Step 1: Human sample
Human ovarian tissues were surgically removed from a female patient with cervical cancer before chemotherapy and/or radiation therapy in order to preserve her fertility.
Step 2: The cryopreservation of human sample (human ovarian cortex tissues)
Human ovarian cortex tissues were frozen and thawed by using the conventional method (Control) (Ref. No. 3) or supercooling (S.C.) procedures (S.C. No.1 and S.C. No. 2).
Used three supercooling machines < 1), 2), and 3) >, Program freezer and Conventional Liquid nitrogen tank were as follows.
Our supercooling machine under variable magnetic field.
DIC-217 (Magiquoal supercooling machine without a variable magnetic field, DIC-217 (Magiquoal supercooling machine, Japan, http://www.magiquoal.com/corporate/index.html).
Conventional supercooling machine under variable magnetic field (ABI CO., LTD., Chiba, Japan) (Ref.1).
Program freezer
Conventional program freezer (Planner Kryo 360-3.3, Depex, Belgium).
- Conventional Liquid nitrogen tank.
The S.C. procedure was as follows. The human ovary was rapidly transported to the laboratory in Leibovitz L-15 medium (Life Technologies, Merelbeke, Belgium). The human ovarian cortex was carefully dissected to obtain 40 small pieces (10 × 10 mm), which were then incubated for 30 min in a cryoprotective solution (Leibovitz medium supplemented with 1.5 M dimethylsulphoxide, 0.1 M sucrose, both provided by Sigma Aldrich, Bornem, Belgium and 10% patient’s serum at 4.0°C). Each piece of the human ovarian cortex was cryopreserved in one 2-ml vial (Simport, Merck Eurolab, Leuven, Belgium), containing 1.4 ml of the cryoprotectant solution. The cryopreservation procedure as S.C. procedure was performed using our three supercooling machines < 1), 2), and 3) >. The following procedures were used for the machine of 1) or 3): started at 4.0°C, 1.0°C/min to –5.0°C, 20 min of soaking in a supercooled state under a variable magnetic field (2kHz, 0.3mT), then manual seeding, 1.0°C/min to –40.0°C, 10.0°C/min to –140.0°C and then plunged into liquid nitrogen. The pieces of the human ovarian tissue were thawed on the day of the experimental procedure following a rapid protocol: vials were placed at room temperature for 2 min and then in water at 25.0°C for 2 min. The human ovarian tissue was washed stepwise for 5 min each in progressively lower concentrations of the cryoprotectant solution (1.5, 1, 0.5 and 0 mol/l). On the other hand, as for the machine of 2), the following procedures were used:started at 4.0°C, 20 min of soaking in a supercooled state at –5.0°C in the supercooling machine (DIC-217: Magiquoal supercooling machine without a variable magnetic field, DIC-217,Japan). After that, the pieces of the human ovarian tissue were transferred to conventional program freezer (Planner Kryo 360-3.3, Depex, Belgium). And, the pieces were cooled at 1.0°C/min to –40.0°C, 10.0°C/min to –140.0°C within the conventional program freezer, and then plunged into liquid nitrogen. The pieces of the human ovarian tissue were thawed on the day of the experimental procedure following a rapid protocol: vials were placed at room temperature for 2 min and then in water at 25.0°C for 2 min. The human ovarian tissue was washed stepwise for 5 min each in progressively lower concentrations of the cryoprotectant solution (1.5, 1, 0.5 and 0 mol/l).
Furthermore, as for the conventional method (Control) by using only Program freezer (Planner Kryo 360-3.3, Depex, Belgium: Ref.No. 3), the human ovary was rapidly transported to the laboratory in Leibovitz L-15 medium (Life Technologies, Merelbeke, Belgium). The human ovarian cortex was carefully dissected to obtain 40 small pieces (10 × 10 mm), which were then incubated for 30 min in a cryoprotective solution (Leibovitz medium supplemented with 1.5 M dimethylsulphoxide, 0.1 M sucrose, both provided by Sigma Aldrich, Bornem, Belgium and 10% patient’s serum at 4.0°C). Each piece of the human ovarian cortex was cryopreserved in one 2-ml vial (Simport, Merck Eurolab, Leuven, Belgium), containing 1.4 ml of the cryoprotectant solution.
The following procedure was used: started at 4.0°C, 2.0°C/min to –7.0°C, 10 min of soaking, then manual seeding, 0.3°C/min to –40.0°C, 10.0°C/min to –140.0°C and then plunged into liquid nitrogen. The pieces of the human ovarian tissue were thawed on the day of the experimental procedure following a rapid protocol: vials were placed at room temperature for 2 min and then in water at 25.0°C for 2 min. The human ovarian tissue was washed stepwise for 5 min each in progressively lower concentrations of the cryoprotectant solution (1.5, 1, 0.5 and 0 mol/l).
Step 3: Gene expression profiles of human oocytes within human ovarian cortex tissues
The expressions of human oocyte marker genes (Ref. No. 2) for human oocytes within human ovarian cortex tissues were investigated. The presence of each indicated mRNA was assessed in total RNA samples by conventional RT-PCR using a SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen) and Platinum Taq polymerase (Invitrogen). Details for PCR primers used to analyze gene expression in total RNA samples prepared from human cells and tissues were according to the method of White YA, et al. (Ref. No. 2).
Step 4: Immunoanalyses
For assessment of human oocytes expression of DDX4, KIT, YBX2 and LHX8 within human ovarian cortex tissues, human ovarian cortex tissue was fixed in 4% PFA, paraffin-embedded and sectioned (6-μm) prior to high temperature antigen retrieval using 0.01 M sodium citrate buffer (pH 6.0). After cooling, sections were washed and blocked for 1 h at 20 °C using TNK buffer (0.1 M Tris-HCl, 0.55 M NaCl, 0.1 mM KCl, 0.5% BSA, and 0.1% Triton-X100 in PBS) containing either 1% normal goat serum (for subsequent detection of DDX4-COOH in human ovary, YBX2 and LHX8 in human ovary) or 1% normal donkey serum (DDX4-NH2 in human ovary and KIT in human ovary). Sections were then incubated with a 1:100 dilution of primary antibody (in TNK buffer with 1% normal serum) overnight at 4 °C washed in PBS, and incubated for 30 min at 20 °C with a 1:500 dilution of goat anti-rabbit IgG conjugated to Alexa Fluor 568 (DDX4-COOH detection in human ovary), goat anti-rabbit IgG conjugated to Alexa Fluor 488 (LHX8 detection in human ovary) or donkey anti-goat IgG conjugated to Alexa Fluor 488 (DDX4-NH2 detection in human ovary and KIT detection in human ovary). For assessment of YBX2 expression in human ovary, primary antibody was detected using a goat anti-rabbit biotinylated IgG for 1 h at 20 °C, followed by reaction with streptavidin-conjugated Alexa Fluor 568. After washing with PBS, sections were cover-slipped using Vectashield containing DAPI (Vector Labs).
Step 5: Human follicle activation and the transplantation of human oocytes inside oviduct
Pairs of cortical cubes were treated with or without bpV(pic) (100 mM) for 24 h in MEM-α medium containing 10% human serum albumin (Mitsubishi Tanabe Pharma), 1% antibiotic/antimycotic solution (Invitrogen), and 0.3 IU/mL FSH before transplantation of human oocytes inside the oviduct. Next, the human oocytes were transplanted within oviduct and were grown during 24 weeks within the oviduct.
Step 6: Human Follicle Counting
Follicles were only counted when the dark-staining nucleolus was seen within the nucleus of the oocytes to prevent recounting of the same follicle.