7. Decant the cortex strips from the cryo vials into a sterile Oosafe
sample container.
8. Transfer the cortex strips in the first well of the six-well plate,
with medium containing 0.75 M sucrose.
9. The incubation time in this solution is 15 min on an orbital
shaker (200 cycles/min) at room temperature (RT).
10. After 15 min, transfer the cortex strips to the well with medium
containing 0.375 M sucrose.
11. The incubation time in this solution is again 15 min on an
orbital shaker (200 cycles/min) at RT.
12. Transfer the cortex strips in the well with medium containing
0.125 M sucrose, and incubate the cortex strips also in this
solution for 15 min on an orbital shaker (200 cycles/min)
at RT.
13. Wash the cortex strips in the well containing 10 mL DPBS/
HSA (i.e., well 4) during 10 min. Thereafter transfer the cortex
strips to the well with 5 mL DPBS/HSA (i.e., well 6), and
incubate in this solution for 5 min.
14. Immediately transport the thawed ovarian cortex strips to the
operation theater. Transport is performed in a 15 mL Corning
Falcon High-Clarity Polypropylene Centrifuge Tube containing the remaining 5 mL DPBS/HSA washing solution.
15. The thawed ovarian cortex strip(s) should be immediately
transplanted by the surgeon (e.g., in a preprepared peritoneal
pocket). This peritoneal pocket should be prepared during the
thawing procedure of the ovarian cortex tissue (i.e., such that
the transplantation/transfer into the peritoneal pocket can take
place exactly 1 h after starting of the thawing procedure).
3.6 Determination
of Ovarian Cortex
Potential Before
and After
Cryopreservation
The success of fertility preservation by cryopreservation, i.e., the
chance of achieving a later pregnancy and the birth of a healthy
child from the transplants, depends on the density of primordial
follicles within the ovarian cortex of a given patient at the time of
collection and before the start of oncological therapy. The better
the ovarian reserve, the better the chances. Ovarian reserve can
relatively precisely be estimated before starting gonadotoxic therapy on the basis of the sonographic determination of the AFC
(antral follicle counts), the size of the ovary, the AMH (anti-Mu ¨llerian hormone) concentration, the age, and the additional determination of the primordial and primary follicle density in the
transplants. For the later method, a suitable quantitative detection
method is required. For this purpose, standardized biopsy punches
(e.g., 6 Â 2 mm biopsies) are taken during the preparation of the
tissue from different cortical areas using biopsy punches. These are
then divided into two equal portions with three biopsies each.
Cryopreservation and Thawing of Human Ovarian Tissue
495
sample container.
8. Transfer the cortex strips in the first well of the six-well plate,
with medium containing 0.75 M sucrose.
9. The incubation time in this solution is 15 min on an orbital
shaker (200 cycles/min) at room temperature (RT).
10. After 15 min, transfer the cortex strips to the well with medium
containing 0.375 M sucrose.
11. The incubation time in this solution is again 15 min on an
orbital shaker (200 cycles/min) at RT.
12. Transfer the cortex strips in the well with medium containing
0.125 M sucrose, and incubate the cortex strips also in this
solution for 15 min on an orbital shaker (200 cycles/min)
at RT.
13. Wash the cortex strips in the well containing 10 mL DPBS/
HSA (i.e., well 4) during 10 min. Thereafter transfer the cortex
strips to the well with 5 mL DPBS/HSA (i.e., well 6), and
incubate in this solution for 5 min.
14. Immediately transport the thawed ovarian cortex strips to the
operation theater. Transport is performed in a 15 mL Corning
Falcon High-Clarity Polypropylene Centrifuge Tube containing the remaining 5 mL DPBS/HSA washing solution.
15. The thawed ovarian cortex strip(s) should be immediately
transplanted by the surgeon (e.g., in a preprepared peritoneal
pocket). This peritoneal pocket should be prepared during the
thawing procedure of the ovarian cortex tissue (i.e., such that
the transplantation/transfer into the peritoneal pocket can take
place exactly 1 h after starting of the thawing procedure).
3.6 Determination
of Ovarian Cortex
Potential Before
and After
Cryopreservation
The success of fertility preservation by cryopreservation, i.e., the
chance of achieving a later pregnancy and the birth of a healthy
child from the transplants, depends on the density of primordial
follicles within the ovarian cortex of a given patient at the time of
collection and before the start of oncological therapy. The better
the ovarian reserve, the better the chances. Ovarian reserve can
relatively precisely be estimated before starting gonadotoxic therapy on the basis of the sonographic determination of the AFC
(antral follicle counts), the size of the ovary, the AMH (anti-Mu ¨llerian hormone) concentration, the age, and the additional determination of the primordial and primary follicle density in the
transplants. For the later method, a suitable quantitative detection
method is required. For this purpose, standardized biopsy punches
(e.g., 6 Â 2 mm biopsies) are taken during the preparation of the
tissue from different cortical areas using biopsy punches. These are
then divided into two equal portions with three biopsies each.
Cryopreservation and Thawing of Human Ovarian Tissue
495