Achieving oocyte survival and stable spindles after vitrification using closed pulled straws regardless of zona status

Dah-Ching Ding^{a, b}, Tang-Yuan Chu^{a, b}, Yu-Hsun Chang^{b, c}

^a Department of Obstetrics and Gynecology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan
^b Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan
^c Department of Pediatrics, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan

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Abstract
Objectives

Embryo cryopreservation is a well-established technique at in vitro fertilization centers but the best protocol for vitrification of oocytes and zona manipulation remains inconclusive. This study aimed to determine if the closed pulled straws (CPS) method and zona drilling for vitrification provide a higher survival rate for thawed oocytes.

Materials and Methods

Female MF1 mice were superovulated by injection of gonadotropins. Cumulus-oocyte complexes were derived from excised fallopian tubes and the oocytes were divided into the following three groups: (1) one-hole zona drilling by laser, (n = 40); (2) intact zona (n = 48); and (3) zona digestion by pronase (n = 43). The control group consisted of 40 nonvitrified oocytes. After thawing, surviving oocytes were stained for spindles and chromosomes after 1-hour and 3-hour incubations, and compared to controls.

Results

There were no significant differences in the survival rates among Groups 1 (34/40, 85%), 2 (34/48, 71%), and 3 (30/43, 70%), but there were significant differences compared with the control oocytes (100%). After 1-hour and 3-hour incubations, vitrified oocytes in the three groups did not have significantly fewer normal spindles than the controls (1 hour: Group 1, 70.5%; Group 2, 64.7%; and Group 3, 66.6% vs. control 90%). There was also no significant difference in the percentage of oocytes with a normal spindle shape between the 1-hour and 3-hour recovery times (3 hours: Group 1, 88.2%; Group 2, 88.2%; and Group 3, 80%; control, 95%).

Conclusions

The zona pellucidum with CPS method has no effect on spindle injury and oocyte survival. Sufficient culture time for the recovery of the meiotic spindle is imperative for fertilization of vitrified oocytes. A CPS vitrified oocyte has the advantages of high survival and preserved good spindles.

Keywords
Cryopreservation; Oocyte; Spindle; Vitrification; Zona pellucida