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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-025-03548-9.

Introduction

Human oocytes recovered from ovarian follicles following controlled ovarian stimulation (COS) form the basis for treatment of infertility and fertility preservation. For over four decades, assisted reproductive technologies (ARTs) have transformed the discipline of reproductive medicine due to improvements in COS [1]. More recently, the purview of human ARTs has extended fertility options by overcoming the effects of advanced maternal age on oocyte quality through the adoption of individual oocyte cryopreservation to delay reproductive timing and/or utilizing oocytes donated by young women voluntarily undergoing COS [2, 3]. Despite these advances, and the current commercial impacts on access to reproductive care, human oocytes remain one of the most enigmatic and poorly understood cell types, in large part due to the lack of available research material.

While most oocytes retrieved from women undergoing COS attain maturation to metaphase II, only a subset of successfully fertilized oocytes is capable of generating blastocysts, with even fewer resulting in term pregnancies following embryo transfer [4]. Moreover, mature oocytes are not recovered from many patients undergoing COS due to age, poor gonadotropin responsiveness, or genetic factors, prompting clinics to resort to so-called rescue in vitro maturation (rIVM) [5–7]. The reasons for the variable incidence of oocytes either failing to mature, so-called meiotic incompetence, or failing to undergo partial or full developmental competence remain unclear and are likely to involve many factors including the follicle environment.

Among these, the microenvironment of the follicle is a major determinant of both oocyte quality and ovulation induction–mediated transmission of signals to elicit cytoplasmic and nuclear maturation [8, 9]. Ovum pickup constitutes a major alteration in the chemical and physical environment of the oocyte and its companion cumulus cells including withdrawal from a steroid hormone–rich milieu. Recent advances in human oocyte biology during maturation and following fertilization continue to emphasize the need for research materials to better understand the biological underpinnings of clinical ART failures, especially as they pertain to the oocyte [10]. Maternal age is widely known to be an additional important factor and as Nicholas and colleagues have recently shown, the customary practice of so-called rescue IVM can lead to improved embryo development following intracytoplasmic sperm injection (ICSI), evidencing that some oocytes judged immature at retrieval have the potential for fertilization and development even though causes for failure to mature await further research [11]. Some genetic factors underscoring maturation failure have come to light with the discovery of mutations linked to specific stages of meiotic maturation arrest [12]. Understanding the underlying causes for failure to mature following COS will improve clinical efficiency and could provide fundamental insights into basic human oocyte biology if a resource of experimentally tractable research materials were to become available. Furthermore, access to viable human oocytes could greatly enhance progress in stem cell research. Pluripotent stem cells derived from unfertilized human eggs have several advantages over human embryonic stem cells, including, fewer ethical considerations, one half the tissue antigen complexity, and a potential source of stem cell therapies for ovulating women. Strikingly, the efficiency of human parthenogenetic blastocyst development remains extremely low, ranging from 10%-20%, even when the starting material are high-quality mature MII eggs. 

Here, we explore a distinct source of human eggs for research purposes, an opportunity made possible by the growth of the “egg banking industry.” Retrieval following COS and storage by vitrification form the foundation for patients to delay their own reproduction (autologous) or serve as donors for patients willing to purchase human oocytes for personal use (heterologous). As noted above, and despite the younger age of patients seeking storage of mature oocytes, COS yields immature oocytes at the time of retrieval that typically are designated as discard material following Institutional Review Board review and patient consent. Whether this “discard” material might constitute a valid research resource has yet to be explored, although the practice of rIVM has been utilized for clinical purposes with some success [13].

The prospect of accessing and utilizing immature oocytes prompted our consideration of developing an ambient temperature transport system capable of maintaining viability and research potential. While studies with mouse and bovine GV–arrested oocytes have shown the feasibility of transport to support meiotic progression to MII stages without compromising subsequent developmental competence [14, 15], such an approach has not been undertaken for human oocytes. Here, we report our initial efforts to define and evaluate ambient temperature transportation of human oocytes. Our results establish conditions that support viability and meiotic progression that will hopefully inform and guide future research into the basic biology of human oocytes.

Materials and methods

Oocyte collection and transport

Oocytes were obtained from young donors who underwent controlled ovarian stimulation at The World Egg and Sperm Bank (TWESB) in Phoenix, AZ. Oocytes determined to be immature at the time of retrieval (germinal vesicle, GV or metaphase I, MI) were donated to the Bedford Research Foundation (BRF) by the medical and laboratory teams of TWESB. The oocyte donation request form signed by all TWESB donors included a stipulation that oocytes deemed unsuitable for cryopreservation could be used for this research. The consent form was also reviewed and approved by the Ethics Advisory Board and Human Subjects Committee of the BRF. Oocytes were shipped during regularly scheduled retrievals following routine ovarian stimulation protocols between the months of April and October 2022.

All donors underwent controlled ovarian stimulation (COS) following 10 days of pituitary suppression using rFSH (Gonal-F; Merck Serono, or Follistim, Organon) (75–300 IU/day) with or without urinary hMG (Menopur, Ferring Pharmaceuticals) or low-dose hCG (10 IU/day; Ferring Pharmaceuticals). Gonadotropin stimulation continued until one follicle reached 14 mm and serum estradiol levels approached > 400 pg/ml. When the lead follicle reached 15–17 mm, Lupron was used for ovulation induction (8 mg, Abbott Laboratories) and oocyte retrieval was conducted 32–36 h later by ultrasound-guided transvaginal ovum pickup (OPU). Cumulus cells were removed by pipetting in hyaluronidase prior to evaluation of oocyte maturation status and quality. All MIIs were selected for cryopreservation by vitrification, whereas any immature (GV, MI) or dysmorphic oocytes were placed in ambient temperature transport medium (ATTM, described below) prior to loading into the shipment vehicle.

The transport protocol was designed to minimize the clinical team’s effort required to forward oocytes to the BRF. Cryotubes (Nunc, 1.5 ml) with 1.0 ml of ATTM (see below) were placed into one-half of a Styrofoam rack from 15-ml conical tubes, with the other half of the rack used as a cover to create a closed container. The tube rack was surrounded by insulating material and placed into a larger Styrofoam transport box. For each day of scheduled oocyte retrieval, TWESB embryology personnel received transport tubes containing ATTM by 10 am on the day of OPU. Oocytes not suitable for cryopreservation, as described above, were transferred to Nunc tubes within 4–6 h of collection, placed in Styrofoam transport boxes, and returned to the BRF by overnight shipping, arriving at approximately 10 am the day after OPU (estimated time in ATTM = 20–24 h). Boxes were equipped with temperature sensor strips to monitor internal temperature extremes over a range of 32–45 °C, and in only one shipment was the temperature found to exceed 45 °C.

Experiment 1: Upon receipt, samples were divided into two and processed for immediate fixation (A group) or placed in culture medium for short-term culture (20–24 h) and then fixation (AC group). Details of the experimental design are illustrated in Fig. 1a. Experiment 2: Upon receipt, samples were transferred to GEM supplemented with 2% SR, 0.8 mM pyruvic acid (Fisher Scientific, cat # AC132140250) with or without the addition of 17β-estradiol (1000 ng/ml) and progesterone (500 ng/ml), and placed into long-term culture (3–5 days) for time-lapse imaging prior to fixation (Fig. 1b).

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

A base culture medium was used throughout unless specified otherwise that is referred to as “GEM,” Gamete-Embryo medium [16–19] comprised of calcium and magnesium-free modified Ham’s F-10 medium prepared as a powder in proprietary batches by GIBCO (Cat ME050030, patent pending) and reconstituted with embryo-tested water (Sigma, cat # W1503) supplemented with 22 mM NaHCO₃, 0.75 mM calcium lactate (Sigma, cat # L4388), 5 mM glucose (Sigma, cat # G6152), 0.02 mM EDTA (Sigma, cat # ED-100G), 0.6 mM MgSO₄ (Sigma, cat # M2643), 50 mg/L streptomycin (Sigma, cat # S1277), and 100 mg/L penicillin (Sigma, cat # P3032) [16–19]. To limit meiotic progression and approximate the intrafollicular conditions and steroidal environment oocytes are subjected to, we developed a transport medium based upon previous studies aimed at maintaining meiotic arrest [6, 8, 14, 15]. For transport of samples, we designed ambient temperature transport medium (ATTM) consisting of GEM-HEPES (GEM plus 25 mM HEPES, pH 7.4) supplemented with 2% (v/v) knockout serum replacement (SR, Gibco, cat # 10828–010), 0.4 mM N⁶, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dibutyryl-cAMP, Sigma, cat # D0627), 100 ng/ml β-estradiol (Sigma, cat # E2758), 50 ng/ml progesterone (Sigma, cat # P8783), and 0.4 mM caffeine (Sigma, cat # C0750). ATTM with added zinc (ATTM-zinc) contained an additional 1 μg/ml of ZnSO₄ (Acros Organics, cat # 389802500), whereas oocytes used for extended culture were transferred to GEM supplemented with 2% SR and 0.8 mM pyruvic acid (Fisher Scientific, cat # AC132140250) with or without the addition of 17β-estradiol (1000 ng/ml) and progesterone (500 ng/ml) as indicated below.

Oocyte evaluation following transport and culture

Upon receipt, oocytes were counted and divided into groups for either immediate fixation, short-term culture (20–24 h, with or without time-lapse imaging) followed by fixation, or placed into long-term culture (with time-lapse imaging) for 3–5 days followed by fixation. At the end of culture, oocyte groups were fixed and processed as described below.

Fluorescence imaging was used to determine meiotic status and activation according to previously published criteria for human oocytes [20]. All experiments were terminated by fixation of oocyte groups in 4% paraformaldehyde in PBS (Chem Cruz, cat # sc-281692) for 20 min at room temperature; samples were prepared for fluorescence labeling by washing three times in PBS wash (Dulbecco’s phosphate-buffered saline (Sigma, cat # D1408) supplemented with 0.1% (v/v) Triton-X-100, 0.01% (v/v) Tween-20, and 1% (w/v) bovine serum albumin (Sigma A-331)). To evaluate meiotic or activation status, oocytes were labeled with 2 µg/ml of Hoechst 33258 (Invitrogen, cat # H3569) and 280 nM of Acti-stain 488 fluorescent phalloidin (Cytoskeleton, cat # PHDG1) for 1 h at room temperature in the dark, further washed three times with PBS wash, mounted on slides with Vectashield Vibrance antifade mounting medium (Vector Laboratories, cat # 30304), and stored at 4 °C until imaging.

Differential interference contrast (DIC) and fluorescent images were taken in an epifluorescence microscope (Zeiss Axiovert 200) with a Plan-Neo Fluor objective (20 × magnification, NA 0.5) or Olympus Ach objective (60 ×, NA 0.8) using an ORCA-ER digital camera (Hamamatsu) controlled by Metamorph software (version 7.10.2.240).

Time-lapse (TL) imaging was used to evaluate the behavior of transported oocytes under experimental conditions as described above. Individual oocyte groups were maintained at 37 °C (6% CO₂) in organ culture dishes on a Cytosmart imaging system (Axion biosystems) capturing sequential brightfield images at 15-min intervals; image data files for each monitored dish were compiled into QuickTime movies for detailed analysis.

Statistical analysis

Data were analyzed using GraphPad Prism version 10.1.0 for Windows (GraphPad Software, Boston, MA, USA, www.graphpad.com). Data met the assumptions of normality and homoscedasticity. Comparisons between groups were conducted using either unpaired t-tests or multiple unpaired t-tests, as specified for each case, with statistical significance defined as p < 0.05.

Results

Experiment 1: Survival and meiotic progression of transported human oocytes

All human oocytes (351 from 38 groups), transported overnight in ambient temperature transport medium (ATTM) with or without added ZnSO₄ (1 μg/ml), were assessed for morphological appearance on arrival (Fig. 2a). Viability characteristics analyzed included intactness of the zona pellucida (ZP), size of the perivitelline space, overall shape, and oocyte surface contour (Fig. 2a). Morphological characteristics on arrival were similar, independent of the presence of added zinc during transport (Fig. 2a) but varied following overnight incubation when removed from dbcAMP, caffeine, and added ZnSO₄ (Fig. 2b).

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

Time-lapse (TL) imaging confirmed both the high level of viability and meiotic competence of transported oocytes. The survival rate on arrival of the 38 analyzed groups was > 96% with no further loss of viability observed during overnight culture. A total of 5 nonviable oocytes were found in the groups with no added zinc during transport, not statistically significantly different from the 6 nonviable oocytes following transport with added zinc (Table 1). Additionally, a small fraction of the oocytes presented either normal or abnormal cleavage on arrival and after overnight culture (Table 1). The rare nonviable and cleaved oocytes seen were excluded from subsequent analyses leaving for further analysis a total of 158 oocytes in the group with added zinc and 154 oocytes in the group without added zinc in the transport medium.

Table 1.

Effect of ambient temperature transport on viability. Oocytes transported either in the presence or absence of added zinc in the ambient temperature transport medium (ATTM) were fixed, classified as dead, abnormally cleaved, or normally cleaved. The oocytes listed in this table represent the total number (n), encompassing both those fixed at arrival and those fixed following 20–24 h of incubation in standard culture medium. “N” represents the number of oocyte groups that were received for analysis

ATTM-zinc	# Dead	Abnormal cleavage	Normal cleavage	n (total eggs)	N (# of groups)
No	5	9	1	169	20
Yes	6	16	2	182	18

Once removed from ATTM, annotation of individual oocytes by TL imaging allowed for unequivocal documentation of meiotic stage and progression, as well as activation potential as indicated by polar body extrusion, the appearance and dissolution of pronuclei, and cleavage activity (Videos 1S and 2S and Fig. 2b). Cytoplasmic movements of organelles were observed in most oocytes, as were waves of cortical contraction and protrusive membrane events, observations consistent with both survival and functionality of transported oocytes as reported by others for fertilized human eggs [21]. Once fixed and processed for fluorescence labeling, oocytes of all groups were classified into germinal vesicle (GV), metaphase I–arrested oocytes (MI), metaphase II–arrested oocytes (MII), or activated (presenting pronuclei) based upon the distribution of chromatin within the cell (Hoechst 33342) and the membrane-associated f-actin (Acti-stain 488) cortical cytoskeleton (Fig. 3a). The percentages of GV, MI, MII, and activated oocytes in each group are presented in Table 2 and in Fig. 3b.

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

Table 2.

Effect of zinc on meiotic status during and following ambient temperature transport. Oocytes fixed either at arrival (Group A) or after culture for 20–24 h in standard culture medium (Group AC) were classified as germinal vesicle (GV), metaphase I–arrested oocytes (MI), metaphase II–arrested oocytes (MII), or activated (showing pronuclei or cleavage) as described in the “Results” and Fig. 2. The percentage of oocytes is indicated in parenthesis (%). “n” indicates the total number of oocytes analyzed; “N” indicates the number of transport groups analyzed. Statistical comparisons between A and AC were performed using multiple unpaired t-tests for each zinc condition (with or without added zinc). Without zinc: no significant differences were found in the proportions of GV, MI, or MII oocytes. A significant difference in activated oocytes was observed after culture (AC vs A; p = 0.00094; marked with *). With zinc: no significant differences were found in the proportions of GV and MI oocytes. Significant differences were found in the proportion of MII oocytes (p = 0.0369; marked with ^#) and in the proportion of activated oocytes (p = 0.000051; marked with **). In addition, a significant difference was found between activated oocytes with and without zinc during transport (multiple unpaired t-test; p = 0.04277; marked with a, b)

ATTM-zinc	Group	GV (%)	MI (%)	MII (%)	Activated (%)	n (total eggs)	N (# of groups)
No	A	15 (20.1)	20 (24.9)	45 (53.4)	2 (1.6)	82	15
Yes	A	9 (12.2)	15 (20.1)	48 (62.7)	3 (5.0)	75	15
No	AC	4 (8.5)	8 (11.2)	43 (56.1)	17 (24.3)*, a	72	13
Yes	AC	3 (4.6)	11 (12.4)	32 (36.5)#	37 (46.5)**, b	83	14

Oocyte status upon arrival (A) was similar between oocytes transported with or without added ZnSO₄. Mature MII oocytes were the prevalent representative stage, followed by MI and GV stages (Table 2 and Fig. 3b). Fewer than 5% of oocytes exhibited signs of activation upon arrival (Table 2 and Fig. 3b). In the group without added zinc during transport, the percentage of GV and MI tended to decrease following overnight culture (Fig. 3b): 20.1 to 8.5% (multiple unpaired t-test, p = 0.256), and 24.9 to 11.2% (multiple unpaired t-test, p = 0.159) respectively, although not statistically significantly different from each other, indicating the ambient temperature transported oocytes were capable of resuming meiosis following transfer out of ATTM (Table 2). The percentage of MII remained the same, 53.4 to 56.1% (multiple unpaired t-test, p = 0.828, Table 1), whereas activated oocytes increased 15-fold following overnight incubation, from 1.6 to 24.3% (multiple unpaired t-test, p = 0.00094, Table 2, Fig. 3b). Taken together, these data suggest many mature MII-stage oocytes underwent spontaneous activation overnight after arrival, and MI oocytes progressed to MII (Table 2, Fig. 3b).

In the group with added ZnSO₄ during transport, the percentage of GV and MI also tended to decrease following overnight culture: 12.2 to 4.6% (multiple unpaired t-test, p = 0.1079) and 20.1 to 12.4% (multiple unpaired t-test, p = 0.3668), respectively, also not statistically different from each other nor from the oocytes transported without added ZnSO₄ (Table 2, Fig. 3b). In contrast, however, the percentage of MII oocytes in the zinc group decreased during overnight culture from 62.7 to 36.5% (multiple unpaired t-test, p = 0.0369, Table 2, Fig. 3b, movie S2) and the increase of activated oocytes following overnight incubation was ninefold from 5 to 46.5% (multiple unpaired t-test, p = 0.000051, Table 2, Fig. 3b). Moreover, the increased percent of activation after overnight culture of oocytes transported with added zinc (46.5%) was significantly greater (multiple unpaired t-test, p = 0.04277) when compared to oocytes transported overnight in the absence of added zinc (24.3%). Together, these data suggest that oocytes transported with zinc have a similar incidence of progression from GV/MI to MII when compared to transport without zinc but demonstrate an approximate doubling of spontaneous activation from MII, including cleavage activity, as shown in movie S2.

Experiment 2: Time-lapse (TL) imaging reveals dynamic behaviors of transported human oocytes during extended culture

Oocytes transported in ATTM with or without added ZnSO₄ (1 ug/ml) were subdivided into four groups (total of 81 oocytes) that were continuously monitored for up to 5 days following transfer out of ATTM into fresh GEM with or without steroid supplementation, as summarized in Fig. 1b and Table 3, and demonstrated in Videos 3S through 6S (see supplement).

Table 3.

Spontaneous cleavage activity monitored by TL imaging. Human oocytes transported in the presence or absence of added zinc and/or steroids (estradiol [E2] and progesterone [P]) and subjected to prolonged culture with TL imaging exhibit varying degrees of fragmentation or cleavage. Transport in ambient temperature transport medium (ATTM) without added zinc is associated with less cleavage activity while pronucleus (PN) formation and cleavage activity are prevalent in a subset of oocytes transported in the presence of zinc independent of steroid supplementation. Group refers to the arbitrary group number assigned to each condition (see Fig. 1b). “# Frag on arrival” indicates the number of oocytes that were fragmented upon arrival. “# GV on arrival” refers to the number of oocytes at the germinal vesicle stage upon arrival. “First PN” indicates the number of oocytes that formed a pronucleus, with the time of PN appearance shown in parenthesis. “Cleaved at 30 h” and at “Cleaved at 91–146 h” refer to the number of oocytes that cleaved within the first 30 h or between 91 and 146 h of culture, respectively

ATTM-zinc	Group (# of ooctyes)	# Frag on arrival	# GV on arrival	Cultured in E2/P	First PN	Cleaved at 30 h	Cleaved at 91–146 h
No	1 (20)	2 (10%)	3	No	1 (25 h)	0	0
No	2 (19)	4 (21%)	3	Yes	1 (2.0 h)	0	0
Yes	3 (23)	1 (4%)	5	No	3 (8 h)	3	5
Yes	4 (19)	2 (11%)	4	Yes	3 (6 h)	3	5

By longitudinally tracking single oocytes over an extended period in culture, TL imaging revealed distinct activities that varied according to the conditions under which transport and extended culture were carried out.

Oocytes transported in the absence of zinc were heterogeneous at the start of culture and overall showed few signs of meiotic progression, as evidenced by GVBD or polar body emission (16/20, Group 1 Video 3S; 15/19 Group 2 Video 4S). Spontaneous activation was observed whether steroid supplemented or not, as evidenced by the appearance of pronuclei in 4 oocytes from each group. Notably, oocytes transported without zinc show no signs of cleavage activity despite maintaining cytoplasmic streaming and membrane protrusive activity. We conclude based on this initial analysis that in the absence of zinc, only a fraction of oocytes is capable of spontaneous activation, most likely a sign of “post-ovulatory aging”, and these oocytes are unable to undergo cleavage activity other than fragmentation. These findings were confirmed by fluorescence imaging of the oocytes imaged by TL, as shown in Fig. 4.

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

In sharp contrast, the presence of zinc in transport medium influenced all dynamic behaviors upon extended culture based upon TL imaging and analysis of fixed samples (Fig. 4). Cleavages resulted in more symmetrical and equally sized blastomeres and analysis of fixed samples confirmed the presence of both mononuclear (Fig. 4 a and c) and binuclear cells (Fig. 4d).

In Group 3 (Video S5), which was transported in zinc but lacked steroid supplementation during extended culture (n = 23), clear evidence of meiotic progression was observed in 3 of 5 GV stage oocytes at 19, 22, or 25 h of culture that spontaneously activated and cleaved, respectively, to 2-cell stages (20, 23, 32 h) and 4-cell stage (33, 43, or fragmented at 72 h), and one of these oocytes progressed to the 8-cell stage at 52 h of culture. The remaining oocytes in this group showed a range of behaviors that included failure to mature (GVs, MIs) or spontaneous activation to 2-cell with no further cleavage activity. A similar pattern of spontaneous activation was observed in Group 4 (n = 19) where steroid supplementation of zinc transported oocytes was tracked (Video S6). Here too, 3 out of 5 MII-stage oocytes displayed signs of initial activation by the appearance of pronuclei at 6, 12, and 18 h, followed by cleavage to the 2-cell stage at, respectively, 26, 32, and 25 h, and 4-cell stage at 39, 46, and 43 h. Each of these now activated eggs underwent fragmentation at 50, 72, and 51 h of culture. As shown in Fig. 4, variations in the number of nuclei present in individual blastomeres confirm that while TL analysis confirms the kinetics of activation and cleavage in the subsets of oocytes demonstrating spontaneous activation followed by progressive cleavage, failure to coordinate karyokinesis with cytokinesis is evident in most such eggs.

Collectively, the results of TL imaging, coupled with fluorescence analysis of oocytes at the end of extended culture, not only confirm the viability of transported samples but also establish an effect of zinc in the transport medium on the long-term survival and meiotic competence of a subset of oocytes.

Discussion

Advances in contemporary ARTs have resulted in the collection of supernumerary oocytes for embryo production and storage by cryopreservation. With a byproduct of conventional COS being the retrieval of immature oocytes that are either subjected to rIVM or discarded, we postulated that this material might offer a novel research resource for human oocytes besides cryopreservation. The present study asked whether ambient temperature transported human oocytes could survive and maintain meiotic competence. We demonstrate here conditions of ambient temperature transport that are permissive and supportive of human oocyte viability and further the capability to undergo spontaneous activation and initial cleavage stages of early development. Importantly, we show that inclusion of zinc in the transport medium is essential for long-term survival, allowing MII oocytes to engage in cellular processes typically associated with fertilization. While only a fraction of transported oocytes undergo spontaneous activation, this work provides a foundation for creating a resource with which to better understand the biology of the human oocyte and develop human parthenogenetic stem cell lines.

To explore the behavior of transported oocytes, some were cultured for an additional 96–124 h and monitored by time-lapse imaging. This analysis revealed three outcomes of dynamic behaviors that were evaluated by imaging of chromatin and cytoskeletal status in oocytes fixed after time-lapse monitoring. The original starting material exhibited a range of meiotic stages that either remained as arrested MIIs, or arrested GVBD/MI, or initiated spontaneous activation (see Table 2, pronuclear score (PN) and Fig. 4). The appearance of single pronuclei was taken as evidence of spontaneous activation with prominent boundaries of interphase nuclei with multiple nucleolar-like bodies; and reorganization of f-actin to the egg cortex was detected with focal zones of actin noted at blastomere boundaries with cases of both symmetric and asymmetric patterns of cytokinesis, the latter reminiscent of fragmentation as commonly observed following IVF or ICSI [22]. Besides the transformation from condensed M phase chromatin into pronuclei with nucleolar-like bodies, the finding of subsequent cleavage to 2-, 4-, and even 8-cell stages of development illustrates the ability of some oocytes to undergo spontaneous activation or parthenogenesis. What could this result reflect with respect to the influence of zinc exposure during transport?

Zinc is recognized as a major determinant in female reproductive physiology [23]. Many studies implicate zinc storage and fluxes in progression through meiotic maturation [24–27]. Moreover, the impact of zinc also emerges at the time of fertilization and into early cleavage stage development due to dynamic changes initiated at the time of fertilization [28, 29]. It is beyond the scope of this preliminary work to speculate as to the underlying effects of zinc of human oocyte transport and behavior but recent work on the mouse does implicate it in modulation of calcium fluxes known to be instrumental in the process of egg activation [30].

Our data demonstrates the feasibility of transporting immature oocytes obtained from young donors subjected to COS. We further show widespread outcomes over short- and long-term culture with respect to meiotic competence expression and spontaneous activation. Such variability is in part due to patient differences and their response to COS, that to a certain extent may have been minimized by exposure to zinc. To fully establish experimental utility of this material, future work will require a more systematic approach in identifying ATTM components that improve both meiotic competence and the somewhat surprising result of spontaneous activation. Spontaneous or parthenogenic activation has been reported in the human for freshly collected oocytes [31] or oocytes subjected to rIVM [20] without the use of agents known to artificially trigger egg activation. Whereas only a small proportion of oocytes exhibited spontaneous activation, this finding, if more oocytes retained this capability, raises the possibility of investigating the underlying causes of maturation arrest, fertilization failure, and early developmental problems that continue to compromise patient outcomes [4, 7, 21].

This work is subject to several limitations. As noted above, the heterogeneity in response and behavior of transported oocytes is likely traceable to the fact that the grouping for experiments required us to pool samples from several different donors upon arrival. Adding to this concern is the fact that neither patient age (although young donors) nor variations in COS protocols were available to us. Together, patient-to-patient variations are therefore likely to at least partially contribute to the wide range of behaviors observed.

In conclusion, this work documents both the transportability and potential utility of human oocytes for fundamental research purposes, among which include the derivation of parthenote stem cells, the overarching goal of further exploration of the spontaneous activation behavior reported here.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

MGG, AAK, DFA: conceptualization; MGG, MK, AAK, DFA: investigation and methodology; MGG, AAK, DFA: writing. All authors gave final approval for publication. All authors read and agreed to the published version of this manuscript.

Funding

This work has been supported by the Bedford Research Foundation and The ESHE Fund (DFA).

Data availability

All images, videos, and associated data can be obtained from the corresponding author upon reasonable request.

Declarations

Ethical approval

All TWESB oocyte donors signed an oocyte donation request form that included a stipulation that oocytes deemed unsuitable for cryopreservation could be used for this research. The consent form was also reviewed and approved by the Ethics Advisory Board and Human Subjects Committee of the Bedford Research Foundation.

Conflict of interest

The authors declare no competing interests.