Ambient temperature transport of human oocytes: an unexpected research resource

Abstract

Purpose: This study aimed to determine the viability and meiotic competence of human oocytes deemed not suitable for clinical use following controlled ovarian stimulation of young egg donors receiving treatment at an egg bank.

Methods: A total of 432 oocytes were shipped at ambient temperature overnight, in a medium containing caffeine and dibutyryl cyclic-AMP to limit meiotic cell cycle progression, and estrogen and progesterone to mimic the intrafollicular environment. In some experiments, transport medium was also supplemented with 1 µg/ml ZnSO₄. Oocytes were either fixed immediately upon arrival or cultured for 20-24 or up to 143 h followed by fixation. Time-lapse imaging and fluorescence imaging were used to establish viability, meiotic status, and spontaneous activation.

Results: Greater than 95% of transported oocytes retained viability, whether transported with or without added ZnSO₄, exhibiting meiotic progression and/or spontaneous activation following overnight culture. Time-lapse imaging and fluorescence imaging revealed a higher incidence of spontaneous activation and subsequent cleavage activity for up to 5 days in culture in samples transported in ZnSO₄.

Conclusions: Under the experimental conditions described here, immature human oocytes retain viability and meiotic competence following ambient temperature transport, providing a novel and experimentally tractable resource for future research in human oocyte biology and the development of human parthenote stem cells.

Keywords: Ambient temperature transport; Human oocyte; Parthenote stem cells; Spontaneous activation; Zinc.

Fig. 1

Diagram illustrating the experimental design. a Major steps involved in the experimental workflow of experiment 1. (1) Oocytes were collected from young donors by ovum pickup. The collected oocytes were assessed for maturation status and categorized as either mature (MII) or immature (GV, MI, dysmorphic). Mature oocytes were selected and remained at TWESB for cryopreservation; (2) Immature oocytes were placed in either ambient temperature transport medium (ATTM) or ATTM supplemented with 1 μg/ml ZnSO₄ (ATTM-zinc); (3) Oocytes were transported overnight at room temperature; (4) Upon arrival, each tube was divided into two groups. One group (Group A) was fixed immediately; (5) The second group (Group AC) was cultured for 20–24 h in culture medium, then fixed and stained. b Major steps involved in the experimental workflow of experiment 2. (1) Oocytes were collected from young donors by ovum pickup. The collected oocytes were assessed for maturation status and categorized as either mature (MII) or immature (GV, MI, dysmorphic). Mature oocytes were selected and remained at TWESB for cryopreservation; (2) Immature oocytes were placed in either ambient temperature transport medium (ATTM) or ATTM supplemented with 1 μg/ml ZnSO₄ (ATTM-zinc); (3) Oocytes were transported overnight at room temperature; (4) Upon arrival, oocytes were placed in either standard culture medium or culture medium supplemented with estradiol (E2) and progesterone (P); (5) Culture dishes were placed in Cytosmart imaging system for time-lapse imaging and incubated for up to 5 days. Images were captured every 15 min. Experimental groups: Group 1, oocytes transported in ATTM, cultured in standard medium; Group 2, oocytes transported in ATTM, cultured in medium supplemented with E2 and P; Group 3, oocytes transported in ATTM-zinc, cultured in standard culture medium; Group 4, oocytes transported in ATTM-zinc, cultured in medium supplemented with E2 and P

Fig. 2

A General condition of living oocytes following transportation in media lacking (left) or containing added ZnSO₄ (right) using Hoffmann contrast optics; note most oocytes lack adherent cumulus cells and exhibit a prominent perivitelline space. B Sequence of images from time-lapse movies (see Supplement Videos S1 and S2) comparing meiotic status over the first 24 h of extended culture. Top panel shows GV, MI, and MII oocytes transported in the absence of added ZnSO₄ (n = 4) with one GV and one MI progressing to MII while others remain unchanged. The lower panel of oocytes transported with added ZnSO₄ show activation of 2 oocytes that were MIIs at the time of arrival (see PN, top and bottom). Elapsed time stamps are shown for each panel series

Fig. 3

a Representative images of meiotic stages (GV, MI, MII) or activated oocytes transported and either fixed immediately upon arrival or following 24 h in culture; individual oocytes were scored with respect to meiotic or activation status by comparing DIC images (top panel) with companion f-actin profile (middle panel, Acti-stain 488 nm) and chromatin patterns (Hoechst 33342). GV stages typically displayed a condensed chromatin ring around the nucleolus (left column), whereas MI and MII stages were distinguished by the presence or absence of polar bodies (note some cumulus cell nuclei remain adherent, middle two columns), while activated eggs exhibited second polar bodies (right column, lower frame at 9 o’clock position) and solitary pronuclei (see text) or multiple micronuclei showing signs of chromatin decondensation and well-demarcated nuclear boundaries. Scale bar = 25 µm. b The quantitation of meiotic or activation status at the time of arrival (A) or following 24 h of culture (AC) for transport without (left graph) or with added ZnSO₄ (right graph). Note that while under both transport conditions the fraction of immature oocytes (GV and MI stages) decreases, in the presence of added ZnSO₄, both the incidence of MIIs at arrival and the percentage of eggs undergoing spontaneous activation are increased relative to eggs transported in the absence of added ZnSO₄. Statistical comparisons between A and AC were performed using multiple unpaired t-tests for each zinc condition (ATTM or ATTM-zinc). ATTM: A significant difference in activated oocytes was observed after culture (AC vs A; p = 0.00094; marked with a, b). ATTM-zinc: Significant differences were found in the proportion of MII oocytes (p = 0.0369; marked with a, b) and activated oocytes (p = 0.000051; marked with c, d). Details on the number of oocytes analyzed are provided in Table 2

Fig. 4

Characterization of spontaneously activated oocytes transported in the presence of added ZnSO₄ that were fixed following extended culture and time-lapse imaging. Nomarski DIC (left column), f-actin and chromatin (middle column), and chromatin patterns (right column) demonstrate various cleavage patterns from 4-cell stage symmetric with decreased chromatin content per blastomere (a), to asymmetric first cleavage and interphase nucleus in smaller blastomere (b, note chromosome fragmentation in larger of the two blastomeres) as aberrant extremes. “Normal” apparent cleavage stages were observed up to 8-cell (c) or 2-cell (d) and in such cases, binucleate blastomeres were observed. Fragmented eggs were not uncommon typically exhibiting variably sized fragments of which few if any contained chromatin (e). Videos S3 and S4 further show the range of cleavage found in eggs transported without added ZnSO₄. Videos S5 and S6, from which the examples for this figure were taken, track the cleavage behavior of eggs transported with added ZnSO₄. Scale bar = 25 µm