Abstract
Purpose
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.
Methods
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.
Results
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.
Conclusion
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.
Keywords: Universal warming procedure, Vitrification, Warming, Oocyte cryopreservation, Kitazato vitrification kit, Irvine vitrification kit
Introduction
Assisted reproductive (AR) centers receive oocytes, embryos, and whole tissues transported from other centers, cryopreserved with various protocols. Regulations specify the use of FDA/CE marked thawing media approved for human AR, and each cryopreservation kit brand recommends the use of its own thawing kit. However, because the shelf life of these media is usually short, it is expensive to keep available the reciprocal solution for every brand’s cryopreservation kit. Furthermore, in some cases, these kits have been withdrawn from the market for various reasons. In this scenario, the possibility of using a “Universal Warming” method to thaw any cell or tissue irrespective of the freezing protocol may simplify the management of thawing procedures [1].
Over the last decades, human oocytes, embryos, and ovarian tissue have been cryopreserved by two main methods as follows: vitrification (VIT) or slow freezing (SF) [2, 3]. Although VIT has nowadays overtaken SF for a number of reasons, including the higher survival rate guaranteed [3, 4], slow-frozen oocytes, embryos, and especially ovarian tissue are still stored in cryobanks worldwide.
In a proof-of-concept study, we demonstrated the possibility of warming slow frozen oocytes using a single “Universal Warming” protocol based on subsequent steps with 1 M and 0.5 M concentration of any extracellular cryoprotectant (ECCP) [1]. The efficacy of this protocol was confirmed by a multi-center study performed on 400 slow-frozen oocytes [5, 6]. Since the oocyte is the most sensitive human reproductive cell to cryoinjury, we believed that our basic research studies would pave the way for the clinical use of this warming protocol for any cell, irrespective of the cryopreservation method used for freezing. Today, oocytes and embryos are vitrified using the different kits available on the market; these ready-to-use VIT kits contain vitrification/warming (VIT/WARM) solutions with only slight differences in their composition, although often the quantity and types of the exact components of these kits are not declared. Furthermore, in different countries, regulatory/commercial/availability differences may influence the choice of the VIT/WARM kit and ultimately this affects clinical practice. Although we can hypothesize that combining different VIT/WARM kits is feasible, nevertheless, it remained to be demonstrated that a WARM solution of a given brand can be used to warm oocytes and embryos vitrified with another brand. We have recently demonstrated in the first clinical study on “Universal Warming,” performed on 800 patients (1370 embryos) that this protocol is feasible and efficient for cleavage stage embryos and blastocysts [7] and that it is also applicable to vitrified oocytes [8].
The aim of the present article is to demonstrate that it is possible to streamline vitrified oocyte exchange for a transnational egg donation program using the “Universal Warming” protocol, by analyzing the cryosurvival, fertilization, blastulation, implantation, and live birth rates obtained by using different WARM kits—containing 1 M and 0.5 M of ECCP—to warm oocytes vitrified with the same single brand.
Materials and methods
Study design
Retrospective multi-center observational study on a cohort of 238 patients enrolled in egg donation programs from 02/03/2017 to 19/09/2018; the study was promoted and coordinated by GynePro Medical Centers and approved by its Institutional Review Board (approval number 22.02.2017). Donated oocytes were vitrified in Spain, at IMER Valencia and then warmed in two centers in Italy where ICSI and embryo transfer (ET) were performed: GynePro Medical Centers Bologna, and Center for Reproductive Medicine, European Hospital Rome. The total number of oocytes warmed was 1.898 (1201 in Bologna and 697 in Rome), and 238 ETs were performed (152 in Bologna and 86 in Rome). Vitrification was performed with Cryotop carrier (Kitazato, Japan) using vitrification kit (Kitazato), and warming was carried out with two different kits: Kitazato warming kit and Vit Kit®-Thaw (FujiFilm Irvine, US); these two kits were used routinely by the two Italian AR centers during the period of the study and the warming was simply performed using the next available warming kit on a “first-in-first-out” basis (kits with earliest expiration date are used first). For data analysis, the warmed oocytes were assigned to 2 groups as follows:
KK (Kitazato/Kitazato) 939 oocytes
KI (Kitazato/Irvine) 959 oocytes
The primary endpoint of the study was the survival rate (number of oocytes surviving/number of oocytes warmed). Warmed oocytes were considered to have survived in the absence of negative characteristics: dark or contracted ooplasm, vacuolization, cytoplasmic leakage, abnormal perivitelline space, and cracked zona pellucida. After 1–2 h post-warm culture, the surviving oocytes were checked again and inseminated by ICSI. The secondary endpoints were the following: fertilization rate (fertilized oocytes/injected oocytes); blastulation rate (blastocysts obtained/fertilized oocytes); implantation rate (gestational sacs/transferred blastocysts); and live birth rate (pregnancies leading to births/embryo transfers). Clinical pregnancy was defined as the presence of a gestational sac with or without fetal heart beat (FHB) at ultrasound examination, 2 weeks after positive hCG testing.
Donor oocytes
Vitrified oocytes from young donors were obtained due to a cooperation agreement between Italian centers (GynePro and European Hospital) and the Spanish gamete cryobank (IMER) as regulated by Italian legislation [9, 10].
Controlled ovarian stimulation, oocyte retrieval, and vitrification
Controlled ovarian stimulation, transvaginal ultrasound-guided oocyte retrieval, and oocyte decumulation were performed as described elsewhere [11, 12]. Oocyte vitrification was performed according to the Kitazato protocol [13, 14].
Oocyte transportation and warming
The oocytes were transported from Spain to the Italian AR centers by an authorized road courier; the use of air transport was avoided to minimize potential physical shocks during shipping [9, 15]. Standard operating procedures (SOP) were shared between the 3 centers to reduce transportation-related risks, as described by Parmegiani et al. [9]. The oocytes were warmed in accordance with the “Universal” protocol as described in the following sections.
Composition of vitrification/warming kits
Kitazato kits contain trehalose as ECCP and are supplemented with hydroxypropyl cellulose (HPC) [16]. Irvine kits contain sucrose as ECCP and are supplemented with dextran serum supplement (DSS). The basic medium is TCM199 for both kits [13, 14]. The cryoprotectant cocktail comprises 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycole (EG) in the equilibration solution, and 15% DMSO and 15% EG in the vitrification solution [13]. Both Irvine and Kitazato WARM kits involve subsequent steps with 1 M and 0.5 M concentration of ECCP.
Universal warming procedure
The “Universal Warming” protocol is slightly different from the instructions for use (IFU) suggested by Irvine, and similar (in volumes and timing) to IFU by Kitazato and other manufacturers. Carrier washing before warming procedure was performed in certified sterile liquid nitrogen (SLN2) to minimize virus/bacterial/fungi/mold contamination [17]. SLN2 was produced by a specifically designed device, Nterilizer™ N-Bath as described elsewhere [1, 18]. The solutions used for oocyte warming are as follows: 4 mL of 1 M ECCP warming solution (1 M WS), 300 μL of 0.5 M ECCP warming solution (0.5 M WS) and 300 μL + 300 μL of washing solution (basic medium—see Composition of vitrification/warming kits—Material and methods). Procedures are described below.
The first warming step was performed with the 1 M ECCP warming solution at 37 °C. The vial containing this solution was pre-warmed to 37 °C for at least 60 min before use and kept closed throughout.
The other solutions were brought to room temperature (20°-25 °C) for at least 30 min before use; the contents of each vial of the kits were well mixed by gentle inversion several times before use, and aseptically dispensed into a six-well multi dish (OOPW-SW02 Sparmed, Denmark or Reproplates Kitazato): 300 μL of 0.5 M into well 1, 300 μL of washing solution into well 2 and a further 300 μL into well 3.
A pre-warmed petri dish (OOPW-TF03, Sparmed or Reproplates Kitazato) was then filled with 4 mL of the warmed 1 M ECCP solution.
The vitrification carrier device containing the oocytes (Cryotop, Kitazato) was opened in certified sterile liquid nitrogen (SLN2) [1, 2] provided by N-Bath (Nterilizer, Italy); the SLN2 insulated container was placed close to the stereomicroscope for rapid manipulation.
The strip of the vitrification carrier was immediately plunged into the petri dish containing the warmed 1 M solution (the oocytes float from the carrier to the top of the 1 M solution dish and are kept in this solution for 1 min until they start to shrink).
Using a pipette containing some of the 1 M solution, the oocytes were transferred from the petri dish to well 1 of the 6-well dish (300 μL of 0.5 M ECCP) for 3 min, minimizing exposure to light (the oocytes remain shrunken for the whole duration of this step).
Then, drawing up some 0.5 M solution from well 1, the oocytes were transferred to well 2 (300 μL of washing solution) for 5 min.
Finally, the oocytes were placed in well 3 for the last washing (1 min) and subsequently moved to a dish of pre-equilibrated culture medium and incubated at 6% CO2–5%O2 incubator at 37 °C for 1–2 h prior to ICSI.
ICSI and embryo culture
The warmed oocytes were inseminated by ICSI and cultured as described elsewhere [2]. Embryo culture was performed at low oxygen tension (5%) with embryoscope (Vitrolife, Sweden). ET was performed at blastocyst stage. The embryos not transferred during the oocyte thawing cycle were vitrified and their performances after warming for future transfer attempts are not analyzed in this article.
Endometrial preparation and embryo transfer
Preparation of the endometrium for the embryo transfer (ET) was performed as described elsewhere [12]. Embryo transfer was carried out after 5 days (day 5) from progesterone administration [19]. Embryo implantation was defined as the presence of a gestational sac with or without fetal heartbeat (FHB) at ultrasound examination, 2 weeks after positive hCG testing.
Statistical analysis
Continuous variables are presented as mean ± standard deviation. Categorical variables are presented as absolute and relative frequencies. Normality of distribution of continuous variables was assessed with a Kolmogorov-Smirnov test (with Lillefor correction). Between-group differences of normally distributed continuous variables were assessed with parametric statistics (Student’s t test), whereas non-parametric statistics (Mann-Whitney Rank Sum Test) were employed when the normality test was not passed. Between-group differences in frequencies were assessed using the χ2–method with Yates correction if needed or Fisher exact test when frequencies were less than 5 in one of the two groups. Data analyses were performed in SPSS Statistics package (version 23, IBM Co., Armonk, NY, USA) and in R 3.4.2. Two tailed P values less than 0.05 were considered significant.
Results
Patient and donor population and number of warmed oocytes and transferred embryos per patient were comparable (Table 1). Mean number of motile sperm after sperm preparation was comparable (22.3 × 106 ± 3.6 vs 16.7 × 106 ± 3.1; P = 0.361). Couples with less than 1 × 106 of motile spermatozoa in the raw semen were not considered for this study. 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); and live birth rate was 52.5% (62/118) in KK and 45.0% (54/120) in KI (Table 2). Pregnancy and multiple gestation rates were statistically comparable: pregnancy 58.5% KK (69/118) vs 55.8% KI (67/120) P = 0.779, multiple gestation: 15.9% KK (11/69) vs 25.4% KI (17/67) P = 0.251. Miscarriage rate was statistically comparable (P = 0.762): KK 10.1% (7/69) vs KI 19.4% (13/67), despite the lower miscarriage rate in KK.
Table 1.
Patient population, number of warmed oocytes, and transferred embryos
| Group KK | Group KI | P Value | |
|---|---|---|---|
| Mean oocyte donor age (±SD) at oocyte retrieval | 24.2 (±3.4) | 23.7 (±3.1) | 0.192 |
| Mean female patient age (±SD) at embryo transfer | 43.0 (±3.9) | 43.2 (±4.2) | 0.672 |
| Mean male patient age (±SD) at ICSI | 44.8 (±6.0) | 45.1 (±7.0) | 0.713 |
| Mean number of warmed oocytes per patient (±SD) | 7.9 (±0.6) | 7.9 (±0.7) | 0.701 |
| Mean number of transferred embryos per patient (±SD) | 1.6 (±0.5) | 1.5 (±0.5) | 0.084 |
Table 2.
Clinical results
| Group KK | Group KI | P Value | |
|---|---|---|---|
| No. of surviving oocytes / warmed oocytes (%) (Min-Max%) | 795/939 (84.6) (37.5–100) | 787/959 (82.1) (25.0–100) | 0.145 |
| No. of fertilized oocytes / injected oocytes (%) (Min-Max%) | 602/795 (75.7) (20.0–100) | 633/787 (80.4) (16.6–100) | 0.027 |
| No. of obtained blastocysts / fertilized oocytes (%) (Min-Max%) | 352/602 (58.5) (25.0–100) | 366/633 (57.2) (33.3–100) | 0.861 |
| No. of pregnancies / embryo transfers (%) (Min-Max%) | 69/118 (58.5) (0.0–100) | 67/120 (55.8) (0.0–100) | 0.779 |
| No. of gestational sacs / transferred blastocysts (%) (Min-Max%) | 80/193 (41.5) (50.0–100) | 84/183 (45.9) (50.0–100) | 0.444 |
| No. of pregnancies leading to births / embryo transfers (%) (Min-Max%) | 62/118 (52.5) (0.0–100) | 54/120 (45.0) (0.0–100) | 0.301 |
| No. of multiple gestation /pregnancies (%) (Min-Max%) | 11/69 (15.9) (0.0–100) | 17/67 (25.4) (0.0–100) | 0.251 |
Discussion
In AR laboratories worldwide, oocytes are vitrified using the different kits available on the market; these ready-to-use VIT kits contain VIT/WARM solutions with only slight differences in their composition. Combining different brands of VIT/WARM kits with 1 and 0.5 M of ECCP in the WARM solution has been shown to be feasible in proof-of-concept and pilot clinical studies [1, 5, 6]. This “Universal Warming” could streamline vitrified oocyte exchange for a transnational egg donation, but before now, the efficiency of this protocol had not been demonstrated in large multi-center studies. We performed a pilot study on a small data set from just one “warming” center in 2018 [8] and, given the successful outcome of this, believed that a larger-scale study adding further data (1.898 compared with 820 in 2018) and involving more than one center would boost the validity of this procedure. Thus, in the present article, we report the cryo-survival and the clinical performance of almost two thousand oocytes vitrified in Spain with one brand of culture media (Kitazato) and then transported and subsequently warmed in two AR centers in Italy with two brands (Irvine-Fujifilm and Kitazato).
The cryo-survival rate was comparable between the two groups studied. Mean donor, recipient, and male partner ages were comparable between the study groups as were the mean number of oocytes warmed, and number of transferred embryos per patient. Mean number of motile sperm after sperm preparation was comparable (22.3 × 106 ± 3.6 vs 16.7 × 106 ± 3.1; P = 0.361). Couples with less than 1 × 106 of motile spermatozoa in the raw semen were not considered for this study. Clinic-related differences in the handling of the oocytes and patient screening and management (overall patient health, existence of any gynecological issues in the recipient female population, blood tests, uterine health, etc.) were avoided by the agreement to use of standard operating procedures (SOP) between the AR centers. Thus, the two groups show no initial bias or confounding factors. Fertilization was significantly higher in the KI group, in which the oocytes were warmed with a kit of a different brand (Irvine) than that used for vitrification (Kitazato). Nevertheless, we would not wish to overemphasize this difference in such a short-term outcome measure and would prefer to be cautious in hypothesizing any potential positive effect of the different ingredients in the warming kit used, also because all the other clinical parameters in our analysis were statistically comparable. Analyzing this outcome measure in the two warming clinics, fertilization rate was 73.0% KK vs 80% KI in Bologna (P = 0.001) and 92.5 KK vs 78.8% KI in Rome (P = 0.017). Despite shared SOP between the centers and the uniformity of semen parameters, the survival rate is variable between groups and clinics. This is probably due to the small size of the data when split in subgroups. An overview of the total fertilization rate between groups perhaps minimizes the influence of confounding factors stemming from any single donor or male partner. Furthermore, fertilization rate was not significantly different between KI and KK groups in the pilot study of 2018 performed on 820 oocytes. Given these slightly irregular results the total potential positive effect for fertilization rate when using the Irvine kit to warm oocytes vitrified with the Kitazato kit is worthy of future studies with even larger datasets. In fact, one aim of our study is to encourage other groups to publish their own data regarding the combination of different brands of vitrification and warming kits, in order to validate in the literature this ever more common procedure due to the increasing number of shipped oocytes and embryos worldwide.
In the present study, blastulation, implantation, and live birth rates were comparable and aligned with the competency values for these key performance indicators [20, 21]. The KK group had a slightly lower implantation rate but a higher live birth rate; these trends were not statistically significant but they could be worth addressing in larger studies.
Cryosurvival and clinical results are aligned with data reported in the largest study on egg donation with oocyte vitrification/warming performed in the same laboratory [22]. The survival rate observed in our study is around 6% lower than the 90% reported by Cobo and colleagues; this is acceptable considering that transportation of vitrified oocytes may reduce survival rate by up to 20% [9, 17, 23]. As previously described, the oocytes were transported from Spain to the Italian AR centers by an authorized road courier; the use of air transport was avoided to minimize potential physical shocks during shipping [9, 15]. Furthermore, standard operating procedures (SOP) were shared between the 3 centers to reduce any risk related with transportation. Despite these precautions, shipped oocyte survival rate was found to be reduced, probably due to the multiple manipulations required for this process, as previously described by Parmegiani et al. [1]. More specifically, survival was 10% lower when compared with mean survival of oocytes vitrified and warmed in each of the 3 centers, and not undergoing transportation (90%). The other KPIs, particularly live birth rate (37.6% in Cobo’s study), are comparable.
This is the first multi-center large-scale clinical study to compare the effect of two warming solutions on Kitazato vitrified and shipped oocytes. Its main limitation is that it is a retrospective observational study; a further limitation is that only oocytes from young donors were included. The clinical application of the Universal Warming protocol with 1 and 0.5 M ECCP had previously been tested on slow frozen oocytes [1] to demonstrate that this procedure was potentially applicable to any cryopreserved cell, irrespective of the freezing protocol. After this pioneering study, we confirmed the reproducibility of this procedure with a multi-center study [5, 6]. The question which remained to be answered following these basic research studies was the clinical efficiency of the Universal Warming protocol. The clinical application of our warming protocol was reported for the first time in a Chinese study; Shun and co-authors [24] performed 11 warming cycles on slow frozen embryos and obtained 5 clinical pregnancies. In a recent study, we showed the clinical efficiency of Universal Warming on 1370 vitrified human embryos in a systematic way [7]. Reporting that survival and implantation did not vary between 16 study groups, we demonstrated the clinical feasibility of combining different kits for vitrification and warming, with 1 and 0.5 M ECCP. After this study on embryos, the way was paved for studies to confirm the clinical efficiency of this protocol on any type of cell using different combinations of kits from different manufacturers. In the present study, we confirm the efficiency of Universal Warming on oocytes. More technically, we compared the efficiency of warming solutions with Sucrose (Irvine -Fujifilm) or Trehalose (Kitazato) as ECCP, supplemented by dextran serum supplement (DSS) (Irvine -Fujifilm) or hydroxypropyl cellulose (Kitazato) for the warming of oocytes vitrified with trehalose + hydroxypropyl cellulose (Kitazato). Sucrose is the most commonly used ECCP in cryopreservation protocols; disaccharide trehalose is employed by certain species to survive in extreme conditions [25] and is used as an osmotic agent in some cryopreservation protocols [26]. Hydroxypropyl cellulose (HPC) is a replacement for human albumin (HA) or serum substitute supplement (SSS) or dextran serum supplement (DSS) for use in cryoprotectant solutions to protect oocytes against injury during vitrification [16, 27]. The results obtained from this this study do not allow us to identify if one ECCP is better than another for warming: the comparable survival rate seems to indicate that sucrose and trehalose are both efficient for warming oocytes vitrified with trehalose.
Comments
Every brand of vitrification kit recommends the use of its own warming kit. However, it is good practice, in AR clinics, to have alternative options for carrying out any procedure and to have some flexibility in the choice of media, especially in the case of biological material exchange between different countries. This is because there may be regulatory or market differences affecting product availability, which can influence clinical practice. To give an example, in July 2017, the CE mark for Kitazato kit was accidentally withdrawn, and this bureaucratic hitch was interpreted in different ways in Spain and Italy: its use was permitted in Spain but not in Italy. In this type of context, the application of “Universal Warming” through the combination of different vitrification/warming kits can solve the immediate clinical problem, but this action may have potential legal consequences for AR centers, until this protocol has been further validated. For this reason, there is an urgent need in the literature for more studies on a larger scale.
Conclusion
The use of a “Universal Warming” protocol with ready-to-use warming kits containing 1 and 0.5 M of ECCP simplifies vitrified oocyte and embryo exchange between AR centers in different countries, to overcome potential regulatory/commercial/availability differences affecting clinical practice. AR centers can choose the warming kit (containing 1 and 0.5 M of ECCP) best suited for their needs, irrespective of the brand used at vitrification, depending on price, stock, and distributor reliability. Furthermore, the widespread use of a “Universal Warming” protocol should encourage manufacturers to declare the exact components and concentrations in their kits, information which is currently often not specified.
Acknowledgments
The authors wish to thank Ms. Maggie Baigent for revising the manuscript. Lodovico Parmegiani wishes to thank all the embryologists worldwide who have shared with him in these last years their opinions and impressions about “Universal Warming”.
Authors’ contributions
LP: study design, execution, analysis and manuscript drafting
MGM: study analysis, statistics
AA: study execution
VC: study execution
GEC: study execution
RV: study execution
MTV: study execution
LAQ: study execution, manuscript revision and critical discussion
EG: study execution, manuscript revision and critical discussion
MF: study execution, manuscript revision and critical discussion
Compliance with ethical standards
Conflict of interest
LP is shareholder of Nterilizer Srl and reports fees from Origio-Coopersurgical, Merck, and Irvine-Fujifilm outside the submitted work. Co-authors have no interest to declare.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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