To compare the amino acid differences of changes of frozen-thawed early-stage human embryos and fresh cultured early-stage human embryos.
To compare the amino acid differences of changes of frozen-thawed early-stage human embryos and fresh cultured early-stage human embryos.
Discarded embryos and their in vitro culture medium of patients who underwent in vitro fertilization-embryo transfer (IVF-ET) at the Research Center for Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University, from September 2010 to April 2011 were collected. Amino acid levels were determined by high performance liquid chromatography.
The amino acid differences of changes in the culture medium of fresh embryos (661.50 μmol/L) were significantly higher than in the medium of post-thawed embryos (232.00 μmol/L) at 0.5 h (P < 0.001). At 1 and 2 h, no significant difference of change was found in all amino acids. Differences in the concentration of amino acids between post-thawed embryos and blank control medium were already present beginning at 1 h.
The level of amino acid metabolism of frozen-thawed early-stage human embryos has already recovered from the state of metabolic stagnation during cryopreservation at 1 h of incubation after thawing, and the amino acid metabolism level at that time approximates that in fresh embryos before freezing. This may be established as the optimal embryo transfer time in IVF-ET.
Frozen-thawed embryo transfer (FET) is an important complementary method to in vitro fertilization-embryo transfer (IVF-ET). FET can increase the patient's cumulative pregnancy rate per oocyte retrieval cycle and reduce the incidence of ovarian hyperstimulation, and it is of greater importance for patients who are not suitable for a fresh embryo transfer cycle due to endometrial or other factors.[1-3] FET has been utilized for nearly 30 years, but the success rate of FET at most reproductive centers is still low.[1, 4] Studies have found that the factors affecting the success rate of FET are primarily the quality of the frozen-thawed embryo, number of embryos transferred, endometrial synchronization, recovery of the frozen-thawed embryos, and culture time in vitro.[5, 6] Of these, determining the quality of embryos, fresh or frozen-thawed, is primarily based on morphological assessment and this has been shown to be a relatively poor predictor of embryo health and success rate.[7, 8] As for the duration of in vitro culture after thawing, day-3 embryos (D3) (3 days after oocyte retrieval) are often transferred after 2–4 h of incubation after thawing. However, there is no theoretical basis for this timing of transfer, and amino acid metabolism in early-stage frozen-thawed human embryo is still not clear.
Amino acids play an important role in early embryonic development, and participate in a number of important physiological processes. Twenty amino acids are involved in protein synthesis during embryonic development.[10, 11] Among these, glutamine is converted into glutamate via the aminotransferase, and then decomposes to α-ketoglutarate and participates in the formation of adenosine triphosphate (ATP) via the citric acid cycle. Glutamine also has other functions including providing carbon and nitrogen for the de novo synthesis of purine and pyrimidine, serving as a reducing agent to protect cells from oxidative stress damage, and regulating glucose metabolism. Aspartic acid participates in metabolic regulation through the malate-aspartate shuttle mechanism, playing an important role in lactate metabolism. Methionine plays an important role in metabolism regulation and nucleotide synthesis, and primarily regulates metabolism through the methionine cycle, thus participating in DNA methylation. Histidine can be converted into histamine by histidine decarboxylase, and histamine plays a role in embryo implantation. Arginine is used to synthesize nitric oxide under the action of NO synthase, thereby participating in multiple signal transduction pathways in the embryo.
Amino acid metabolism is also related to the developmental potential of embryos, and many recent studies have examined amino acid differences of changes in developing embryos and found that it can be considered as an independent predictor of the clinical pregnancy rate and live birth rate after embryo transfer.[10-14] While some studies have examined differences between fresh and frozen-thawed embryos with respect to growth rate and incubation time,[7, 15] few have examined differences in amino acid metabolism. Stokes et al. examined amino-acid metabolism from day 2 to 3 in post-thawed embryos and found that an analysis of amino acid differences of changes could be used to predict which embryos will develop to the blastocyst stage regardless of the post-thaw grade.
Thus, the purpose of this study was to use high performance liquid chromatography (HPLC) to detect and compare the amino acid differences of changes of frozen-thawed early-stage human embryos and fresh cultured early-stage human embryos. These data may provide a basis for determining the appropriate timing for the transfer of frozen-thawed embryos.
Discarded embryos and their in vitro culture medium of patients who underwent IVF-ET at the Research Center for Reproductive Medicine, the Sixth Affiliated Hospital of Sun Yat-sen University, from September 2010 to April 2011 were collected. All patients receiving IVF-ET signed written informed consent, and a separate consent agreeing with the use of discarded embryos for research. The inclusion criteria for embryos were as follows: (1) Abnormally fertilized embryos (3PN). The pro-nucleus was checked on day 1 (D1) to determine whether the egg was fertilized. The presence of two pro-nuclei indicated normal fertilization, while the presence of three pro-nuclei indicated 3PN embryos. (2) D3 score; the embryos had 6–10 uniform blastomeres and <20% fragmentation. (3) After vitrification freezing and thawing, all the blastomeres of the embryo survived.
Controlled ovulation hyperstimulation was performed according to the conventional protocol of gonadotropin-releasing hormone agonist (GnRH-a) plus follicle stimulating hormone (FSH) plus human chorionic gonadotropin (HCG) in all patients. The GnRH-a was used for pituitary downregulation in the mid-luteal phase (about day 20 of the menstruation cycle). Gonadotropin (Gn) was used for controlled ovulation hyperstimulation 14 days after the downregulation. Follicle growth was monitored through transvaginal B-mode ultrasound after the administration of Gn. When ultrasound revealed at least one dominant follicle with a diameter ≥18 mm or two dominant follicles with a diameter ≥17 mm, intramuscular injection of HCG 10000 IU was performed at about 9 pm on that day, and transvaginal ultrasound-guided oocyte retrieval was performed after 36 h.
On the day of oocyte retrieval, standard semen analysis was carried out according to the World Health Organization (WHO) laboratory manual for the examination of human semen and sperm, and mobile sperm was collected using density gradient centrifugation and the sperm swim-up technique.
Oocytes were retrieved through the aforementioned ultrasound-guided puncture and were washed three times with human tubal fluid (HTF) medium (Quinn's Advantage fertilization HTF medium; Quinn's, SAGE, USA). Next, they were cultured in HTF medium until fertilization. The culture conditions were 5–6% CO2 at 37°C. Routine IVF insemination was carried out 38 to 40 h after injection of HCG.
The morning of D1 after oocyte retrieval, the granulosa cells were stripped off and the single fertilized egg was transferred into a 20-μL droplet of balanced and oil-covered cleavage medium (Quinn's, SAGE). The pronucleus was observed under microscopy to determine whether the oocyte was fertilized. Appearance of two pronuclei indicated normal fertilization, and appearance of three pronuclei (3PN) indicated abnormal fertilization. On the morning of D3 after oocyte retrieval, the embryo was observed and the morphological score was assessed according to the scoring and classification standard. Abnormally 3PN fertilized, discarded embryos on D1 that developed to D3 with 6 to 10 uniform blastomeres and <20% fragmentation were selected as study materials.
On D3, the single embryo was transferred into a 20 μL droplet of balanced and oil-covered blastocyst culture medium (Quinn's, SAGE) at 10 o'clock and cultured alone. To determine the metabolism level of the embryo before freezing, the embryos were randomly divided into three groups (0.5 h, 1 h, and 2 h) and a 15-μL sample of medium was collected from the culture at 0.5 h, 1 h, and 2 h after incubation and placed in a 0.6 mL EP tube, respectively. The embryos of the 0.5-h and 1-h groups were placed in the other drops of culture medium until 2 h when all media samples were frozen and stored at −80°C until needed. The medium in the same dish that was not used for embryo culture was collected for use as a blank control. Vitrification freezing was performed for all embryos 2 h after culture.
Thawed embryos were washed five times with culture medium, and then were placed in 20 μL of blastocyst medium and cultured continuously. For the thawed embryos, a 15-μL sample of medium was collected from the culture 0.5 h, 1 h, and 2 h after thawing and placed in a 0.6-mL EP tube, respectively. At the same time, 15 μL of medium in the same dish that was not used for embryo culture was collected for use as a blank control. The media samples were stored at −80°C until needed.
Embryo transfer was performed by specific personnel using a pipette with matching size. The inner diameter of the pipette was 140 μm, and the pipette was replaced after each embryo transfer. The culture medium transferred with the embryo was minimized during transfer (i.e. the amount of medium was the smallest amount required for transfer).
Vitrification solution A was HEPES solution containing 20% SPS, solution B contained 7.5% EG (Sigma) and 7.5% DMSO (Sigma), and solution C contained 15% EG, 15% DMSO, and 1.0 M sucrose. Embryos were rinsed in solution A for 1 min, and were then transferred into solution B and immersed for 2 min. Finally, they were transferred into solution C, then placed onto cryotip within 1 min, and then frozen in liquid nitrogen. Embryos were in solution C for no more than 30 s before being placed in liquid nitrogen.
The cryotip with the embryo was removed from the liquid nitrogen, and they were placed in 1.0 M sucrose thawing solution, 0.5 M sucrose solution, 0.33 M sucrose solution, and 0.2 M sucrose solution for 1 min, 1 min, 2 min, and 3 min, respectively. Next, they were put in HEPES solution with 10% SPS for 5 min. The embryos were individually transferred into 20 μL of blastocyst medium, and cultured in an incubator. All of the thawed embryos were cultured to 24 h after thawing, and were observed again to confirm that they had further development.
Frozen specimens were sent to the Guangzhou Analytic Testing Center, which is recognized by the Chinese state-level qualification (CMA) and the National Accreditation Service (CNAS), for chromatographic analysis. The management system conforms to ISO/IEC17025 and ISO/IEC 17020 requirements, and operation is in accord with ISO 9001 standards. Amino acid standards were purchased from Sigma (A9781: Amino acid standards for protein hydrolysates analytical standard; Sigma-Aldrich, St Louis, MO, USA). The standards were first dissolved in 0.1 M HCl as directed by the manufacturer, and serial dilutions were made as needed. Detection of all samples was done by the same staff, and they used the amino acid standard solutions alone for a pre-run test. The test samples were thawed at room temperature, and type 1100 HPLC (Agilent, Santa Clara, CA, USA) was used to detect the concentrations of 20 free amino acids in the specimens. The detection method was the pre-column derivatization and reversed-phase HPLC method, as previously described.[12, 13]
The Hypersil ODS column (Agilent) was used for HPLC (4.0 × 125 mm, particle size 5 μm). The composition of mobile phase A was 10 mmol/L Na2HPO4−/NaH2PO4+ pH 7.2 buffer (PBS) containing 0.5% (φ) tetrahydrofuran (THF). The composition of mobile phase B was PBS, methanol, and acetonitrile in a 50:35:15 volume ratio. The linear gradient was within 0–25% (mobile phase B increased linearly from 0% to 100%). The flow rate was 1.0 mL/min, column temperature was 40°C, detection time was 0–20.5 min, excitation wavelength was 340 nm, and emission wavelength was 450 nm for detection of primary amino acid derivatives. Proline and cysteine cannot be measured by reverse phase HPLC. Instead, taurine (which is byproduct of decarboxylated cysteine) and alanyl-glutamine (which can be degraded into alanine and glutamine) were measured. The amino acids were separated sequentially according to the time each amino acid stayed on column. A sample validatory chromatogram is shown in Figure 1.
The chromatograms of the test samples were compared to those of the amino acid standards with known concentrations, and the area under the curve (AUC) was converted into the concentrations of the amino acids of interest. The ‘external spike’ method was used to measure samples. Sample contents were obtained from the following formula: [(amino acids standards plus samples) minus (amino acids standards only)]. Embryos acted as their own self-control (i.e. amino acid metabolism of an individual embryo before and after cryopreservation was compared). Changes in the concentrations of amino acids in the culture medium relative to the control medium were calculated by deducting the median amino acid concentration in the control medium at a certain time point from the median amino acid concentration in the culture medium of the thawed embryo at the same time point.
The amino acid differences of changes were calculated as ‘turnover’ as previously described.[8, 12, 13] In brief, the increased or decreased amount of amino acid was obtained by deducting the concentrations of 20 amino acids in the control medium at a certain time point from the concentrations of 20 amino acids in the culture medium of the embryo at the same time point (if increased, the concentrations in the medium were greater than those in the control; if decreased, the concentrations in the medium were lower than those in the control). The absolute values of the increase or decrease in the concentrations of 20 amino acids were added to obtain the amino acid differences of changes. The amino acid differences of changes at different time points before embryo freezing and after embryo thawing was compared; However, we did not normalize the results as turnover as concentrations(moles)/embryo/h.
Continuous data were presented as mean ± SD or median with inter-quartile range (IQR; 25th and 75th percentile) depending on the normality of data. The differences in concentration of amino acids among the three time points in the control group were assessed by Kruskal–Wallis test. Differences in the amino acid concentrations between frozen-thawed embryos and fresh embryos at the three time points and amino acid differences of changes were determined by Wilcoxon sign rank test. All statistical assessments were evaluated at a two-sided alpha level of 0.05. Statistic analyses were performed using SAS version 9.2 statistics software (SAS Institute Inc., Cary, NC, USA).
At total of 54 fresh embryos from 54 couples were randomly divided into three groups (n = 18 in each group). The median age of the females was 31 (22, 34) years, and that of the males was 33 (30, 36.5) years. The median duration of infertility of the 54 couples was 1.5 (1, 3.5) years, and in seven couples infertility was due to tubal factors, in four couples it was due to tubal factors and endometriosis, in one couple it was due to tubal factors and a male fertility issue, in one couple it was due to male fertility, in one couples infertility was due to other problems. In two couples, the reasons for infertility were not known.
The concentrations of 20 amino acids were measured at 0.5, 1, and 2 h after incubation (three time points) (Table 1, Fig. 2). HPLC analysis showed that in the control culture medium, the concentrations of aspartate, taurine, alanyl-glutamine, glycine, serine and valine showed no significant difference (all, P > 0.05), while the concentrations of the other 14 amino acids were significantly different.
|Time after (μmole/L)|
Figure 3 presents the differences of changes in the concentration of amino acids between post-thawed embryo culture medium and blank control culture medium at 0.5, 1, and 2 h. Results were presented as median, and the difference of concentration of amino acid was defined as the median concentration in post-thawed embryo culture medium minus the median concentration in the blank control group culture medium at each time point. As shown in Figure 3, the difference was obviously increasing beginning at 1 h.
The amino acid differences of changes in the culture medium of fresh or post-thawed embryos at 0.5, 1, and 2 h are summarized in Table 2 and Figure 4. The median differences of changes in fresh embryo culture medium (661.50 μmol/L) were significantly higher than the median differences of changes in post-thawed embryo culture medium (232.00 μmol/L) at 0.5 h (P < 0.001). At 1 and 2 h, no significant differences of changes were found in all amino acid. Of note, however, is that the amino acid differences of changes in the culture medium of post-thawed embryos at 1 h were higher than that at both 0.5 h and 2 h.
|Time (h)||Amino acid differences of changes (μmol/L)||P-value|
|Fresh embryos||Frozen-thawed embryos|
|0.5||661.50 (392, 855)||232.00 (142, 336)||<0.001*|
|1||438.50 (328, 504)||507.50 (238, 730)||0.167|
|2||301.00 (145, 470)||257.00 (180, 404)||0.924|
In this study, frozen-thawed 3PN embryos demonstrated metabolic recovery from metabolic stagnation during cryopreservation beginning at 1 h after thawing, and exhibited similar metabolism compared to that of 3PN embryos that had not been frozen.
FET is an indispensable component of assisted reproductive technology (ART), and many factors may affect success of FET. Of these factors, determining the time for frozen-thawed embryo recovery and in vitro culture after thawing are important factors affecting the success rate of FET in that they may help choose the appropriate timing of transplantation. Currently, most FET centers transfer D3 thawed embryos after 2 to 4 h of in vitro culture, but there is no relevant theoretical basis for that timing of transfer. This practice is only based on the belief that embryos should need a certain amount of time to recover from the frozen state after thawing.
Studies have begun to examine embryo metabolism in an attempt to improve the success rates of ART.[9, 10] Houghton et al. examined amino acid turnover during in vitro culture and found that the turnover patterns of alanine, arginine, methionine, glutamine, and asparagine could predict blastocyst potentiality at >95%. Brison et al. examined the depletion and appearance of amino acids in embryo culture medium of 53 IVF cycles and found that the turnover of asparagine, glycine, and leucine were independently correlated with clinical pregnancy and live birth. In a study of embryo metabolism following cryopreservation, Stokes et al. examined amino acid turnover in human embryos from day 2 to 3 of development. The authors found marked differences in the turnover of glutamine, alanine, glycine, glutamate, arginine, and lysine between thawed embryos that arrested prior to blastocyst formation and those that developed to the blastocyst stage. In addition, it was possible to predict with 87% accuracy which embryos would develop to the blastocyst stage by using the sum of glutamine, glycine, and alanine turnover.
The results of this study suggest that at 1 h and 2 h after thawing, concentrations of free amino acids in the embryo culture medium were increased compared to the blank control at the corresponding time points. It indicates that embryos cultured in vitro will secrete some amino acids into the culture medium. This study added the absolute values of amino acid increases and decreases, and calculated the amino acid differences of changes, which may better reflect the embryonic amino acid metabolism.
The amino acid differences of changes in the culture medium at 0.5 h of incubation after embryo thawing were lower than that of incubation of fresh embryo. Few amino acids in the embryo culture medium were depleted compared to the corresponding fresh embryo counterpart such as aspartate, glutamate, serine, glutamine, alanine, and valine for the 0.5 h time point. It was found that the amino acid differences of changes in the culture medium at 1 h of incubation after embryo thawing was higher than that at 0.5 h and 2 h of incubation after embryo thawing. We speculate that after thawing, embryos require more amino acids for metabolism in order to adapt to the stress from the in vitro culture environment. After adapting to the environment, amino acid metabolism slows down. However, with the continuous development of an embryo cultured in vitro, the embryonic need for amino acids will gradually increase. Generally, embryos are considered to be in a state of metabolic stagnation during cryopreservation. This study found that at 0.5 h of incubation after thawing, the amino acid differences of changes in the embryo culture medium was still significantly lower than that in culture medium of fresh embryos at 0.5 h of incubation. However, there was no significant difference in the amino acid differences of changes between the culture medium of the frozen-thawed embryos at 1 h of incubation after thawing and that of the fresh embryos at 1 h of incubation, suggesting that frozen-thawed early-stage human embryos begin amino acid metabolism within 1 h after thawing, and at that time they have already recovered from the state of metabolic stagnation during cryopreservation. Moreover, their amino acid metabolism has also approached the level before embryo freezing.
In order to exclude the influence of the culture environment, this study compared the amino acid concentrations in the blank control medium (not used for embryo culture) at different time points. It was found that in the blank control, the concentrations of most amino acids were significantly different at the first three time points, but by the end of 24 h (data not shown) all but three amino acids (glutamine P = 0.019, tyrosine P = 0.046, and tryptophan [P = 0.030] were noted) showed significantly different concentrations, suggesting that the amino acid composition of the culture medium will not change significantly if the culture medium is placed under the culture conditions for <24 h. At the same time, the results suggest that the changes in the amino acid concentrations in the embryo culture medium after initiating the culture of thawed embryos would be caused by the metabolism of the frozen-thawed embryos, and not by the incubation environment. The possible reasons for the concentration difference in the amino acids in the blank control might be that the sample size of the blank control was small in this study.
Our study materials were abnormal fertilized embryos, and these embryos often have chromosomal abnormalities. Edirisinghe et al. compared the growth rate of fresh and frozen-thawed embryos based on whether the embryo chromosomes were normal. They found that regardless of whether the embryos were fresh or frozen-thawed at day 3, the growth rate was similar in both embryos with normal and abnormal chromosomes. However, for embryos at D4, the growth rate of fresh embryos with normal chromosomes was greater than that of embryos with chromosomal abnormalities; this phenomenon, however, was not observed in frozen-thawed embryos. A recent study by Picton et al. examined the association between amino acid turnover and chromosome aneuploidy in human embryos in vitro and found that asparagine, glycine, and valine turnover was significantly different between genetically normal and abnormal embryos on days 2–3 of culture and by days 3–4 the profiles of serine, leucine, and lysine differed between euploid versus aneuploid embryos. In this study, the concentrations of aspartate, taurine, alanyl-glutamine, glycine, serine and valine showed no significant differences among blank control culture medium at three time points, while the concentrations of the other 14 amino acids were significantly different. The median differences of changes in fresh embryo culture medium (661.50 μmol/L) were significantly higher than the median differences of changes in post-thawed embryo culture medium (232.00 μmol/L) at 0.5 h. Thus, we must consider our use of 3PN embryos a limitation of the study as in is not certain whether the amino acid differences of changes would be affected by the genetic status of the embryo, or if the results will be applicable to normal embryos.
In conclusion, the results of this study indicate that the level of amino acid metabolism of frozen-thawed early-stage human embryos has already recovered from the state of metabolic stagnation during cryopreservation at 1 h of incubation after thawing, and the amino acid metabolism level at that time approximates that in fresh embryos before freezing.
This study was supported by the National Natural Science Foundation of China (grant no.81070495), the Natural Science Foundation of Guangdong Province (grant no. 9151008901000018).
The authors declare that they have no competing interests.