2.1 Ethics statement

Animal experimental protocols were approved by the Peking University Laboratory Animal Administration Committee, and experiments were performed according to the Peking University Guidelines for Animal Experimentation. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

The study was performed under a protocol approved by the institutional review boards from the Peking University Shenzhen Hospital. All the study participants provided written informed consent at enrolment.

2.2 Animal

54 female nude mice (BALB/C nude mice, 6–7 weeks of age) were obtained from the experimental animal center of Southern Medical University. The animals were housed in a high-efficiency particulate air-filtered positive pressure room. Cages were filter topped and animals had free access to food and water, under12 ​h light and 12 ​h darkness conditions. Groups of 5–6 mice were housed in one cage. Upon arrival from the breeding company, the mice were allowed to get acclimated for one week. All procedures, tests, and injections were performed under a laminar flow hood in a positive pressure room.

2.3 Tissue preparation

Ovarian tissue was obtained from 4 informed patients with various forms of the benign ovarian disease: the mean age of the women was 29.6 ​± ​1.5 years. Normal ovarian tissues rather than diseased parts were collected and transported to the laboratory within 24 ​h in sterile 15 ​ml Corning tubes, which contained about 5 ​ml Leibovitz-15 medium supplemented with penicillin/streptomycin (0.1 ​mg/ml) and 10% human serum albumin (HSA), and were pre-warmed to 37 ​°C. After removal of the medulla, the cortex was sliced into pieces 1–2 ​mm thick and 10–20 ​mm long with a scalpel blade under a stereomicroscope.

2.4 Vitrification-warming protocol

The vitrification protocol was based on a two-step method in an open carrier. Ovarian samples were placed in a 60 ​mm dish containing 15 ​ml vitrification solution Ⅰ (VSⅠ) consisting of 10% dimethyl sulfoxide (DMSO), 10% ethylene glycol (EG), 0.1 ​M sucrose, 0.1 ​M trehalose, 6% HSA, M199 medium, and Hepes at room temperature for 15min. Subsequently, the samples were transferred into a second 60 ​mm dish containing 15 ​ml vitrification solution Ⅱ (VSⅡ) consisting of 20% DMSO, 20%EG, 0.3 ​M sucrose, 0.1 ​M trehalose, 6%HSA, M199 medium, and Hepes at room temperature for 10min. Then the ovarian slices were transferred onto a sterile cryotissue straw or self-made simple cryotissue solid grids. The straws or grids were immediately plunged into liquid nitrogen and transferred into cryotubes for storage.

The tissues were warmed at room temperature for 30s and immediately into thawing solution (TS) Ⅰ (2.5%EG ​+ ​2.5%DMSO ​+ ​0.5 ​M sucrose ​+ ​0.2 ​M trehalose ​+ ​6% HSA ​+ ​M199) at 37 ​°C for an additional 1 ​min. The tissues were then washed stepwise in thawing solution Ⅱ, Ⅲ and Ⅳ, TS Ⅱ (0.5 ​M sucrose +0.2 ​M trehalose ​+ ​6% HSA ​+ ​M199) for 3 ​min, TS Ⅲ (0.5 ​M sucrose ​+ ​6% HSA ​+ ​M199) for 5 ​min, TS Ⅳ (0.25 ​M sucrose ​+ ​M199) for 5 ​min. Finally, the warmed samples were rinsed in M199 medium.

2.5 Transplantation into nude mice

Fifty-four recipient mice were used for the experiment and all they underwent oophorectomy, which was performed under narcosis with ketamin irrespective of the stage of the oestrus cycle. During surgery, mice were kept on a warming plate. The incision site was disinfected with 75% alcohol and covered with a one-way sterile towel. Both ovaries were removed by a small body wall incision that was sutured with absorbable thread.

54 mice were divided into two group: vitrified ovarian xenotransplant Group (VOX, n ​= ​45) and non-transplant control group (n ​= ​9). Xenografting of the ovarian cortex was performed 14 days after oophorectomy. The ovarian tissue pieces were placed in an intramuscular pocket of the back muscle.

The transplanted mice were grouped into vitrified/warmed grafted-4 weeks (VOX-4w, n ​= ​15), vitrified/warmed grafted-6weeks (VOX-6w, n ​= ​15) and vitrified/warmed grafted-12 weeks (VOX-12w, n ​= ​15) according to the time after transplantation.

2.6 Gonadotropin stimulation

Mice received every other day i.p. injections of human menopausal gonadotropin (hMG, Menogon, Ferring, Kiel; 2 IU Follicle-stimulating hormone （FSH）/2 IU luteinizing hormone （LH） per animal/two days) from day 14 after transplantation until 4 weeks, 6 weeks, or 12 weeks. The no transplanted group received the hormonal treatment for the same period as the transplanted group.

2.7 Oestrus cycle stage determination

Vaginal smears were taken once a week from all mice starting at day 14 after transplantation using sterile pipettes, to examine if follicular tissues survived the transplantation and could produce enough estrogens to cornify the vaginal epithelium. Vaginal cells were left to dry after being smeared on a microscopic slide and were then stained with methylene blue. The epithelium cells were classified into one of the four following categories: a) pro-estrous, b) estrous, c) metestrous, d) diestrous. The animals were weighed and killed respectively at 4 weeks, 6 weeks, and 12 weeks after transplantation, and serum was collected for E2 measurement by E2 ELISA Kit purchased from Elabscience Biotechnology with a sensitivity of 9.38 ​pg/L.

Uterus was collected after its periphery tissues were carefully trimmed and were weighted after being fixed in 4% paraformaldehyde. The uterus/weight ratio was used to evaluate the grafts function recovery. Grafts were recovered and fixed in Paraformaldehyde for 24 ​h.

2.8 Morphological evaluation

Vitrified-warmed samples were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 6 ​h at 4 ​°C for light microscopic (LM) evaluation and some samples with high numbers of follicles were selected for TEM. For ovarian cortical pieces from VOX-4w, VOX-6w, and VOX-12w, they were also fixed respectively in 2.5% glutaraldehyde in PBS (pH 7.4) for 6 ​h at 4 ​°C for LM and some typical samples were picked for TEM.

For LM, after routine paraffin embedding, the samples were serially sectioned (4 ​μm) and every tenth section was stained with hematoxylin and eosin and examined microscopically. The developmental stages of the follicles were evaluated according to the regulation defined by Gougeon.⁹

For TEM, the fixed samples were then post-fixed with 1% osmium tetroxide in the same buffer for 2 ​h. After dehydration in an ascending series of ethanol, specimens were placed in propylene oxide and embedded in LX-112, then cut into 0.5 ​μm sections and stained with toluidine blue. Ultra-thin sections (60 ​nm) were contrasted with uranyl acetate and lead citrate and examined by using a transmission electron microscope. The ultrastructural analysis was performed according to subcellular criteria reported by Hreinsson J.⁷

2.9 Statistical evaluation

SPSS 22.0 was used for data evaluation. Nominal data were compared by using the t-test and it was considered statistically significant when the p value was less than 0.05.