3 Methods
3.1 Removal
of Ovarian Tissue
for Cryopreservation
Before laparoscopy starts, it must be ensured that the infection
status of the patient is negative; otherwise processing under Good
Manufacturing Practice (GMP) criteria is not possible for any tissue, which is a hurdle to subsequent cryopreservation. The German
FertiPROTEKT network recommends screening for anti-HIV1/
2 p24 antigen, HBs-Ag, and anti-HBc; HBV-PCR in case of positivity; as well as anti-HCV-IgG and Treponema pallidum TP (syphilis pathogen)—based on the regulations of the German law for
transplantation (“TPG,” }4, Appendix 3: Required laboratory tests
and test procedures in accordance with }4, No. 1a,d). The serological determination of the actual concentration of anti-Mu ¨llerian
hormone (AMH) in serum is also helpful before the start of cytotoxic therapy, as it is an indicator for the specification of the fertility
reserve. The amount of ovarian tissue that should be removed varies
according to the expected gonadotoxicity of the planned oncological therapy, i.e., the higher the gonadotoxic risk of the therapy,
the more ovarian tissue should be removed. In the case of a high
gonadotoxic risk or in prepubertal girls (due to the small size of the
ovaries), the removal of an entire ovary may even be considered (see
Notes 2 and 3).
3.2 Preparation
of Medium
for (Overnight)
Transportation
1. Aliquot 20 mL of Custodiol in 30 mL PS Multipurpose Containers with a 25 mL pipette under sterile conditions.
2. Mark the tubes with a label for the date of preparation, date of
expiry, name of the producer, and LOT and REF numbers of
the used Custodiol solution.
3. The ovarian tissue should be transferred immediately after
removal into a sterile holding and transportation medium.
Transport to the laboratory of cryobank must be carried out
at 4–8
C. If the preparation/storage and cryopreservation of
the ovarian tissue cannot take place directly at the clinic that
does the surgery, it is possible to cooperate with specialized,
external, and centralized cryobanks for fertility preservation.
Transportation to an external cryobank is carried out in special
shipping containers, which ensure that the cold chain is not
interrupted (see Fig. 1 and Note 4).
4. Store the aliquots at 4
C.
3.3 Preparation
of Medium
for Cryopreservation
of Ovarian Tissue
1. Remove 50 mL medium from 500 mL MHM-C under sterile
conditions.
2. Substitute with 50 mL of CryoSure-DMSO using a sterile
pipette.
3. Mix the prepared medium gently for 25 min on a shaker.
Cryopreservation and Thawing of Human Ovarian Tissue
489
3.1 Removal
of Ovarian Tissue
for Cryopreservation
Before laparoscopy starts, it must be ensured that the infection
status of the patient is negative; otherwise processing under Good
Manufacturing Practice (GMP) criteria is not possible for any tissue, which is a hurdle to subsequent cryopreservation. The German
FertiPROTEKT network recommends screening for anti-HIV1/
2 p24 antigen, HBs-Ag, and anti-HBc; HBV-PCR in case of positivity; as well as anti-HCV-IgG and Treponema pallidum TP (syphilis pathogen)—based on the regulations of the German law for
transplantation (“TPG,” }4, Appendix 3: Required laboratory tests
and test procedures in accordance with }4, No. 1a,d). The serological determination of the actual concentration of anti-Mu ¨llerian
hormone (AMH) in serum is also helpful before the start of cytotoxic therapy, as it is an indicator for the specification of the fertility
reserve. The amount of ovarian tissue that should be removed varies
according to the expected gonadotoxicity of the planned oncological therapy, i.e., the higher the gonadotoxic risk of the therapy,
the more ovarian tissue should be removed. In the case of a high
gonadotoxic risk or in prepubertal girls (due to the small size of the
ovaries), the removal of an entire ovary may even be considered (see
Notes 2 and 3).
3.2 Preparation
of Medium
for (Overnight)
Transportation
1. Aliquot 20 mL of Custodiol in 30 mL PS Multipurpose Containers with a 25 mL pipette under sterile conditions.
2. Mark the tubes with a label for the date of preparation, date of
expiry, name of the producer, and LOT and REF numbers of
the used Custodiol solution.
3. The ovarian tissue should be transferred immediately after
removal into a sterile holding and transportation medium.
Transport to the laboratory of cryobank must be carried out
at 4–8
C. If the preparation/storage and cryopreservation of
the ovarian tissue cannot take place directly at the clinic that
does the surgery, it is possible to cooperate with specialized,
external, and centralized cryobanks for fertility preservation.
Transportation to an external cryobank is carried out in special
shipping containers, which ensure that the cold chain is not
interrupted (see Fig. 1 and Note 4).
4. Store the aliquots at 4
C.
3.3 Preparation
of Medium
for Cryopreservation
of Ovarian Tissue
1. Remove 50 mL medium from 500 mL MHM-C under sterile
conditions.
2. Substitute with 50 mL of CryoSure-DMSO using a sterile
pipette.
3. Mix the prepared medium gently for 25 min on a shaker.
Cryopreservation and Thawing of Human Ovarian Tissue
489