Ting Tian1, Gang Zhao2, Dan Han1, Kaixuan Zhu3, Dawei Chen4, Zhiguo Zhang4, Zhaolian Wei4, Yunxia Cao4, Ping Zhou5. 1. Center for Reproductive Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China. 2. Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, China. 3. Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, China. 4. Center for Reproductive Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China. 5. Center for Reproductive Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China zhoup_325@163.com.
Abstract
STUDY QUESTION: Is sucrose more effective than trehalose in human ovarian tissue cryopreservation? SUMMARY ANSWER: The effect of sucrose as a cryoprotective agent (CPA) was not significantly different from that of trehalose in human ovarian tissue cryopreservation. WHAT IS KNOWN ALREADY: Sugars have the ability to keep the cell membrane intact and can decrease the toxicity of CPAs. Sucrose is the most commonly used non-permeable CPA, while trehalose is rarely used in human ovarian tissue cryopreservation. Although various methods are utilized to evaluate the efficiency of human ovarian tissue cryopreservation, few studies have evaluated the effect of cryopreservation from the viewpoint of biomechanics. STUDY DESIGN, SIZE, DURATION: A total of 15 ovarian tissue samples were collected from 15 patients (20-41 years old) with benign ovarian tumors or malignancies, and each was dissected into six slices. Two slices were taken as the fresh control group. The remaining four slices were vitrified using different vitrification protocols. After warming, samples in each group were either fixed for histological evaluation or destined for stress relaxation test. PARTICIPANTS/MATERIALS, SETTING, METHODS: The CPA solutions for the control and vitrified groups were composed of EDS and EDT (E, ethylene glycol; D, dimethylsulphoxide; S, sucrose; T, trehalose). The stress relaxation experiments were carried out at room temperature using a dynamic mechanical analyzer. Ovarian tissue samples were assessed for both their morphology and viscoelasticity. Stress relaxation data (SRD) were calculated as a percentage, representing the ability to maintain the initial stress after stretching. The percentage of morphologically normal follicles was compared between groups, which was represented by morphologic preservation ratio. MAIN RESULTS AND THE ROLE OF CHANCE: The morphologic preservation ratio of the primordial follicles in the fresh control group (87.58%) was higher than that in group S (72.33%) (P = 0.000) and group T (79.56%) (P = 0.002). Although not statistically significant, compared with the S group, vitrification with T suggested a trend toward a higher morphologic preservation ratio of the primordial follicles. The SRD in the fresh control group (0.6433 ± 0.7233) was significantly different from that in group S (0.5200 ± 0.8331, P = 0.000) or in group T (0.5667 ± 0.6415, P = 0.000). However, no significant difference was found between groups S and T. LIMITATIONS, REASONS FOR CAUTION: Experimental samples were directly exposed to the air, which will result in a discrepancy in the viscoelastic properties between experimental tissues and in vivo tissues. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggested a trend toward a higher morphologic preservation ratio of the primordial follicles after vitrification in trehalose compared with sucrose, which may provide a basis for further optimizing human ovarian tissue vitrification. In addition, it was possible to evaluate the effect of ovarian tissue cryopreservation from a biomechanics perspective. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the grants from the Medical Scientific Research Subject, Health Ministry of Anhui Province (2010B014) and National Basic Research Program of China (973 Program) (2012CB944704), and the National Natural Science Foundation of China (Nos. 51276179 and 51476160). The authors declare that there is no conflict of interests regarding the publication of this original paper.
STUDY QUESTION: Is sucrose more effective than trehalose in human ovarian tissue cryopreservation? SUMMARY ANSWER: The effect of sucrose as a cryoprotective agent (CPA) was not significantly different from that of trehalose in human ovarian tissue cryopreservation. WHAT IS KNOWN ALREADY: Sugars have the ability to keep the cell membrane intact and can decrease the toxicity of CPAs. Sucrose is the most commonly used non-permeable CPA, while trehalose is rarely used in human ovarian tissue cryopreservation. Although various methods are utilized to evaluate the efficiency of human ovarian tissue cryopreservation, few studies have evaluated the effect of cryopreservation from the viewpoint of biomechanics. STUDY DESIGN, SIZE, DURATION: A total of 15 ovarian tissue samples were collected from 15 patients (20-41 years old) with benign ovarian tumors or malignancies, and each was dissected into six slices. Two slices were taken as the fresh control group. The remaining four slices were vitrified using different vitrification protocols. After warming, samples in each group were either fixed for histological evaluation or destined for stress relaxation test. PARTICIPANTS/MATERIALS, SETTING, METHODS: The CPA solutions for the control and vitrified groups were composed of EDS and EDT (E, ethylene glycol; D, dimethylsulphoxide; S, sucrose; T, trehalose). The stress relaxation experiments were carried out at room temperature using a dynamic mechanical analyzer. Ovarian tissue samples were assessed for both their morphology and viscoelasticity. Stress relaxation data (SRD) were calculated as a percentage, representing the ability to maintain the initial stress after stretching. The percentage of morphologically normal follicles was compared between groups, which was represented by morphologic preservation ratio. MAIN RESULTS AND THE ROLE OF CHANCE: The morphologic preservation ratio of the primordial follicles in the fresh control group (87.58%) was higher than that in group S (72.33%) (P = 0.000) and group T (79.56%) (P = 0.002). Although not statistically significant, compared with the S group, vitrification with T suggested a trend toward a higher morphologic preservation ratio of the primordial follicles. The SRD in the fresh control group (0.6433 ± 0.7233) was significantly different from that in group S (0.5200 ± 0.8331, P = 0.000) or in group T (0.5667 ± 0.6415, P = 0.000). However, no significant difference was found between groups S and T. LIMITATIONS, REASONS FOR CAUTION: Experimental samples were directly exposed to the air, which will result in a discrepancy in the viscoelastic properties between experimental tissues and in vivo tissues. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggested a trend toward a higher morphologic preservation ratio of the primordial follicles after vitrification in trehalose compared with sucrose, which may provide a basis for further optimizing human ovarian tissue vitrification. In addition, it was possible to evaluate the effect of ovarian tissue cryopreservation from a biomechanics perspective. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the grants from the Medical Scientific Research Subject, Health Ministry of Anhui Province (2010B014) and National Basic Research Program of China (973 Program) (2012CB944704), and the National Natural Science Foundation of China (Nos. 51276179 and 51476160). The authors declare that there is no conflict of interests regarding the publication of this original paper.