Successful cryoloop vitrification and subsequent in vitro maturation of mouse preantral follicles

Abstract

The purpose of this study was to assess follicular viability and competence through in vitro maturation (IVM) of cryoloop vitrified mouse preantral follicles. Early mouse preantral follicles were isolated and vitrified using the cyroloop vitrification technique. After thawing, the preantral follicles and oocytes were cultured and in vitro matured for 10 d to the metaphase two stage (M2). Oocytes were assessed for viability at 2 and 10 d of IVM and compared to a control group of freshly isolated preantral follicles undergoing IVM. Of vitrified follicles, 94.0% (345/367) were recovered after thawing. The survival rate after the first two-days of IVM culture was 82.3% (284/345) for the cryoloop vitrified follicles and 100% for the control follicles (437/437). The percentage of oocytes in the cryoloop group that developed to M2 was 70.2% (174/248), comparable to that of the control group at 68.7% (241/351) (p value 0.12). Our results indicate that cyroloop vitrification is a viable and practical technique for cryopreservation of mouse preantral follicles. Human oocyte cypreservation by means of cryoloop vitrification may prove to be useful as a possible treatment modality for human fertility preservation.

Keywords:

• fertility preservation
• in vitro maturation
• oocytes
• preantral follicle
• vitrification

Introduction

Improved cancer detection and treatments have prompted the medical community to emphasize quality of life issues for cancer survivors [Presidents Cancer Panel 2003; Breast Cancer Progress Review Group for the National Cancer Institute 2005]. Chemotherapy and radiotherapy induced gonadal toxicity and its resultant infertility/subfertility is a consequential but often underappreciated effect of cancer treatment [Oktay and Sonmezer 2007]. While sperm banking has been a clinically successful option available for men, fertility preservation options for women facing premature ovarian failure and infertility/subfertility are more complicated and limited [West et al. 2009].

Over the past decade, several fertility preservation techniques have been developed for female cancer patients including embryo, oocyte, and ovarian tissue banking. In vitro fertilization and embryo cryopreservation has proven successful in female cancer patients, however, its use is restricted by the need for a sperm source, the creation of embryos, and the potential concerns of tumor growth in hormonally sensitive cancers [Mukaida et al. 2006; Nakashima et al. 2010; Takahashi et al. 2005]. Other fertility preservation techniques including ovarian tissue banking and transplantation have shown limited success in animal models, as ovarian function is generally short-lived due to ischemic tissue injury [Aubard et al. 1999; Bedaiwy and Falcone 2004]. Concerns regarding the possible reintroduction of malignant cells into the patient with the reimplantation of previously cryopreserved tissue have also been raised [Donnez et al. 2005]. These apprehensions, and others, highlight the need for alternative approaches.

Compared to the number of mature oocytes obtained following a cycle of controlled ovarian hyperstimulation, the reservoir of preantral follicles in ovarian tissue offers a significantly larger and potentially successful opportunity for fertility preservation. Preantral follicle isolation and IVM may be a more practical strategy than other current treatment approaches as it avoids a long delay in the initiation of cancer therapy, the dilemma of embryo creation, and the reintroduction of tissue and potential cancer cells into the patient [Jurema and Nogueira 2006]. Up until now, cryopreservation of immature and mature follicles has been technically difficult due to the number and variety of cells involved and the delicate nature of the follicular structure. Slow cooling cryopreservation protocols of primary follicles have reportedly yielded a 78% survival rate with only 61% of follicles able to resume meiosis in culture [Carroll et al. 1990].

Vitrification of oocytes and embryos has proven to be a successful innovation that has improved cryopreservation success [Balaban et al. 2008]. Vitrification involves the ultra rapid freezing of a cell or tissue in a small volume of cryoprotectant, avoiding the formation of intracellular ice crystals. Several methodological variations of oocyte and embryo vitrification have been reported including the use of cryoloop vitrification [Takahashi et al. 2005]. In this study, we evaluated the viability and competence of cryoloop vitrified mouse preantral follicles by IVM and compared them to a cohort of freshly obtained control preantral mouse follicles (non-cryopreserved) also undergoing IVM.

Results

Figure 1 illustrates the survival rates of both cohorts of preantral oocytes. A total of 804 mouse early preantral oocytes were isolated from 45 mice. From these, 367 mouse preantral follicles were vitrified using the cryoloop technique. They were compared to 437 fresh follicles. Of those follicles vitrified, 94.0% (354/367) were recovered after thawing. Normal follicles at d 20 appeared mushroom-like with the cumulus-oocyte-complex in the center of the follicle (Fig. 2). Although the follicles recovered from vitrification appeared morphologically normal, some of the follicles underwent degeneration during the following in vitro culture. Examples are shown in Figure 3. The survival rate after the first two-days of IVM culture was 82.3% (284/354) for the cryoloop vitrified follicles, while 100% of the control follicles (437/437) survived during the same time period. The survival rate after 10 d of culture was 70.7% (248/354) for cryoloop vitrified follicles compared to 80.3% (351/437) for the control group. If the survival rate is calculated from the base number of follicles that survived the 2-d culture, the survival rate of the cryoloop vitrified follicles is higher at 87.3% (248/284) than that of the control cohort at 80.3% (351/437).

Figure 1. Survival Rates of In Vitro Matured Follicles from Vitrified/Thawed Cohort and Controls.

Figure 2. Isolated Preantral Mouse Oocytes. A) Cumulus oocyte complex. B) Oocyte complex with cumulus cells removed through digestion with hyaluronic acid.

Figure 3. Vitrified and In Vitro Matured Preantral Mouse Oocytes. A) Preantral oocytes within cryoloop. B) Thawed oocytes. C) Oocyte after 2 days of in vitro maturation. D) Oocyte after 10 days of in vitro maturation.

Following 17 h of final culture in hCG containing medium, 70.2% (174/248) of the surviving cryoloop vitrified IVM oocytes reached the M2 stage, while 68.7% (241/351) of the surviving control IVM oocytes reached M2, which was not statistically significant (P = 0.12). In the remaining oocytes that failed to reach the M2 stage, 57.8% (26/45) had reached the germinal vesicle break down (GVBD) stage in comparison to 50.0% (21/42) of the control IVM oocytes.

Discussion

While life expectancies of cancer patients have improved with current treatment regimens, their fertility potential continues to be damaged by cytotoxic therapies. Although fertility preservation techniques can be employed, these protocols are not universally applicable and posses a number of financial, ethical, and procedural disadvantages. The isolation, vitrification, and IVM of a preantral follicle is a promising protocol that avoids many of the pitfalls of previously discussed fertility preservation procedures including a long delay in cancer therapy, the dilemmas of embryo creation, and the reintroduction of tissue and potential cancer cells into the patient [Jurema and Nogueira 2006]. Despite previously reported successes, oocyte cryopreservation and vitrification may damage the delicate nature of the oocyte complex [Jurema and Nogueira 2006]. This study demonstrates that cryoloop vitrification and subsequent in vitro maturation of preantral follicles is feasible and deserves additional study as a possible treatment modality.

Previously, vitrification of human embryos has been proven successful with embryo survival rates after vitrification of 86 to 97% and reported pregnancy rates of 44% [Mukaida et al. 2006; Takahashi et al. 2005]. Oocyte survival after conventional straw vitrification has also been reported at approximately 80 to 86% with pregnancy rates of 14 to 41% [Almodin et al. 2010; Liebermann et al. 2003; Noyes et al. 2010]. Compared with conventional straw vitrification, the cryoloop technique uses a smaller volume of vitrification solution during cryopreservation and reduces the exposure of the follicle to potentially damaging cryoprotectant. The cryoloop technique has been utilized to vitrify mouse and human embryos with minimal impact on embryonic developmental potential or implantation rates [Sheehan et al. 2006]. Survival thaw rates of 85% have been reported in human embryos after cryoloop vitrification with subsequent clinical pregnancy rates of 44%, supporting our conclusions that cryoloop vitrification is a successful and promising technique in reproductive medicine [Desai et al. 2007]. Mature human oocytes vitrified by the cryoloop technique show a higher percentage of cortical granules than those mature oocytes vitrified by traditional cryoleaf procedures, however, the impact of cryoloop vitrification upon oocyte developmental potential is unknown [Nottola et al. 2009]. Despite the theoretical advantage of a smaller amount of cryoprotectant and previous favorable cryoloop embryo outcomes, the use of this technique for the vitrification of preantral oocytes has yet to be reported [Sheehan et al. 2006]. Based on our study, it appears that cryoloop vitrification holds promise as a practical, economical, and successful technique for the preservation and subsequent in vitro maturation of preantral follicles.

In the present study, we cryopreserved preantral mouse oocytes by utilizing the cryoloop vitrification technique and evaluated their developmental potential with in vitro maturation. We have previously reported successful IVM of mouse follicles, but the present study demonstrates a lack of effect of vitrification upon IVM outcomes [Carrell et al. 2005]. Ninety-four percent of cryoloop vitrified oocytes were recovered after thawing, a far larger percentage than has been previously reported using other cryopreservation techniques [Liebermann et al. 2003]. Although certain follicles recovered from vitrification appeared morphologically normal under the inverted microscope, some may have been compromised by the vitrification procedure or suffered minor damage during the isolation protocol. Those minor damages may not interfere with initial oocyte survival rates but instead may compromise the developmental potential of the follicles as assessed by IVM. Interestingly, we found the 10-d survival rate of the cryoloop vitrified follicles was higher (87.3%) than that of the control cohorts (80.3%), if the rate is based on the number of the 2-d follicles. Additionally, the percentage of the M2 oocytes in the cryoloop vitrification group (70.2%) was comparable to that of the oocytes in the control group (68.7%). Finally, examining our overall 10-d survival rate based on the number of oocytes initially isolated, our 10-d survival rate of 67.6% (248/367) is higher than previous calculated reports of 51% using different vitrification protocols [dela Pena et al. 2002].

Altogether, the results of our study suggest that cryoloop vitrification may be another viable tool for human oocyte preservation. Future studies will investigate the embryo quality and pregnancy rates of vitrified, thawed, and in vitro matured preantral oocytes. Additional investigations using human preantral oocytes are required before application of this technique, however, it is hoped that these studies of oocyte vitrification and maturation will result in new options for fertility preservation.

Materials and methods

Animal and Chemicals

All protocols and treatments were performed with Institutional Animal Care and Use Committee (IACUC) approval. B6D2 F1 (C57BL/DBA F1 hybrid) mice were bred in an on-site laboratory facility and were housed in a temperature and light controlled room at 23 – 25^o C, on a 12h light/dark cycle and fed with ad lib pellet food and water. All chemicals and reagents were purchased from Sigma (St. Louis, MO, USA), unless otherwise specified.

Follicle Isolation

Early preantral follicles were mechanically isolated from 12 to 14 d old B6D2F1 females as described previously. Only 100 – 130 µm diameter follicles with a round shape, a central oocyte location, the absence of space between the oocyte and surrounding granulosa cells, and the identification of complete basal lamina morphology were selected for this study. Forty-five mice were used to isolate 804 preantral follicles for control and experimental groups.

Preantral Follicle Vitrification and Warming

The vitrification and thawing protocols used in this study were previously described by Mukaida et al. [2001] with small modifications described below. For cryoloop vitrification, three early preantral follicles were exposed to Vitrification Solution 1 (HEPES-MEM medium pH 7.4 containing 7.5% (V/V) DMSO and 7.5% (V/V) ethylene glycol, 0.5% BSA) for 2 min. At the completion of 2 min, one cryoloop with cap was dipped into Vitrification Solution 2 to create a thin layer of the solution on the nylon loop, while the follicles were transferred to Vitrification Solution 2 (HEPES-MEM medium containing 15% DMSO, 15% ethylene glycol, 0.5% BSA, 10 mg/ml Ficoll 70, 0.696 M sucrose) and incubated for 30 s. Thereafter, all three follicles were loaded to the cryoloop (Hampton Research, Aliso Viejo, CA, USA) and immediately plunged into liquid nitrogen. Follicles vitrified in this study were frozen for 3 hr to 14 d.

In the thaw protocol, the cryoloop and vial were removed from liquid nitrogen and placed in Warming Solution 1 at 37°C (HEPES-MEM medium supplement with 0.33 M sucrose, 0.5% BSA). The follicles immediately fell from the loop into the warming solution. After 2 min in Warming Solution 1, the follicles were transferred to Warming Solution 2 (HEPES-MEM medium supplemented with 0.2 M sucrose and 0.5% BSA). Three min later, the follicles were washed twice with holding medium (HEPES-MEM medium supplemented with 10% fetal calf serum FCS, 100 mU/ml penicillin 100 ug/ml streptomycin) and once with culture medium (α-MEM medium pH 7.4 with Glutamax (Invitrogen Corporation, Cat. 32561-037) supplemented with 5% FCS, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium (ITS; BD Biosciences, 354350), 100 mIU/ml recombinant hFSH (Gonal-F, Serono), 50 mIU/ml penicillin, and 50 µg/ml streptomycin. IVM of the follicles followed (Fig. 3).

Preantral Follicle and Oocyte In Vitro Maturation

Follicles were individually cultured in culture medium droplets under mineral oil. Twenty droplets in one 60 x15 mm-dish were cultured for 2-10 d in culture medium with 5% CO₂ at 37°C. Half of the culture medium was replaced by fresh medium every other day. Freshly isolated follicles were cultured under identical circumstances. Survival rates were based on quantification of the number of nonviable, degenerated follicles viewed under the inverted microscope. Degenerated follicles were defined as those follicles that were no longer attached to the bottom of the culture dish and had demonstrated a reduction of follicular size (d 2) as well as follicles that had extruded the oocyte (d 11) and/or had a dissolving oocyte complex within the follicular cells. Normal follicles at d 20 appeared mushroom-like with the cumulus-oocyte-complex in the center of the follicle (Fig. 2). On the afternoon of d 10, the culture medium was replaced with the maturation medium (α-MEM medium with Glutamax supplemented with 5% FCS, 2.5 U/ml of hCG (Calbiochem, Cat. 230734) and antibiotics) and the follicles were continued to culture for 17 hr. At the end of the maturation, the cumulus-oocyte complexes were collected for oocyte evaluation.

Oocyte Evaluation

The oocytes were denuded of cumulus cells by gentle agitation using a micropipette in a medium containing 80 U/ml hyaluronidase (SAGE Media, Cat. 4007) until all cumulus cells were completely removed (Fig. 2). The oocytes were evaluated with a TE200 Hoffman inverted, modulation contrast microscope. All oocytes with increasing cytoplasmic darkening and granularity were classified as degenerated. Oocytes with a polar body were designated as M2 and those without a germinal vesicle were classified as GVBD while the remainder of the oocytes were classified as germinal vesicle (GV).

Statistical Analysis

Statistical analysis was carried out by the χ² test using STATA/IC 10.0 for Macintosh (CRC, College Station, TX, USA). P < .05 was considered statistically significant.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Abbreviations

IVM	=	in vitro maturation
M2	=	metaphase two stage
GVBD	=	germinal vesicle break down.