Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue.

Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

Cryostorage of human ovarian tissue: evaluating the storage and disposal pattern over a 22-year period in 2475 patients.

RESEARCH QUESTION: What are the parameters of age, indications for ovarian tissue cryopreservation, storage characteristics and reasons for tissue disposal in a large cohort of individuals undertaking cryopreservation? DESIGN: The relevant parameters in a single university centre were revised and digitalized in the period from 2019 to 2021. To assess patients' motivation at the end of storage, patients were contacted by letter, e-mails and telephone calls. RESULTS: A group of 2475 patients with stored ovarian tissue were analysed in the time period between 2000 and 2021; the response rate for contact calls and letters was 28.8% (224/777). Where storage had ended (n = 1155), patients had on average stored for 3.8 years and begun storing at age 30 years; the main indications were breast cancer (53%) and lymphoma (17.5%). Of these participants, 2.5% had a transplantation on site, 10.3% transferred their tissue to another cryobank and 11.5% were deceased. The majority of the group (75.7%) ended their storage due to pregnancy (49.1%), a lack of desire to have children (25.9%), storage fees that were too expensive (8.9%), death (8.5%), recurrence of cancer (8.5%), lack of a partner (4%) and fear of surgery in the future (3.1%); 6.7% retrospectively regretted ending storage. CONCLUSIONS: The pregnancy rate of 49.1%, resulting from ovarian tissue that was not removed during surgery for scheduled ovarian tissue cryopreservation supports the clinical approach of removing and cryopreserving only 25-50% of one ovary. It is proposed that interdisciplinary counselling should be implemented not only prior to fertility preservation, but also when intending to end storage.

The effect of high-throughput vitrification of human ovarian cortex tissue on follicular viability: a promising alternative to conventional slow freezing?

BACKGROUND: The standard procedure most frequently used for ovarian tissue cryopreservation (OTC) is slow freezing, while vitrification has been proposed as promising alternative and has built an impressive catalog of success in fertility laboratories regarding cryopreservation of oocytes and embryos. METHODS: We developed and evaluated a high-throughput protocol for vitrification of human ovarian tissue suitable for clinical processing. Follicular viability was assessed via calcein staining prior and after cryopreservation analyzing ovarian tissue of a cohort of 30 patients. RESULTS: We found no significant differences regarding follicular viability between slow frozen and vitrified cortex tissue samples 24 h after thawing and rapid warming. Follicular viability of thawed and rapid warmed samples was not significantly different in comparison to fresh samples, indicating high proportions of follicular survival rates with both methods. CONCLUSIONS: High-throughput vitrification is a promising option in a clinical setting. More research is required to determine the status of other tissue-specific quality indicators potentially influencing on autotransplantation.

In vitro growth (IVG) of human ovarian follicles in frozen thawed ovarian cortex tissue culture supplemented with follicular fluid under hypoxic conditions.

BACKGROUND: Despite its clinical success rates, transplantation after ovarian tissue cryopreservation (OTC) remains a matter of concern. Certain cancer subtypes may lead to the transfer of malignant cells when transplantation of affected ovarian tissue is conducted. IVG and subsequent isolation of vital follicles obtained from frozen thawed ovarian tissue for further in vitro maturation (IVM) would expand current fertility protection techniques while reducing the risk of retransplanting malignant cells. METHODS: A total of 216 cortical biopsies from 3 patients were included in this study in 4 treatment groups. After freezing, thawing and 8 days of hypoxic tissue culture supplemented with different concentrations of human follicular fluid (HuFF) and follicle-stimulating hormone (FSH), follicles were isolated enzymatically and stained with calcein to determine follicular viability. Numbers and size of vital follicles were assessed by fluorescence microscopy (Ti2, Nikon) and specified by computer assisted, semi-automated measurement (NIS software, Nikon). To estimate the effect of in vitro culture on apoptosis, tissue sections were stained for nicked DNA (TUNEL) prior and after tissue culture. RESULTS: Analysing 3025 vital follicles, we observed significant differences [P < 0.01] regarding follicle size when hypoxic tissue culture was supplemented with HuFF compared with the control group on day 1, individual follicles reached sizes > 100 µm. CONCLUSIONS: The results implicate that HuFF contains valuable factors contributing to significant IVG of follicles in human ovarian tissue and could be regarded as an additional tool in personalized fertility restoration prior to retransplantation of ovarian tissue.

Effect of mild α-chymotrypsin treatment of highly viscous semen samples on fertilization rates.

BACKGROUND: Highly viscous semen reduces sperm motility significantly and can contribute to infertility. When processing semen samples, few techniques exist to induce liquefaction in case of seminal hyperviscosity such as different washing steps and mechanical treatment. The use of α-chymotrypsin seems controversial due to possible negative effects on fertilisation rates after in vitro fertilization (IVF). The main objective of this study was to examine the influence of mild α-chymotrypsin treatment of semen samples on the fertilisation rate after artificial reproductive treatment (ART). METHODS: The fertilization rate of 52 ART cycles was examined following IVF using a low dose of α-chymotrypsin to induce liquefaction of highly viscous semen and was compared to a control group of 88 ART cycles. RESULTS: There was no significant difference in the fertilization rates of α-chymotrypsin treated semen samples compared to the control group; pregnancy rates were unaffected. CONCLUSIONS: The use of mild α-chymotrypsin treatment of semen samples in case of hyperviscosity does not appear to impact negatively on the fertilization rates after ART and could be regarded as an additional method to induce liquefaction of highly viscous semen samples in IVF.