 | |
Ovum Aspiration Needle (Single lumen) | Cook | K-OSN-1730-B-60 | |
Primaria Dish | Corning | 353803 | Corning Primaria Dish 100×20 mm style standard cell culture dish |
Round- bottom tubes | Falcon | 352001 | Falcon 14ml Round Bottom Polystyrene Test tube with snap cap |
Round- bottom tubes | Falcon | 352003 | Oocyte collection tubes/ Falcon 5ml 12×75 Round Bottom Polipropilene Test tube with snap cap |
Rubber Bulb | Sigma Aldrich | Z111589-12EA | |
Sterile glass Pasteur pipettes | Hunter Scientific | PPB150-100PL | Pipette Pasteur Cotonate, 150mm, MEA e CE |
Denudation | |||
Equipment | |||
CO2 incubator | Eppendorf | Galaxy 14S | |
Flexipet adjustable handle set | Cook | G18674 | Stripper holder |
Gilson Pipetman | Gilson | 66003 | p20 |
k-System Incubator | Coopersurgical | G210Invicell | |
Lab Markers | Sigma Aldrich | ||
Laminar Flow Hood | IVF TECH | Grade A air flow | |
Stereomicroscope | Leica | Leica M80 | |
Consumables | |||
Biopur epTIPS Rack | Eppendorf | 30075331 | Micropipettes epTIPS Biopur 2-200 µl |
Human Serum Albumin | thermoFisher Scietific | 9988 | |
Hyaluronidase | Fujifilm Irvine Scientific | 90101 | 80 IU/mL of hyaluronidase enzyme in HEPES-buffered HTF |
IVF culture dish (60 x 15mm) | Corning | 353802 | Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish |
IVF dish 4-well plate with sliding lid | ThermoFisher Scietific | 176740 | Multidishes 4 wells (Nunc) |
IVF One well dish | Falcon | 353653 | Falcon 60 x 15 mm TC treated center-well IVF |
Mineral Oil for embryo culture | Fujifilm Irvine Scientific | 9305 | |
Modified HTF Medium | Fujifilm Irvine Scientific | 90126 | HEPES-Buffered medium |
Rubber Bulb | Sigma Aldrich | Z111589-12EA | 1 mL for pasteur pipettes |
Sterile glass Pasteur pipettes | Hunter Scientific | PPB150-100PL | Pipette Pasteur Cotonate, 150 mm, MEA e CE |
stripping pipette tips (140 µm) | Cook | K-FPIP-1140-10BS-6 | PIPETTE FLEXIPETS PER DENUDING |
stripping pipette tips (130 µm ) | Cook | K-FPIP-1130-10BS-7 | PIPETTE FLEXIPETS PER DENUDING |
stripping pipette tips (170 µm) | Cook | K-FPIP-1170-10BS-5 | PIPETTE FLEXIPETS PER DENUDING |
Vitrification | |||
Equipment | |||
Electronic Timer | |||
Flexipet adjustable handle set | Cook | G18674 | Stripper holder |
Gilson Pipetman | Gilson | F123601 | p200 |
Lab Markers | Sigma Aldrich | ||
Laminar Flow Hood | IVF TECH | Grade A air flow | |
Stainless Container for Cooling Rack | Kitazato | Liquid nitrogen container for vitrification | |
Stereomicroscope | Leica | Leica M80 | |
Consumables | |||
Biopur epTIPS Rack | Eppendorf | 30075331 | Micropipettes epTIPS Biopur 2-200 µL |
Human Serum Albumin | Fujifilm Irvine Scientific | 9988 | |
IVF culture dish (60 x 15 mm) | Corning | 353802 | Corning Primaria Falcon Dish 60 x 15 mm TC Primaria standard cell culture dish |
IVF dish 6-well | Oosafe | OOPW-SW02 | OOSAFE 6 WELL DISH WITH STRAW HOLDER |
Modified HTF Medium | Fujifilm Irvine Scientific | 90126 | HEPES-Buffered medium |
stripping pipette tips (170 µm) | Cook | K-FPIP-1170-10BS-5 | PIPETTE FLEXIPETS PER DENUDING |
Vitrification Freeze kit | Fujifilm Irvine Scientific | 90133-so | 2 Vials of ES (Equilibration Solution, 2 x 1 mL) and 2 Vials of VS (Vitrification Solution, 2 x 1 mL) |
Vitrifit | Coopersurgical Origio | 42782001A | VitriFit Box |
Warming | |||
Equipment | |||
Electronic Timer | |||
Flexipet adjustable handle set | Cook | G18674 | Stripper holder |
Gilson Pipetman | Gilson | F123601 | p200 |
k-System Incubator | Coopersurgical | G210Invicell | |
Lab Markers | Sigma Aldrich | ||
Laminar Flow Hood | IVF TECH | Grade A air flow | |
Stainless Container for Cooling Rack | Kitazato | Liquid nitrogen container for vitrification | |
Stereomicroscope | Leica | Leica M80 | |
Consumables | |||
Biopur epTIPS Rack | Eppendorf | 30075331 | Micropipettes epTIPS Biopur 2-200 µL |
CSCM (Continuos single culture complete) medium | Fujifilm Irvine Scientific | 90165 | IVF culture medium supplemented with HSA |
IVF culture dish (60 x 15 mm) | Corning | 353802 | Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish |
IVF dish 4-well plate with sliding lid | ThermoFisher Scietific | 176740 | Multidishes 4 wells (Nunc) |
IVF dish 6-well | Oosafe | OOPW-SW02 | OOSAFE® 6 WELL DISH WITH STRAW HOLDER |
Mineral Oil for embryo culture | Fujifilm Irvine Scientific | 9305 | |
SAtripping pipette tips (300µm) | Cook | K-FPIP-1300-10BS-5 | PIPETTE FLEXIPETS PER DENUDING |
Vitrification Thaw kit | Fujifilm Irvine Scientific | 90137-so | 4 Vials of TS (Thawing Solution, 4 x 2 mL) + 1 Vial of DS (Dilution Solution, 1 x 2 mL) +1 Vial of WS (Washing Solution, 1 x 2 mL) |