Post-warming survival rates and clinical outcomes of human cleavage stage embryos vitrified/warmed using CryoTouch and Cryotop methods

Vitrification has become the method of choice for cryopreservation of human embryos and gametes. There are multiple commercial media, containing different combinations and concentrations of cryoprotectants, available for vitrification and warming procedures. The aim of this retrospective study was to compare post-warming survival rate and clinical outcomes of cleavage stage embryos vitrified/warmed using two different commercial methods (CryoTouch and Cryotop) during intracytoplasmic sperm injection/frozen embryo transfer (ICSI/FET) cycles. This retrospective study evaluated a total of 173 FET cycles performed on 446 warmed cleavage stage embryos between January 2018 and December 2020. Post-warming embryo survival rate and clinical outcomes including clinical pregnancy, implantation, and live birth rates were calculated. The results showed no significant differences between two groups in terms of post-warming survival rate (p value = 0.5020), clinical pregnancy rate (p value = 0.7411), implantation rate (p value = 0.4694), and live birth rate (p value = 0.5737). Collectively, high successful rates were observed in outcomes of vitrified/warmed cleavage stage embryos using both CryoTouch and Cryotop commercial methods.


Background
Production of excess embryos is prevailing during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. The embryos that are not selected for transfer to the uterus should be cryopreserved to be used in future embryo transfer (ET) cycles [1]. Therefore, embryo cryopreservation provides additional chances for achieving pregnancy without extra exposures to the exogenous gonadotropins used for ovarian stimulation [2]. In addition, embryo cryopreservation facilitates the single embryo transfer (SET) program performed to avoid multiple pregnancies [2,3]. Frozen embryo transfer (FET) cycles are now preferred over fresh ET cycles as they avoid to transfer the embryo into the uterus if the endometrial preparation is suboptimal [4]. The first report of successful pregnancy achieved by a FET cycle was published in 1983 [5]. Since then, embryo cryopreservation has become an undeniable part of the assisted reproductive technology (ART) [4,6].
A successful cryopreservation procedure preserves all the functional and morphological properties of embryonic cells and protects them against damages caused by ice crystal formation and fracture damages, the main concerns during steps of cryopreservation, i.e., cooling and warming [7]. Different cryoprotective agents (CPAs) and cooling-warming rates are being used to avoid detrimental events [8]. There are two common methods for embryo and gamete cryopreservation in ART practice, slow freezing and vitrification [7]. Over the past decades, vitrification has gradually become the method of choice for embryo and gamete cryopreservation because of ease of use and low cost [9,10]. Vitrification has been broadly used for human cleavage and blastocyst stage embryos [6,11].
Vitrification eliminates ice crystal growth by ultrarapid freezing of the cells in a mixture of concentrated CPAs [7]. It has been well established that embryo survival and clinical outcomes are improved by vitrification [7,12]. In addition, vitrification needs no expensive equipment and requires less time in comparison to the slow freezing [7,12]. Based on these reasons, vitrification is being used by almost every fertility treatment center worldwide [13,14].
A great variation exists in vitrification protocols, most of which are cryopreservation devices (Cryotop ® , Cry-oTip ® , Cryolock ® , Cryoloop, Open Pulled Straw, High Security Vitrification Straw, etc.); however, the acceptable result largely depends on the quality of the embryos, the duration of exposure to CPAs, and reduced volume of the solution used for loading the embryos on the device [15]. As the vitrification device and media (solutions) all have enormous impacts on the final results, some studies compare different vitrification methods [16][17][18]. Herein, the vitrification method means the use of a vitrification device and vitrification/warming media from the same manufacturer, and this nomenclature is often based on the name of the vitrification device.
Most of the previous studies have focused on identifying the best cryopreservation method with the highest clinical yields [7,12,14]. Although there are multiple commercial vitrification/warming methods available for ART use, no effort has been made to compare the benefits/risks and cost/efficiency ratios among them so far [15,19]. It is assumed that products provided by different companies may differ in terms of advantages and limitations. In addition, the availability of the product for ART clinics may also impact the clinical results due to the instability of their composition, especially during transit.
The aim of this retrospective study was to evaluate the post-warming survival rate and clinical outcomes of cleavage stage embryos vitrified/warmed using two commercial methods: CryoTouch method (RS Medical, Ravan Sazeh Co., Tehran, Iran) and Cryotop method (Kitazato BioPharma Co., Shizuoka, Japan).

Study population
This retrospective study was conducted to analyze the survival rate of cleavage stage embryos after vitrification/warming performed by CryoTouch and Cryotop methods, and the subsequent clinical outcomes in two infertility clinics located at Erfan Niayesh Hospital Tehran, Iran and Mehr Fertility Research Center, Rasht, Iran, from January 2018 to December 2020. The study protocol was done according to the Ethical Principles for Medical Research and approved by the Institutional Review Board of each center.
The details about patients' basic characteristics, protocol of ovarian stimulation, ICSI procedure, embryo culture and evaluation methods, vitrification and warming protocols, FET protocol, and clinical outcomes were all obtained from registered documents at the mentioned infertility clinics. A total of 978 FET cycles involving embryos vitrified/warmed on day 3 of development were evaluated to enter the study based on the following inclusion/exclusion criteria: The inclusion criteria were age range between 20 and 35 years old, body mass index (BMI) <30 kg/m 2 , serum level of follicle-stimulating hormone (FSH) < 10 mIU/ml on day 3 of menstrual cycle, having more than 8 cleavage stage embryos produced by ICSI procedure, and undergoing the first ICSI/FET cycle with gonadotropin-releasing hormone (GnRH) antagonist protocol for ovarian stimulation.
The exclusion criteria included the presence of anatomical uterine anomalies, space-occupying lesions, hormonal dysregulations, ovarian hyperstimulation syndrome (OHSS), inflammatory disorders, hydrosalpinx, endometriosis, autoantibodies, history of ectopic pregnancy, repeated implantation failure (RIF), miscarriage, and having less than 8 cleavage stage embryos produced by ICSI procedure.

Ovarian stimulation protocol
GnRH antagonist protocol was performed in all patients. Table 2 provides ovarian stimulation characteristics and final outcomes in patients undergoing an ovarian stimulation procedure. First, estradiol valerate (2 mg, PO, BID; Aburaihan Co., Tehran, Iran) was administered from day 21 of the natural cycle and continued until days 2-4 of the subsequent cycle. Then, follicular growth was stimulated by administration of recombinant FSH (150-225 IU, daily; Gonal F, Merck, Germany) from day 2 or 3 of the cycle. The prescribed dose was adjusted based on the follicular growth

Cleavage stage embryo quality evaluation
Cleavage stage embryo quality was assessed by recording morphological parameters according to the literature [20]. The morphological parameters included the number and symmetry of blastomeres, degree of fragmentation (anucleate structures of blastomeric origin), presence of multinucleation, and presence of abnormalities in intracytoplasmic and extracytoplasmic compartments. Highquality cleavage stage embryos were defined as those with 4 blastomeres on day 2 or 6-8 blastomeres on day 3, having symmetric blastomeres, having less than 15% fragmentation, without multinucleation, without intracytoplasmic abnormalities such as vacuoles and inclusion bodies, and without extracytoplasmic abnormalities such as granularities in the perivitelline space and zona pellucida dysmorphism. Otherwise, the embryos were considered as low-quality embryos.

Vitrification and warming procedures
High-quality cleavage stage embryos were selected for the vitrification with two commercial methods and the procedure was performed based on the manufacturer's protocol, CryoTouch method (RS Medical, Ravan Sazeh Co., Tehran, Iran) or Cryotop method (Kitazato BioPharma Co., Shizuoka, Japan). The method means the use of vitrification/warming media and vitrification devices from the same manufacturer. CryoTouch and Cryotop methods had similar protocols for vitrification and warming procedures with a little difference. Briefly, the equilibration was performed in equilibration solution for 7 min in CryoTouch method or 10 min in Cryotop method, both at room temperature. Afterwards, the embryos were placed in vitrification solution for 50-60 s in both protocols. Then, the embryos (maximum 3 in each vitrification device) were immediately aspirated with a minimum volume of the vitrification solution and placed onto the tip of CryoTouch ® or Cryotop ® vitrification devices. The loaded devices were immediately submerged vertically into liquid nitrogen, then placed in a goblet and stored in a liquid nitrogen tank.
In the morning of FET, the embryos were warmed in the corresponding commercial media based on the manufacturer's protocol. Briefly, the vitrification device was quickly removed from liquid nitrogen and immersed in a warming solution (prewarmed at 37 °C). The embryos were detected and immediately transferred to another droplet of warming solution and incubated for 1 min. Subsequently, the embryos were transferred to the dilution solution and incubated for 3 min. In the next step, the embryos were transferred to the washing solution and maintained for 5 min. Ultimately, the embryos were placed in embryo culture medium and incubated at 37 °C in 6% CO 2 until ET.

Embryo survival assay
The vitrified/warmed embryos were considered survived if they had 50 % or more viable blastomeres with no evidence of degenerated morphology.

FET cycle
Hormone replacement therapy (HRT) was performed for preparing the endometrium as a standard protocol. Estradiol valerate (6 mg/day, PO; Aburaihan Co., Tehran, Iran) was administered from days 2-3 of the menstrual cycle and continued up to 8 mg/day until the endometrial thickness reached 8 millimeters. Progesterone (400 mg, suppository, BID; Cyclogest, Actavis, England, UK) was initiated when the endometrial thickness was upper 8 mm. In the presence of a positive result for β-hCG test, the estradiol and progesterone administrations were continued until weeks 6 and 12 of gestation, respectively. ET was conducted using an embryo transfer catheter (Guardia ™ Access, Cook, USA) by an expert gynecologist under the guidance of ultrasound, based on the guideline provided by the American Society for Reproductive Medicine (ASRM). Single or double high-quality cleavage-stage vitrified/warmed embryos were selected for each ET cycle.

Outcome assessment
The embryo post-warming survival rate was calculated as the percentage of survived embryos, based on the mentioned definition, after the warming procedure.
The clinical pregnancy rate was calculated from the number of observed gestational sacs by ultrasonography per embryo transfer.
The implantation rate was calculated from the number of observed gestational sacs by ultrasonography per the number of transferred embryos.
The live birth rate was calculated from the number of live births per embryo transfer.

Statistical analysis
GraphPad Prism (GraphPad Software, USA) was used to analyze all the obtained data. Comparisons of the means were performed by Student's t test. The P value < 0.05 was considered as the level of significance. Data are represented as mean ± standard deviation (SD).

Results
A total of 173 ICSI/FET cycles have entered the study based on the inclusion/exclusion criteria. Among these cycles, 95 vitrification/warming cycles were conducted by the CryoTouch method and 78 vitrification/warming cycles were performed using the Cryotop method. As shown in Table 1, there were no significant differences between the two groups in mean age, BMI, duration of infertility, type of infertility (primary or secondary), serum level of day 3 FSH, serum level of anti-Mullerian hormone (AMH), and sperm count of the spouse. Table 2 represents the characteristics of the studied patients during ovarian stimulation including the total dose of administered gonadotropin, duration of gonadotropin administration, serum levels of luteinizing  hormone (LH), estradiol (E2), and progesterone (P) on trigger day, number of retrieved COCs, and percent of high-quality cleavage stage embryos resulted from ICSI procedure. Based on the obtained data, there were no significant differences in these characteristics between the patients who were undergone ICSI/FET cycles using the CryoTouch or Cryotop methods. A total of 446 cleavage stage embryos were warmed and 173 ET cycles were performed using double high-quality embryos. Figure 1 demonstrates the warmed embryos in two groups. Among 446 embryos, 233 embryos were vitrified/warmed using the CryoTouch method and 213 embryos were vitrified/warmed using the Cryotop method.

Discussion
The present study detected no differences in post-warming survival rate, and clinical pregnancy, implantation and live birth rates of cleavage stage embryos that were undergone vitrification/warming procedures using two commercial methods, the CryoTouch method and Cryotop method. Vitrification is the method of choice for embryo and gamete cryopreservation due to its simplicity, high efficiency, and low cost [12]. Although vitrification provides a high embryo survival rate and good clinical outcomes [6,14], some concerns remain regarding the toxic effects of its ingredients which are used at high concentrations during manufacturing [21]. Most of the commercial vitrification media contain dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), or other polymers, all of which have some degrees of cellular toxicity [22]. The main goal during the formulation of vitrification media is to create a mixture of CPAs with the lowest toxicity [22]. The high concentration of CPAs needed for the best efficiency of vitrification accompanies with the increased probability of cryoprotectant toxicity [8]. Vitrification media of the Cryotop method contain DMSO and EG as CPAs [22,23]. As declared by the company, vitrification media of the CryoTouch method also contain the same components. The observed similarity in embryo survival rate after vitrification/warming using the commercial media of these two companies somehow indicates their equality in efficiency for human embryo vitrification.
It has been revealed that prolonged exposure to CPAs enhances the probability of embryo toxicity during vitrification procedure [24,25]. Based on this, an optimal balance is required between the concentration of used CPAs and the speed of the procedure to obtain the best result. This delicate adjustment will minimize the likelihood of possible toxicity as well as osmotic injury which is prevalent when the embryos are held in media without mineral oil overlay during vitrification procedure [12]. The obvious difference between the vitrification protocols of CryoTouch and Cryotop methods is the incubation time of the equilibration solution as the CryoTouch method needs lesser incubation time compared to the Cryotop method (7 min for CryoTouch versus 10 minutes for Cryotop). This difference in incubation time may be due to different concentrations of cryoprotectants in two methods. However, this difference made no discrepancies in embryo survival rate and clinical outcomes.
Several vitrification protocols have been introduced so far; however, the reported clinical success rates are not homogeneous, possibly due to the technical differences [14,26,27].   [6] evaluated the qualities of 6019 embryos undergone vitrification/warming using the Cryotop method in different developmental stages and concluded that nearly 95% of the cleavage stage embryos had 100% of intact blastomeres. In line with this study, we showed ~90% embryo survival rate using both CryoTouch and Cryotop methods. The Cryotop method for vitrification/warming is being widely used for human embryo and oocyte cryopreservation [13,20,28] and its efficiency is proven [6,13,20,28]. To the best of our knowledge, there is no published data from the Cryo-Touch method despite its routine use in infertility clinics of Iran, and data presented here show its comparable efficiency with the Cryotop method.
The number of reported live births obtained from vitrified/warmed human embryos has enormously increased over the past decades [13,27]. However, different rates have been reported for implantation, clinical pregnancy, and live births. Cobo et al. reported an implantation rate of 34.6%, a pregnancy rate of 41%, and a live birth rate of 39.2 % with vitrified/warmed embryos [6]. Desai et al. performed 200 FET cycles with vitrified/warmed day 3 embryos in patients under 38 years old and reported 24% of implantation rate and 45% of clinical pregnancy rate [29]. Debrock et al. showed 16.1% of implantation rate and 20.7% of live birth rate in a randomized controlled trial conducted using vitrified/warmed embryos [29]. These high variable results in clinical outcomes may be explained by the differences in the possible contributors such as the developmental stage of transferred embryos, the selection criteria for vitrification, protocol of vitrification/warming, the selection criteria for ET, and patients' characteristics and medication protocols. La Marca et al. conducted a retrospective study which included 2617 embryos from 630 patients and showed 30.6% of live birth rate after the first FET on day 2 or 3 of embryonic development [13]. They used the Cryotop method for embryo vitrification/warming. Our results for clinical pregnancy, implantation, and live birth rates were all comparable with previous studies. In addition, we demonstrated no differences in implantation, clinical pregnancy, and live birth rates between embryos vitrified/warmed using Cry-oTouch and Cryotop commercial methods.

Conclusions
In conclusion, similar outcomes were observed in cleavage stage embryos vitrified/warmed using CryoTouch and Cryotop methods. These results provide strong evidence regarding the comparable efficiency of the CryoTouch versus Cryotop method. This is promising for infertility clinics in Iran that are using the CryoTouch method due to its availability, low cost, and prolonged expiration date as they are domestically produced in Iran.