Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate

Background: Phosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments. Methods: Ovarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5–3.0 × 1.5–3.0 × 0.5–0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5–3.0 × 1.5–3.0 × 1.5–2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). Results: For Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative). Conclusion: The presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue

First Reported Use of Radiofrequency Identification (RFID) Technique for Targeted Excision of Suspicious Axillary Lymph Nodes in Early Stage Breast Cancer - Evaluation of Feasibility and Review of Current Recommendations

Background/Aim: The purpose of this study was to evaluate, whether radio frequency identification (RFID) labeling of axillary lymph nodes (LNs) for the use of targeted resection is feasible in primary breast cancer patients with suspicious LNs. Patients and Methods: We analyzed 10 consecutive patients where RFID technique was used for intraoperative detection of suspicious LNs without preceding neoadjuvant chemotherapy (NACT). We compared the specifics of these procedures to 10 consecutive sentinel lymph node biopsies (SLNB) in the cN0 situation. Results: Intraoperative detection rate (DR) for the RFID-labeled target lymph node (TLN) was 100%. Perioperative complications were infrequent and comparable to SLNB. Average time for location of the RFID labeled TLN was quicker than for the SLN. In 71.4% the chip bearing TLN equaled a SLN. Conclusion: The use of the RFID technique for intraoperative localization of axillary LNs for targeted excision seems feasible. RFID technique for targeted axillary dissection (TAD) following NACT should be investigated in a prospective manner

The rate of estrogen receptor-conversion associated with tumor progression in estrogen receptor-positive breast cancer patients following adjuvant Tamoxifen administration

Background Hormone Receptor (HR)-discordance between primary breast cancer and metastasis is a known biological phenomenon. Discordance studies usually comprise a heterogeneous group of HR-positive and negative patients and allow for the comparison of changes in HR-status from the primary to the recurrent disease. However, in a clinical setting, the rate of estrogen receptor-conversion following endocrine therapy with agents such as Tamoxifen (TAM) in estrogen receptor-positive cancers is of primary interest as opposed to total receptor discordance. Aim To investigate the rate of estrogen receptor-conversion associated with tumor progression in estrogen receptor-positive breast cancer patients following adjuvant TAM administration and to compare the results with the meta-analysis data of HR-discordance studies. Methods and Results A retrospective double-center review of biomarkers in 67 estrogen receptor-positive breast cancer patients who underwent TAM treatment in the adjuvant setting. The estrogen and progesterone receptor-status were compared at the time of diagnosis and following relapse and the Disease-free Survival (DFS), mean duration of TAM treatment as well as the operative, radiation, and cytotoxic therapies registered before TAM treatment, were recorded. Initially, all patients were estrogen receptor-positive. The average age at the time of diagnosis was 52.8 +/- 12.4 years. After recurrence, only 47 patients (70.1%) were still estrogen receptor-positive with a highly significant loss of estrogen receptor-expression in 29.9% of cases. The mean duration of TAM treatment was 40.7 +/- 19.9 months. 45 patients (i.e., 67.2%) progressed during the TAM treatment and the remaining 22 patients (32.8%) developed relapse after the TAM treatment had finished. Initially, there were 82.1% progesterone receptor-positive and 17.9% progesterone receptor-negative, but after relapse the progesterone receptor-positive cases diminished significantly to 53.7%, showing a progesterone receptor-loss of 28.4%. Conclusion The rate of estrogen receptor-loss associated with tumor progression following TAM treatment is approximately 30%, which is of clinical relevance in order to evaluate further endocrine efficacy in these patients. This rate of receptor conversion is roughly 6-13% higher compared to the recently published meta-analysis data of discordance studies. This discrepancy could possibly be due to anti-hormonal therapy with TAM accentuating receptor conversion

Long-term (24h) cooling of ovarian fragments in the presence of permeable cryoprotectants prior to freezing: Two unsuccesful IVF-cycles and spontaneous pregnancy with baby born after re-transplantation

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 degrees C for 22-24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at -9 degrees C, cooling from -9 to -34 degrees C at a rate of -0.3 degrees C/min and plunging at -34 degrees C into liquid nitrogen. After thawing in a 100 degrees C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 +/- 1.9% and 96.4 +/- 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the autotransplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 degrees C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing

Improving Breast Conserving Surgery Using the Faxitron (R) OR Specimen Radiography System - A Complication Analysis, Cost Evaluation and Literature Review

Background/Aim: The combination of pre-surgical clip placement and hook-wire guided surgery is considered the gold standard for adequately locating non-palpable lesions during breast conserving surgery. After surgical removal of the segment, radiography is required to confirm clip removal, increasing surgical time, post-surgical complication rates, and cost. Patients and Methods: We performed a retrospective analysis, using the Faxitron (R) in-theater specimen radiography system, of the following primary endpoints: surgical time and complication rates. The secondary endpoints were cost effectiveness and clip-location rates. The Control cohort included breast conserving surgery patients prior to May 2019 (n=150) and the Validation cohort included breast conserving surgery patients after May 2019 (n=53). Results: The analysis showed an improvement in surgical time when using the Faxitron (R) system, which is directly linked to a benefit in cost effectiveness. A numerical benefit in complication rates was also shown. A subgroup analysis showed a significant advantage in surgical time for breast conserving surgery plus sentinel node biopsy and open breast biopsies. Conclusion: Use of the Faxitron (R) system significantly reduces surgical time, which increases cost efficiency while maintaining a low complication rate

Patient with ovarian insufficiency: baby born after anticancer therapy and re-transplantation of cryopreserved ovarian tissue

Background The second major cause of death is cancer. In fact, the effectiveness of anticancer treatments and positive long-term prognosis for young women has increased. However, the problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic and lead to the functional death of ovaries. There is potential key solution to this problem: cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence. Data regarding cryopreservation and re-transplantation of ovarian tissue from patients with ovarian insufficiency is limited. The aim of this treatment was the re-transplantation of cryopreserved ovarian tissue after anticancer therapy of patient with ovarian insufficiency (56 IU/l FSH, 8 ng/l beta-estradiol, 0.1): it was found 1 primordial follicle in 5 mm(3)of tissue. Thirty seven days after the re-transplantation of ovarian tissue, the restoration of the menstrual cycle of Patient W. was noted. Three months after the transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Conclusions Described protocol of conventional cryopreservation of ovarian tissue can be used for treatment of patients with ovarian insufficiency

Comparison of Subpectoral versus Prepectoral Immediate Implant Reconstruction after Skin- and Nipple-Sparing Mastectomy in Breast Cancer Patients: A Retrospective Hospital-Based Cohort Study

Introduction: Implant-based immediate breast reconstruction (IBR) is a common surgical procedure in breast cancer patients. Comparative analysis concerning the placement of implants is still lacking. Hence, we aimed to analyze pre- versus subpectoral IBR in breast cancer patients. Patients: A single-center experience with implant-based IBR following skin/nipple-sparing mastectomy was evaluated. Patient demographics, incidence of major complications, and quality of life assessed with BREAST-Q were compared between the pre- and subpectoral cohort. Results: A total of 63 patients were included in this analysis of whom 29 underwent subpectoral and 34 underwent prepectoral IBR. Median duration of surgery was prolonged in the subpectoral versus the prepectoral group (104 +/- 28 vs. 80 +/- 91 min; p < 0.05). The mean number of major complications was significantly increased in the subpectoral group (1.41 +/- 1.76 vs. 0.47 +/- 0.75 per patient; p < 0.05). Detailed analysis showed a significantly increased incidence of implant dislocation (p < 0.05) and a trend concerning capsular contracture (p = 0.086, not significant) and necrosis (p = 0.092, not significant) in the subpectoral group. Quality of life was equal in both groups. Conclusion: The mean number of major complications in the subpectoral group should be considered when IBR is indicated. Prepectoral IBR seems to be a feasible alternative surgical treatment option with less major complications in selected patients

Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex

Abstract Background The aim of this study was to examine the effectiveness of Tumor Dissociation Enzyme (TDE) on the viability of follicles after digestion of fresh and cryopreserved ovarian cortex fragments (OCFs). Methods Fresh and thawed OCF from 14 patients (29 ± 6 years), sized 20 to 210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1, frozen OCF digested with TDE; Group 2, frozen OCF digested with LiberaseTM; Group 3, fresh OCF digested with TDE; and Group 4, fresh OCF digested with Liberase TM. Evaluation of follicle viability was performed under light microscope after staining with Neutral red. For visualization of viable and dead cells under a confocal laser scanning microscope, the follicles were stained with Calcein AM and ethidium homodimer-1. Results The results showed that the number of retrieved follicles was significantly higher (990 vs 487; P < 0.01) in the TDE-treatment group compared to the Liberase TM-group. The presence of intense neutral red stained follicles was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (70.3% ± +/− 6.22 vs 53,1% ± 2.03 and 94.2% ± 6.6 vs 79.1% ± 2.1; P < 0.01). The percentage of Calcein AM stained follicles of class V1 was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (95.97% ± 7.8 vs 87.87% ± 2.4; 97.1% ± 6.8 vs 91.3% ± 2.3; P < 0.01). Conclusion The enzymatic digestion of ovarian cortex with TDE provides recovery of a higher number of healthy preantral follicles in contrast to earlier described Liberase TM procedure

Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 +/- 6 years old), sized 20-210 mm(3) were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 +/- 2.4% vs. 90.4 +/- 3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 +/- 3.4% vs. 89.7 +/- 4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 +/- 3.4%; Group 2: 91.8 +/- 6.0%; Group 3: 90.5 +/- 6.9%; Group 4: 87.3 +/- 2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 +/- 1.6%; Group 2: 4.4 +/- 1.6%; Group 3: 1.6 +/- 1.1%; Group 4: 2.4 +/- 1.5%), late apoptotic (Group 1: 2.7 +/- 2.4%; Group 2: 44.0 +/- 1.9%; Group 3: 3.1 +/- 1.1%; Group 4: 2.8 +/- 0.7% and necrotic (Group 1: 0.9% +/- 0.1%; Group 2: 2.9 +/- 0.8%; Group 3: 3.4 +/- 4.5%; Group 4: 1.1 +/- 0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex