Sperm and testicular tissue cryopreservation and assisted reproductive technology outcomes in male cancer patients: a 15-year experience.

OBJECTIVE: To explore the characteristics of cancer patients who cryopreserved sperm/testicular tissue samples in the Cryobank of Charité-Universitätsmedizin Berlin between 2004 and 2019, and the ART utilization rate with associated outcomes. METHODS: Retrospective data were available for 506 cancer patients, of which 46 (9.1%) had used their samples for artificial reproductive technologies (ART). Corresponding cycle information was collected from external fertility centers. RESULTS: Our cohort included 53/506 (10.5%) patients aged < 18 years at diagnosis. While adolescents and adults mainly banked sperm, adolescents showed higher rates of testicular tissue cryopreservation before (11.8%, 6/51 vs. 6.4%, 26/406) and after treatment (16.7%, 4/24 vs. 7.8%, 13/167). At study conduction, storage had been ended for 44.8% (269/601) of samples. The majority of samples used for ART were requested within the first 3 years after cryopreservation (71.5%, 28/39, range = 0-12 years). Pregnancy rate was 51.4% (19/37 cycles), resulting in 11 singleton births, 3 twin pairs, and 4 miscarriages. CONCLUSION: With the new advantage of public health insurance coverage of fertility preservation (FP) in Germany, an increased utilization has already been noticed in our center, emphasizing the necessity of further knowledge for individual counseling. Adolescent cancer patients need to be addressed specifically, as these patients show especially low cryopreservation rates.

Survey of Long-Term Experiences of Sperm Cryopreservation in Oncological and Non-Oncological Patients: Usage and Reproductive Outcomes of a Large Monocentric Cohort.

Progress in oncological treatment has led to an improved long-term survival of young male cancer patients over the last decades. However, standard cancer treatments frequently implicate fertility-damaging potential. Cryopreservation of sperm is the current standard option to preserve patient's fertility after treatment, yet long-term data on usage and reproductive experiences is still limited. Natural fertility after treatment and especially in relation to the type of treatment has been poorly analyzed so far. Therefore, we performed a retrospective survey including male patients with an indication for gonadotoxic treatment who cryopreserved reproductive material at our institution between 1994 and 2017. Study questionnaires regarding treatment, material usage, and reproductive outcomes were sent to eligible patients. Additionally, semen analyses of study participants from the time of cryopreservation were evaluated. A total of 99 patients were included in the study. Respondents' median age was 38.0 years. Most frequent diagnoses were testicular cancer (29.3%) and lymphoma (26.3%). A further 8.1% suffered from autoimmune diseases. Testicular cancer patients had a significantly lower pre-treatment median sperm concentration (18.0 million/ml) compared to non-testicular cancer patients (54.2 million/ml). Until November 2020, the determined sperm usage and cumulative live-birth rate per couple were 17.2% and 58.8%, respectively. Most sperm users received treatments with high (40.0%) or intermediate (33.3%) gonadotoxic potential. 20.7% of all patients reported to had fathered at least one naturally conceived child after treatment, this being the case especially if they had been treated with less or potentially gonadotoxic therapies. In conclusion, our findings emphasize the importance of sperm cryopreservation in the context of male fertility preservation. Furthermore, they indicate that the gonadotoxic potential of patients' treatments could represent a predictive factor for sperm usage.

Rebubbling in Descemet Membrane Endothelial Keratoplasty: Influence of Pressure and Duration of the Intracameral Air Tamponade.

PURPOSE: To explore the impact of intracameral air tamponade pressure and duration on graft attachment and rebubbling rates. DESIGN: A prospective, interventional, nonrandomized study. METHODS: setting: Department of Ophthalmology, Charité - Universitätsmedizin Berlin. STUDY POPULATION: One hundred seventeen patients who underwent Descemet membrane endothelial keratoplasty (DMEK). OBSERVATION: Intraocular pressure (IOP) at the end of the surgery, immediately after filling the anterior chamber with air, categorized into low (<10 mm Hg), normal (10-20 mm Hg), and high (>20 mm Hg), and the time until partial removal of the air. MAIN OUTCOME MEASURES: Rebubbling rates and endothelial cell density over a 3-month follow-up period analyzed by a multivariable Cox regression model and an analysis of covariance model. RESULTS: Thirty-two patients required a rebubbling (27% [95% CI 19%-35%]). Nine patients required more than 1 rebubbling (7% [95% CI 3%-12%]). Compared with normal IOP, lower (HR 8.98 [95% CI 1.07-75.41]) and higher IOP (HR 10.63 [95% CI 1.44-78.27]) increased the risk of requiring a rebubbling (P = .006). Independent of the IOP, an air tamponade duration beyond 2 hours reduced the risk of rebubbling (HR 0.36 [95% CI 0.18-0.71, P = .003]). One month after surgery, the mean endothelial cell loss was 13% (95% CI 2%-25%) and 23% (95% CI 17%-29%) in the group with air tamponade duration of below and above 2 hours, respectively (P = .126). At 3 months after surgery, it was 31% (95% CI 17%-42%) and 42% (95% CI 32%-52%) in the respective groups (P = .229). CONCLUSIONS: A postsurgical air tamponade of at least 2 hours with an IOP within the physiological range could help to reduce rebubbling rates.

Validation of Serological Testing for Anti-Treponema pallidum from Postmortem Blood on the Siemens-BEP(®)-III Automatic System.

BACKGROUND: Infectious disease marker testing is obligatory for the release of human tissue for transplantation. Most CE-marked tests are not validated for postmortem blood. In a previous study we have validated the testing for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc. Here, we present the validation of testing for antibodies against T. pallidum, which is the last marker obligatory for tissue release for transplantation. METHODS: 17 samples of postmortem sera and 10 samples of both pre-und postmortem sera were obtained from cornea donors and tested for anti-T. pallidum on the Siemens-BEP-III-System. These sera were spiked with anti-T. pallidum-positive standard sera in concentrations which give low- and high-positive results at the respective dilution. RESULTS: Two of the unspiked postmortem sera were false-positive most likely due to intense hemolysis (free hemoglobin > 50 mg/dl). Of the 25 negative postmortem sera, none of the spiked samples was false-negative after 0, 24 and 60 h. CONCLUSION: There is no indication that postmortem samples give false-negative or false-positive results with the test system and test kits used in cases of low hemolysis. The procedure described might serve as a model for validating other test kits on postmortem samples.

Virus NAT for HIV, HBV, and HCV in Post-Mortal Blood Specimens over 48 h after Death of Infected Patients - First Results.

OBJECTIVE: According to EU regulations (EU directive 2006/17/EC), blood specimens for virologic testing in the context of post-mortal tissue donation must be taken not later than 24 h post mortem. METHODS: To verify validity of NAT in blood specimens collected later, viral nucleic acid concentrations were monitored in blood samples of deceased persons infected with HIV (n = 7), HBV (n = 5), and HCV (n = 17) taken upon admission and at 12 h, 24 h, 36 h and 48 h post mortem. HIV and HCV RNA were quantified using Cobas TaqMan (Roche), HBV DNA was measured by in-house PCR. RESULTS: A more than 10-fold decrease of viral load in samples taken 36 h or 48 h post mortem was seen in one HIV-infected patient only. For all other patients tested the decrease of viral load in 36-hour or 48-hour post-mortal samples was less pronounced. Specimens of 3 HIV- and 2 HBV-infected patients taken 24 h post mortem or later were even found to have increased concentrations (>10-fold), possibly due to post-mortem liberation of virus from particular cells or tissues. CONCLUSION: Our preliminary data indicate that the time point of blood collection for HIV, HBV and HCV testing by PCR may be extended to 36 h or even 48 h post mortem and thus improve availability of tissue donations.

Comparison of in situ Corneoscleral Disc Excision versus Whole Globe Enucleation in Cornea Donors Regarding Microbial Contamination in Organ Culture Medium - a Prospective Monocentric Study over 9 Years.

BACKGROUND: CORNEAS NEEDED FOR KERATOPLASTY CAN BE HARVESTED USING TWO TECHNIQUES: whole globe enucleation and in situ excision of the corneoscleral disc. This study evaluates the rate of microbial contamination of the donor cornea organ culture medium according to the method of retrieval. METHODS: All donor corneas of our cornea bank received between January 1, 2001 and December 31, 2009 put into organ culture and microbio-logically tested were prospectively analyzed for microbial contamination of the organ culture medium. RESULTS: 2,805 donor corneas could be included in this study in total. 975 of them were retrieved by whole globe enucleation (group 1) and 1,830 by in situ corneoscleral disc excision (group 2). 15 corneas of group 1 (1.5%) and 46 corneas of group 2 (2.5%) showed a contamination of the organ culture medium. The difference was shown not to be statistically significant (p = 0.082). CONCLUSION: The rate of microbial contamination in organ-cultured donor corneas does not seem to be dependent on the method of their retrieval.

Validation of the Microbiological Testing of Tissue Preparations Using the BACTEC™ Blood Culture System.

BACKGROUND: Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary. METHODS: Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTEC™ Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTEC™ Standard Aerobic/Anaerobic for HTK and Ringer solution) and subsequently spiked with 10-100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. RESULTS: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. CONCLUSION: The use of BACTEC™ blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.

Validation of Virus NAT for HIV, HCV, HBV and HAV Using Post-Mortal Blood Samples.

OBJECTIVE: Commercial available NAT systems are usually not validated for screening of post-mortem blood samples. NAT testing might be challenging due to inhibitory substances in the cadaveric blood sample that cause false-negative test results. Validation studies have to be performed to show the performance characteristics of the NAT assays for testing cadaveric blood. METHODS: A set of 32 post-mortem serum and plasma samples from cornea donors and 40 control samples from blood donors, serologically and NAT negative for all investigated parameters, were spiked with defined concentrations of WHO reference material and tested for HIV-1, HCV, HBV, and HAV by NAT using DRK Baden-Württemberg-Hesse CE PCR kits. Analytical sensitivity, analytical specificity and reproducibility/precision were validated and compared with each other in both groups of samples. RESULTS: The analytical sensitivity was 100% for control and post-mortem specimens when spiked with virus standards at concentrations of 3 × level of detection (LOD). Invalid results did not occur. The analytical specificity rate for all assays was 100%. Intra-assay variation was analyzed as a function of sample material and sampling time post mortem. Values of % coefficient of variation (%CV) were comparable for serum and plasma but slightly higher for post-mortem samples especially for those samples collected more than 24 h post mortem. CONCLUSION: Based on the presented validation, postmortem donor samples can be tested with the automated DRK Baden-Würtemberg-Hesse NAT system.

A prospective time-course study on serological testing for human immunodeficiency virus, hepatitis B virus and hepatitis C virus with blood samples taken up to 48 h after death.

The transmission of viral and non-viral infectious pathogens continues to be the most serious of the potential adverse effects of allogenic tissue transplantations. EU Directive 2006/17/EC stipulates that cadaveric blood specimens for serology testing in the context of post-mortem tissue donation must be taken not later than 24 h post-mortem. An expanded time slot would significantly improve the availability of tissue donations, but there are no significant data on the stability of infectious serology assays for anti-human immunodeficiency virus (HIV), anti-hepatitis C virus (HCV), hepatitis B virus (HBV) surface antigen (HBsAg) and anti-HBC core antigen (HBc) in samples collected more than 24 h post-mortem. In this prospective study, serum samples of 30 deceased persons were taken upon admission to the Institute of Forensic Medicine (University Hospital Hamburg-Eppendorf, Germany) and at 12, 24, 36 and 48 h post-mortem. All samples were measured twice, first using the Abbott AxSYM system, and then after ~9 months of storage at -70 °C using the BEP III System with Siemens and Ortho reagents. For HIV, six deceased persons with a pre-mortem HIV history were included. All samples (at committal and at 12, 24, 36, 48 h) were reactive. Indeterminate or false-negative results did not occur. For HCV, 17 deceased persons with a pre-mortem HCV history were included; 16 samples were reactive up to 48 h and one was reactive at 36 h post-mortem (48 h sample was not available). Indeterminate or false-negative results did not occur. For HBV, nine deceased persons were included: five samples were initially positive for HBsAg and remained positive up to 48 h, and eight of the samples were reactive for anti-HBc up to 48 h and one up to 36 h post-mortem (48 h sample was not available). Indeterminate or false-negative results did not occur. These data suggest that infectious serological testing may be extended for blood samples of potential tissue donors collected up to 48 h post-mortem to detect antibodies or antigens for HIV, HBV and HCV.

Validation of the Serological Testing for Anti-HIV-1/2, Anti-HCV, HBsAg, and Anti-HBc from Post-mortem Blood on the Siemens-BEP-III Automatic System.

BACKGROUND: Some properties of blood are modified post mortem. These modifications might give false-negative or false-positive results in infectious disease testing. Most CE-marked test equipment for infectious serology testing is not validated for testing post-mortal blood. Validation, however, is obligatory, if the results are used for the release of tissues for transplantation. MATHODS: Samples of pre- and post-mortem sera were obtained from 20 cornea donors, and the results were compared for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc on the Siemens-BEP-III Automatic System. Negative post-mortem sera were spiked with standard sera (PEI anti-HCV IgG, PEI HBsAg ad 1000 standard, anti-HBc IgG (WHO) NIBSC 95/522, PEI anti-HIV-IV) in concentrations which give low- and high-positive results for the respective marker. RESULTS: All pre-mortem sera were negative for all markers. None of the post-mortem samples was false-positive. None of the spiked postmortem samples was false-negative. Technical errors occurred during the validation process but could be detected and eliminated. Serum samples should be centrifuged immediately after collection, and it must be taken into account that post-mortem serum could rarely lead to blockage of pipetting systems due to clotting phenomena. CONCLUSION: There is no indication that post-mortem samples give false-negative or false-positve results with the test system and test kits used. The procedure described might serve as a model for validating other test kits on post-mortem samples.