Abstract
Objective
This study aimed to compare the effects of two brands of commercial vitrification carriers on pregnancy outcomes in freeze–thaw cycles.
Methods
We included 4871 patients who underwent a “freeze all” strategy using the commercial carriers J.Y. straw and OYASHIPS straw in the Reproductive Center of the First Hospital of Jilin University. The pregnancy outcomes of cleavage-stage embryos and blastocysts were studied separately. Detailed data and the safety of children born from mothers with the two types of carriers were also compared.
Results
Patients who used J.Y. straw had similar clinical pregnancy and live birth rates with one and two cleavage-stage embryo transplantation to those who used OYASHIPS straw. In patients who had blastocyst transplantation, the clinical pregnancy rate of one blastocyst transplanted in those who used OYASHIPS straw was significantly higher than that in those who used J.Y. straw (57.85% vs 47.09%). Among children born from mothers who used J.Y. straw, the congenital disability rate was significantly higher than that in those with OYASHIPS straw.
Conclusion
The OYASHIPS straw carrier is cheap and can achieve clinical pregnancy and live birth outcomes comparable to those of J.Y. straw. Therefore, OYASHIPS straw is a good alternative option.
Keywords
Introduction
Embryo and gamete freezing, which is widely used in reproductive centers worldwide, has resulted in many infertile couples having children. 1 After nearly 40 years of development and continuous improvement, embryo cryopreservation technology has become skilled and integral to human-assisted reproductive technology (ART). Embryo cryopreservation can effectively solve the preservation problem of remaining embryos and improve the utilization rate of embryos in a single oocyte retrieval cycle and the cumulative pregnancy rate. 2 The “freeze all” strategy, which has become popular recently, can effectively reduce the risk of ovarian hyperstimulation syndrome and improve the safety of ART. The commonly used freezing methods can be divided into conventional slow freezing and vitrification. Slow freezing dehydrates the embryos with a low concentration of cryoprotectant gradient and slowly cools them down through a programed freezer. Vitrification freezes embryos in high concentrations of cryoprotectant, and rapid cooling to form a vitrified state can avoid cell death caused by ice crystal formation.3–5 Many studies have compared the freezing outcomes of the two methods during the transition from slow freezing to vitrification.5–7 The vitrification technique yields the same pregnancy rate as slow frozen blastocyst transfer. 8 Slow freezing has gradually been replaced by vitrification because of problems, such as the easy formation of ice crystals, expensive equipment, and long-term exposure to protective agents. 9
With the popularization of vitrification and freezing technology, vitrification and freezing carriers have been developed and applied, further improving the effect of vitrification and freezing. This method uses many types of commercial10–12 and non-commercial13–15 vitrification carriers. Regardless of whether the embryos are directly in contact with liquid nitrogen when the embryos are stored in a liquid nitrogen tank after freezing, the carriers can be divided into three categories: fully open carriers, 16 open cooling and closed storage carriers, 17 and closed carriers. 18 The preferential use of open or closed carriers has been the focus of discussion among embryologists 19 , 20 because open carriers are in direct contact with liquid nitrogen during cooling and storage, and there is a risk of mutual transmission. However, despite the potential risk of contamination when storing embryos, 21 , 22 open carriers are widely used because of their high cooling and thawing rates. Furthermore, many studies have shown that the effect of open and closed carriers for vitrification is equivalent.23–25
Currently, many commercial carriers can be used, and the mainstream carriers are Cryoloop, Cryotop, and J.Y. straw. Unfortunately, little is known about the differences in the freeze–thaw effect on pregnancy using different brands of carriers. We retrospectively analyzed the pregnancy outcomes of two open vitrification carriers (J.Y. straw and OYASHIPS straw) used by our center for embryo freezing and thawing cycles to provide evidence for selecting suitable freezing carriers in the future. OYASHIPS straw is half the cost and would be a good choice if it could achieve a similar pregnancy outcome to that of J.Y. straw.
Methods
Study design
This was a retrospective and longitudinal study of couples who underwent embryo transfer in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles at the Reproductive Center of the First Hospital of Jilin University. We included patients who received freeze–thaw embryo transplantation from January 2015 to December 2021, and they were divided into groups according to the carrier used in the embryo freezing procedure. Among them, patients who used the frozen carrier J.Y. straw (JieYing Laboratory Inc., Longueuil, Quebec, Canada) had 2630 cycles (J.Y. group). Patients who used the OYASHIPS frozen carrier (Jiangxi Zixing Biotechnology Co., Ltd., Nanchang, China) had 2241 cycles (OYASHIPS group) (Figure 1). Embryos were divided into two subgroups of the cleavage stage and the blastocyst stage. Exclusion and inclusion criteria were as follows: (1) the couple had normal chromosomes, and the man had no AZF gene microdeletion; (2) IVF/ICSI was performed for the first time and the fresh cycle transplant was canceled; and (3) there were results of the first freeze–thaw cycle. This study was approved by the Ethics Committee of First Hospital of Jilin University (approval number: 2021-741-1), and all patients signed informed consent for embryo vitrification. We de-identified all patients’ details. The reporting of this study conforms to the CONSORT guidelines. 26
Two commercialized vitrification carriers. (a) J.Y. straw (orange) and OYASHIPS (blue) vitrification carriers and (b) An enlarged image of the front end of the carrier rod.
Ovarian stimulation and IVF
Ovarian stimulation was performed according to our previously described methods. 27 A long luteal downregulation protocol was used for experimental subjects and controls. A gonadotropin-releasing hormone agonist (Triptorelin, Ferring, Germany) was administered in the previous cycle’s mid-luteal phase. Human menopausal gonadotropin (Lebaode; Lizhu Ltd., Zhuhai, China) was administered by intramuscular injection from day 3 of the menstrual phase. Subsequently, 5 to 7 days after human menopausal gonadotropin was administered, ovarian follicles were monitored and evaluated by a type-B ultrasound examination and serum estradiol concentrations. The gonadotropin-releasing hormone agonist and human menopausal gonadotropin administration was stopped when two or more ovarian follicles reached ≥18 mm in diameter, and then 10,000 IU human chorionic gonadotropin (hCG) (Lizhu Ltd.) was administered. Oocyte retrieval occurred 36 to 40 hours after hCG administration. IVF or ICSI was performed 3 to 4 hours after oocyte retrieval. Fertilization was observed at 16 to 20 hours after fertilization, and the embryos were observed at 72 and 120 hours. .The embryos that met the freezing requirements were vitrified and stored in liquid nitrogen. Embryo culture at the cleavage stage in most patents with the J.Y. carrier occurred in 5.5% CO2, and blastocyst culture was performed in three gas culture conditions (5% O2, 6% CO2, and 89% N2). Embryo culture in the cleavage stage in most patients with the OYASHIPS carrier was also performed in three gas culture conditions.
Embryo freezing and thawing
After oocyte retrieval, embryo freezing was performed on days 3, 5, and 6. On the third day, two to eight cleavage-stage embryos were chosen according to the patient’s wishes for freezing, and the remaining embryos were cultured with blastocysts. On days 5 and 6, embryos that met the criteria for usable blastocysts were frozen according to Gardner and Schoolcraft’s description. 28 , 29 The freezing and thawing reagents were SAGE (JieYing Laboratory Inc.) and KITAZATO (Kitazato Corporation, Fuji, Shizuoka, Japan). The general embryo freezing procedure comprised placing the embryos in equilibration solution for 5 to 10 minutes and transferring them to the vitrification solution when the embryo shape recovered to >80%. The embryos were placed on freezing carriers and quickly immersed in liquid nitrogen for preservation. The blastocysts were laser shrunken before freezing. When thawing, the carrier was immersed in the thawing solution at 37°C within 1 minute, and then the embryos were transferred to the diluent for 3 minutes and then to washing solutions 1 and 2 for 5 minutes. The embryos were transferred to the embryo culture medium for later embryo transfer. The cultivation environment was 37.0°C and 5% CO2. Cleavage-stage embryos were cultured overnight and then transferred, and blastocysts were transferred approximately 2 hours after thawing.
Endometrial preparation and embryo transfer
The endometrium was prepared using artificial or natural cycles, and embryo transfer was performed under abdominal ultrasound guidance. In the artificial cycle, the patient was examined by B-ultrasound on the second to fourth days of the menstrual cycle. Oral estradiol valerate was taken. After 10 to 14 days of continuous medication, B-ultrasound was repeated. The thickness of the endometrium was measured, and the serum estradiol concentration was measured. The transplantation period was determined when the patient’s endometrial thickness reached 7 mm. A total of 60 mg of progesterone was injected intramuscularly 3 to 5 days before transplantation. In the natural cycle, The patient underwent a B-ultrasound examination on the 9th to 11th days of the menstrual cycle to monitor ovulation and determine the transplantation period. The patient was administered 40 mg progesterone by intramuscular injection 3 to 5 days before transplantation. Blood hCG concentrations were checked on the 14th day after transplantation, and B-ultrasonography was performed for positive (hCG concentrations >5.3 mIU/mL) patients 30 days after transplantation. The presence of intrauterine gestational sacs was recorded as a clinical pregnancy, and the number of gestational sacs was recorded. Patients with a clinical pregnancy were followed up, and miscarriage and continued pregnancy were recorded.
Observational indicators
We collected data on the general information of the patients, the embryo resuscitation survival rate (survived embryos after thawing/total thawed embryos), the positive ß-hCG rate (positive ß-hCG cycles/frozen–thawed embryo transfer cycles), the clinical pregnancy rate (clinical pregnancies/freeze–thaw embryo transfer cycles), ongoing pregnancy cycles, and clinical pregnancy cycles before the follow-up time. Additionally, we collected data on the non-live birth rate (cycles without live births to follow-up/clinical pregnancy cycles to follow-up) and live birth rate (cycles of live births to follow-up/clinical pregnancy cycles to follow-up).
Statistical analysis
IBM SPSS Statistics for Windows, Version 23.0, software (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. All numerical data are presented as the mean value ± standard deviation. Data were tested for normality using the Shapiro–Wilk and Kolmogorov–Smirnov tests. Data with a normal distribution were compared by Student’s t-test, while those that did not were compared by the Mann–Whitney U-test or Kruskal–Wallis test. With regard to categorical variables, data are expressed as the number and percentage of the total number and the total and percentage of the total, and were compared by Pearson’s chi-square test. A two-sided P value <0.05 indicates a statistically significant difference.
Results
Comparison of general data of the patients
A total of 4871 patients who received freeze-thaw embryo transfer were included in the study, including 2630 patients who used the J.Y. carrier and 2241 patients who used the OYASHIPS carrier during freezing. In the J.Y. group, there were 2103 cases of thawing and transfer of cleavage-stage embryos and 527 cases of blastocysts. In the OYASHIP group, 557 cycles were natural cycle transplantation, and 2073 (78.82%) were artificial cycle transplantation. In the J.Y. carrier group, 513 cycles were natural cycle transplantation, and 1728 (77.11%) were artificial cycle transplantation. The mean ages of women were 32.49 ± 4.71 and 32.77 ± 4.29 years, and the those of men were 34.13 ± 5.47 and 34.11 ± 4.89 years in the J.Y. and OYASHIPS groups, respectively. Primary infertility accounted for 59.01% in the J.Y. group and 61.76% in the OYASHIPS group. Among the infertility factors, pelvic tubal factors showed the highest rate in both groups. Regarding the stimulation regimen, the J.Y. group mainly used the gonadotropin-releasing hormone antagonist regimen, and the OYASHIPS group primarily used the gonadotropin-releasing hormone agonist regimen. The main method of fertilization in both groups was IVF. Finally, we also compared the concentrations of female basal hormones between the two groups. Anti-Müllerian hormone, follicle-stimulating hormone, estradiol, prolactin, and progesterone concentrations were significantly different between the groups (all P < 0.01) Table 1.
Comparison of basic clinical information in patients using two carriers.
Data are shown as n, n (%), or the mean ± standard deviation. “/” indicates that data were only described between the two groups without conducting a statistical analysis.
ART, assisted reproductive technology; GnRH, gonadotropin-releasing hormone; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol.
Pregnancy outcomes of cleavage-stage embryos transferred with two types of carriers
The number of cleavage-stage embryos that were frozen and thawed, and the number of embryos that survived in both groups are shown in Table 2. In the statistical analysis of pregnancy outcomes, we divided the transfer of embryos into one or two embryos. In patients with one embryo transferred, the endometrial thickness and the proportion of endometrial morphological types were not different between the two groups. The rates of positive ß-hCG cycles clinical pregnancy cycles and live birth cycles were not significantly different between the two groups. Among them, 18 patients in the OYASHIPS group had not reached the follow-up time and continued to be pregnant. In patients with two embryos transferred, the endometrial thickness and the proportion of endometrial morphological types were not different between the two groups. The rates of positive ß-hCG cycles, clinical pregnancy cycles, and live birth cycles were not different between the two groups Among them, 62 patients in the OYASHIPS group did not reach the follow-up time and continued to be pregnant (Table 2).
Pregnancy outcomes of cleavage-stage embryos transferred with two types of vitrification carriers.
Data are shown as n, n (%), or the mean ± standard deviation. “Rate” refers to calculations described in “Observational indicators.”
One case was lost to follow-up.
hCG, human chorionic gonadotropin.
Pregnancy outcomes of blastocysts transferred with two types of carriers
The number of blastocysts that were frozen and thawed, and the number of blastocysts that survived in both groups are shown in Table 3. The ratio of blastocyst transplantation in the OYASHIPS group was 66.0% (1479/2241), with 415 cycles of live births. In the J.Y. group, the ratio of blastocyst transplantation was only 20.04% (527/2630), with 253 cycles of live births. With regard to pregnancy outcomes, in patients with one blastocyst transferred, the endometrial thickness and the proportion of endometrial morphological types were not significantly different between the two groups. The rate of positive ß-hCG cycles was not different between the two groups. However, the rate of clinical pregnancy cycles was significantly higher in the OYASHIPS group than in the J.Y. group (P = 0.008). A total of 215 patients in the OYASHIPS group did not reach at the follow-up time and continued to be pregnant. The number of live birth cycles was not different between the two groups. In patients with two blastocysts transferred, the endometrial thickness and the proportion of endometrial morphological types were not different between the two groups. The rates of positive ß-hCG cycles, clinical pregnancy cycles, and live birth cycles were not different between the two groups. Among them, 1 patient in the J.Y. group and 137 patients in the OYASHIPS group did not reach the follow-up time and continued to be pregnant (Table 3).
Pregnancy outcomes of blastocyst-stage embryos transferred with two types of vitrification carriers.
Data are shown as n, n (%), or the mean ± standard deviation. “Rate” refers to calculations described in “Observational indicators.”
hCG, human chorionic gonadotropin.
Comparison of neonates born from mothers with two types of vitrified cryopreservation carriers
We compared the birth outcomes and health at 1 year of follow-up of the two groups of carriers. The number of live births in the J.Y. group was 1386, including 750 singletons and 318 twins. The OYASHIPS group had 705 live births, including 491 singletons and 107 twins. The proportion of single embryo transplantation in the OYASHIP group was 55.56% (1245/2241), resulting in 82.11% (491/598) of single live births. The proportion of single embryo transplantation in the J.Y. group was 12.13% (319/2630), with 70.22% (750/1068) of single live births (Table 4).
Comparison between two types of vitrification carriers in live born newborns.
The male-to-female ratio at birth was 0.9971:1 in the J.Y. group and 1.1235:1 in the OYASHIPS group. Birth weight was significantly heavier in the OYASHIPS group than in the J.Y. group in boys and girls (both P < 0.01). The majority of neonates had a normal weight in both groups. The congenital disability rate was significantly higher in the J.W. group than in the OYASHIPS group (P = 0.017). After 1 year of birth, we conducted a follow-up of general health. Four children in the J.Y. group had health problems or died, and those in the OYASHIPS group were all healthy (Table 4).
Discussion
Cryopreservation of remaining embryos in a fresh IVF/ICSI cycle reduces the use of ovulation induction drugs and repeated oocyte retrievals, reducing the clinical risk of IVF and reducing the cost to the patient. Freeze–thaw cycles are an excellent option for patients with failed fresh cycle transplantation, a risk of ovarian hyperstimulation, a poor endometrial environment, and the need to reproduce. With the continuous maturity of embryo freezing technology, the implantation potential of frozen embryos after thawing is similar to that of fresh embryos, and freeze–thaw embryo transfer has become an important supplement to fresh cycle embryo transfer. 30
The principle of embryo cryopreservation is to place the embryo in an ultra-low temperature environment through special protection measures and cooling procedures. Additionally, this cryopreservation uses a low temperature to reduce the level of cell metabolism, inhibit cell life activity, and enable cells to be preserved for a long time, and then resume metabolism after thawing. Cryo/thawing technology is an important factor affecting the outcome of embryo transfer. Vitrification has gradually replaced slow freezing because ice crystals are formed during freezing, which is time-consuming and requires expensive freezing equipment. Whether to use an open or closed carrier has been a controversial choice for embryologists in embryo freezing. Although the existing research evidence has shown no significant difference in the cryopreservation performance of the two types of carriers, 19 open carriers are more important in embryo laboratories. Generally, the goal is to achieve the fastest possible cooling and thawing rates. 24 A study by Aizer et al. showed that the number of cleavage-stage embryos or blastocysts placed on a single cryopreserved carrier affected survival rates, and the best results were obtained when placing three cleavage-stage embryos or one blastocyst. 31
Currently, many commercial carriers are suitable for vitrification and freezing, but there is still no systematic comparison of the cryo/thawing effects of the different carriers. In 2018, Guerrero et al. compared the freezing effects of two closed carriers, High Security Vitrification and SafeSpeed. 18 They showed that both closed carriers achieved comparable outcomes in terms of survival of blastocysts in the vitrification process. AbdelHafez et al. compared the freezing effect of three carriers, which comprised two closed carriers (High Security Vitrification straw and Cryotop) and one open carrier (Cryoloop). 23 They found that the cryopreservation rate of the closed carrier was lower than that of the open carrier, probably because the closed carrier is more technically difficult to operate. Chu et al. compared the freezing and thawing effects of three commonly used open vectors (Cryoloop, Cryotop, and Cryoleaf) in human cleavage-stage embryos, blastocyst-stage embryos, and oocytes. 16 They found that the first-trimester miscarriage rate was significantly higher in Cryoloop than in the other two carriers. However, this difference in results may not have been related to the vitrification carrier because the women in the Cryloop group were significantly older than those in the other groups. Kader et al. wrote a review of the factors affecting the outcome of the human blastocyst vitrification system and summarized the effects of different types of carriers on survival and pregnancy rates. 32 The survival rate ranged from 63% and 100%, and the pregnancy rate ranged from 31% to 56%. Munck et al. compared the freezing effect of open and closed oocyte vitrification devices in an oocyte donation program. 17 They found that the CryotopSC device (open vitrification and closed storage) and the CBSvit device (closed vitrification and closed storage) showed similar survival rates.
No previous studies have compared the freezing efficiency of the J.Y. and OYASHIPS carriers. These two devices are open carriers, but their structures are slightly different. The front end of the OYASHIPS carrier is narrower and thinner than the J.Y. carrier. A thinner front end results in as faster cooling speed and thawing speed. Additionally, the curved surface design of the front end of the OYASHIPS carrier is conducive to loading embryos and absorbing excess liquid, making it easier to freeze. As a result, the amount of liquid carried is less, which is beneficial for temperature conduction. The J.Y. carrier is safe and reliable in clinical practice. However, the cost of the J.Y. carrier is approximately twice that of the OYASHIPS carrier. This study systematically retrospectively compared the freezing efficiency of these two vectors for cleavage-stage and blastocyst-stage embryos. The recovery rates of cleavage-stage embryos and blastocysts after freezing by these two vectors were > 99%, with no significant difference between the two groups. In addition to the embryo recovery rate, the developmental potential after embryo recovery (i.e., the ability of the embryo to continue to grow and implant), can better reflect the extended effect of the freezing process on embryonic development. This study showed no significant difference in the live birth outcomes of frozen embryos thawed by the two carriers, regardless of cleavage-stage embryos or blastocysts. This finding indicated that the freezing effect of the OYASHIPS carrier was not different from that of the J.Y. carrier.
In the assessment of fetal safety, we found that the weight of children born from mothers who used the OYASHIPS carrier was higher than that of J.Y. carrier. Additionally, the congenital disability rate of children born from mothers who used the OYASHIPS carrier was significantly lower than that with J.Y. carrier. With regard to the higher birth weight, one reason for this finding is that the OYASHIP carrier has a higher proportion of single embryo transplantation. Zhu et al. showed that the neonatal birth weight was related to single or twin pregnancies, and the body weight of single births was higher than that of twins. 33 The number of embryos transferred is related to the proportion of full-term birth. We found that the rate of full-term birth was 85.45% in the OYASHIP group and 78.46% in the J.Y. group, which may have led to the higher birth weight in the OYASHIP group. Another reason for our finding may be that the OYASHIP carrier has a higher proportion of blastocyst transplantation. Zhang et al. showed that an extended in vitro culture time might affect birth weight. 34 In addition, another study demonstrated that transplantation with an artificial cycle led to a higher mean birth weight than that from a natural cycle. 35 However, in our study, there was no difference in the proportion of the artificial cycle between the two groups. Notably, Castillo et al. showed that the incubator culture environment could affect perinatal outcomes. 36 Unfortunately, we could not trace the specific proportion of this change in our study. We will continue to follow body weight in the future. A reason for the difference in the congenital disability rate between the groups may be that the OYASHIPS group had a higher non-live birth rate, including ectopic pregnancy, spontaneous abortion, and medical abortion/introduction, than the J.Y. group.
In summary, the OYASHIPS carrier is safe and available, and the cost is more than half that of the J.Y. carrier. However, this study has some limitations. We did not perform a prospective study, and the data were not propensity scored. In future studies, we will focus on the efficiency and safety of different vitrification carriers and choose the most suitable carrier.
Footnotes
Author contributions
HBZ, LLL, HGZ, and QX contributed to the concept of the study, data collection and analysis, and writing the manuscript. YTJ and RZL contributed to statistical analysis. All authors read and approved the final version of the manuscript.
Declaration of conflicting interests
The authors declare that there is no conflict of interest.
Funding
This study was supported by the Science and Technology Department of Jilin Province, China. (20210502001ZP).
