Technology for Biobanking of Epididymal Spermatozoa from Patient with Obstructive Azoospermia: Case Report about Baby Born after Conventional Freezing Only with a Nonpermeable Cryoprotectant 360 kDa Polyvinylpyrrolidone.

This publication reports, for the first time, the birth of a healthy child after intracytoplasmic sperm injection (ICSI) of motile spermatozoa after conventional ("slow") freezing of epididymal spermatozoa using 5% polyvinylpyrrolidone (PVP) of high molecular weight (360 kDa). Cryopreservation solution with 10% PVP was added to 30 µL of spermatozoa suspension in a 1:1 ratio, with a final PVP concentration of 5%. Then, polycarbonate capillaries for oocyte denudation with a diameter of 170 µm were filled with 60 µL of the resulting sperm suspension. After that, the capillaries were placed for 10 minutes at a height of 15 cm above liquid nitrogen and immersed into liquid nitrogen. To warm the spermatozoa, the capillaries were immersed in a water bath at a temperature of 40°C for 30 seconds. Oocyte fertilization was performed by ICSI. Zygotes were cultured in vitro for 5 days to the blastocyst stage. More than 100 spermatozoa were obtained after percutaneous epidydimal sperm aspiration, of which 80% were motile. After cryopreservation, storage for 3 months in liquid nitrogen, and thawing, 72% of the total sperm cells remained motile. Ten oocyte-cumulus complexes were found after follicle puncture, and eight metaphase II stage oocytes were fertilized using ICSI. After 18 hours, two pronuclei were found in seven (88%) of the oocytes. An analysis of the morphological characteristics of 5-day-old embryos showed that four (57%) of them reached the blastocyst stage. One embryo was transferred, and the remaining embryos were cryopreserved (vitrified). The onset of pregnancy was detected on the 14th day after embryo transfer, and one healthy girl (3300 g) was born at term.

Pelvic Exenteration for Recurrent Vulvar Cancer: A Retrospective Study.

Pelvic exenteration (PE) is one of the most radical surgical approaches. In earlier times, PE was associated with high morbidity and mortality. Nowadays, due to improved selection of suitable patients, perioperative settings, and postoperative care, patients' outcomes have been optimized. To investigate patients' outcomes and identify possible influencing clinical and histopathological factors, we analysed 17 patients with recurrent vulvar cancer who underwent PE in our department between 2007 and 2022. The median age was 64.9 years, with a difference of 40 years between the youngest and the oldest patient (41 vs. 81 years). The mean overall survival time was 55.7 months; the longest survival time reached up to 164 months. The achievement of complete cytoreduction (p = 0.02), the indication for surgery (curative vs. palliative), and the presence of distant metastases (both p = 0.01) showed a significant impact on overall survival. The presence of lymphatic metastases (p = 0.11) seems to have an influence on overall survival (OS) time. Major complications appeared in 35% of the patients. Our results support the existing data for PE in cases of recurrent vulvar cancer; for a group of selected patients, PE is a treatment option with good overall survival times and acceptable morbidity.

Shifting Trends in Obstetrics: An 18-year Analysis of Low-risk Births at a German University Hospital.

BACKGROUND/AIM: At the beginning of the 21st century, obstetric medicine took a turn from interventional to restrictive in low-risk birth. The present study examined the changes in peripartum management over the past 20 years at the Women's University Hospital Cologne. The attitudes of the becoming mother and physicians towards anesthesia, episiotomy, and vaginal-operative deliveries were compared and the factors influencing the duration of birth over the past 20 years were examined. PATIENTS AND METHODS: In this retrospective study, the low-risk singleton birth of 955 in 2000/2001 and 944 births in 2018 at the Women's University Hospital Cologne were analyzed. RESULTS: The age of women who tended to give birth has significantly increased at present compared to 20 years ago. In 2018, labor was induced significantly more often than in 2000/2001. The rate of vaginal operative deliveries has fluctuated between 15% and 20% in the last 20 years. Forceps are no longer used. The use of episiotomy has taken a fundamental turn in the last 20 years. Prophylactic episiotomy is not performed anymore, most vaginal operative deliveries take place without the episiotomy. The birth duration has been significantly shortened at present compared to 20 years ago. CONCLUSION: Pregnancy and childbirth over the last years are not considered as a disease, but as a natural course, and the trend of minimizing interventions in low-risk delivery has a positive effect on childbirth.

Maternal and Perinatal Outcome After Induction of Labor Versus Expectant Management in Low-risk Pregnancies Beyond Term.

BACKGROUND/AIM: Due to still controversial discussion regarding appropriate termination of low-risk singleton pregnancies beyond term, this retrospective study aimed to evaluate maternal and perinatal outcomes depending on gestational age and obstetric management. PATIENTS AND METHODS: This is a retrospective cohort analysis including 3.242 low-risk singleton deliveries at the Department of Obstetrics of the University Hospital of Cologne between 2017 and 2022. According to current national guidelines, the cohort was subdivided into three gestational groups, group 1: 40+0-40+6 weeks, group 2: 40+7-40+10 weeks and group 3>40+10 weeks. RESULTS: In our cohort, advanced gestational age was associated with higher rates of secondary caesarean sections, lower rates of spontaneous vaginal deliveries, higher rates of meconium-stained amniotic fluid and depressed neonates with APGAR < 7 after 5 min. Analyzing obstetric management, induction of labor significantly increased the rate of secondary sections and reduced the rate of spontaneous deliveries, while the percentage of assistant vaginal deliveries was independent from obstetric management and gestational age. Induction of labor also significantly enhanced the need for tocolytic subpartu and epidural anesthesia and caused higher rates of abnormalities in cardiotocography (CTG), which also resulted in more frequent fetal scalp blood testing; however, the rate of fetal acidosis was independent of both obstetric management and gestational age. CONCLUSION: Our study supports expectant management of low-risk pregnancies beyond term, as induction of labor increased the rate of secondary sections and did not improve perinatal outcome.

Pelvic Exenteration for Recurrent Endometrial Cancer: A 15-Year Monocentric Retrospective Study.

Treatment options for recurrent endometrial adenocarcinoma are limited. In those cases, secondary surgical procedures such as pelvic exenteration form the only possible curative approach. The aim of this study was analyzing the outcomes of patients who underwent pelvic exenteration during the treatment of recurrent endometrial cancer intending to identify prognostic factors. More than 300 pelvic exenterations were performed. Fifteen patients were selected that received pelvic exenteration for recurrent endometrial adenocarcinoma. Data regarding patient characteristics, indication for surgery, complete cytoreduction, tumor grading and p53- and L1CAM-expression were collected and statistically evaluated. Univariate Cox regression was performed to identify predictive factors for long-term survival. The mean survival after pelvic exenteration for the whole patient population was 22.7 months, with the longest survival reaching up to 69 months. Overall survival was significantly longer for patients with a curative treatment intention (p = 0.015) and for patients with a well or moderately differentiated adenocarcinoma (p = 0.014). Complete cytoreduction seemed favorable with a mean survival of 32 months in contrast to 10 months when complete cytoreduction was not achieved. Pelvic exenteration is a possible treatment option for a selected group of patients resulting in a mean survival of nearly two years, offering a substantial prognostic improvement.

Endometrial Cancer. Guideline of the DGGG, DKG and DKH (S3-Level, AWMF Registry Number 032/034-OL, September 2022). Part 1 with Recommendations on the Epidemiology, Screening, Diagnosis and Hereditary Factors of Endometrial Cancer, Geriatric Assessment and Supply Structures.

Summary The S3-guideline on endometrial cancer, first published in April 2018, was reviewed in its entirety between April 2020 and January 2022 and updated. The review was carried out at the request of German Cancer Aid as part of the Oncology Guidelines Program and the lead coordinators were the German Society for Gynecology and Obstetrics (DGGG), the Gynecology Oncology Working Group (AGO) of the German Cancer Society (DKG) and the German Cancer Aid (DKH). The guideline update was based on a systematic search and assessment of the literature published between 2016 and 2020. All statements, recommendations and background texts were reviewed and either confirmed or amended. New statements and recommendations were included where necessary. Aim The use of evidence-based risk-adapted therapies to treat women with endometrial cancer of low risk prevents unnecessarily radical surgery and avoids non-beneficial adjuvant radiation therapy and/or chemotherapy. For women with endometrial cancer and a high risk of recurrence, the guideline defines the optimum level of radical surgery and indicates whether chemotherapy and/or adjuvant radiation therapy is necessary. This should improve the survival rates and quality of life of these patients. The S3-guideline on endometrial cancer and the quality indicators based on the guideline aim to provide the basis for the work of certified gynecological cancer centers. Methods The guideline was first compiled in 2018 in accordance with the requirements for S3-level guidelines and was updated in 2022. The update included an adaptation of the source guidelines identified using the German Instrument for Methodological Guideline Appraisal (DELBI). The update also used evidence reviews which were created based on selected literature obtained from systematic searches in selected literature databases using the PICO process. The Clinical Guidelines Service Group was tasked with carrying out a systematic search and assessment of the literature. Their results were used by interdisciplinary working groups as a basis for developing suggestions for recommendations and statements which were then modified during structured online consensus conferences and/or additionally amended online using the DELPHI process to achieve a consensus. Recommendations Part 1 of this short version of the guideline provides recommendations on epidemiology, screening, diagnosis, and hereditary factors. The epidemiology of endometrial cancer and the risk factors for developing endometrial cancer are presented. The options for screening and the methods used to diagnose endometrial cancer are outlined. Recommendations are given for the prevention, diagnosis, and therapy of hereditary forms of endometrial cancer. The use of geriatric assessment is considered and existing structures of care are presented.

Digital droplet PCR-based quantification of ccfHPV-DNA as liquid biopsy in HPV-driven cervical and vulvar cancer.

PURPOSE: More than 99% of cervical cancers and up to 40% of vulvar cancers are human papillomavirus (HPV) related. HPV 16 and 18 are the most relevant subtypes. Novel technologies allow the detection of minimal amounts of circulating cell-free HPV DNA (ccfHPV-DNA). The aim of this study was to evaluate ccfHPV-DNA assessed by droplet digital PCR (ddPCR) as a biomarker for molecular therapy monitoring in early, advanced, relapsed and metastatic HPV-driven cervical and vulvar cancer. METHODS: Inclusion criteria of the study were histologically proven HPV 16/18-driven cervical and vulvar cancer with first diagnosed disease, newly diagnosed recurrence, or progression of disease. Blood samples were taken pre- and post-therapeutically. Circulating cell-free HPV DNA was quantified using ddPCR and the results were correlated with clinical data. RESULTS: The mean copy number of ccfHPV-DNA was 838.6 (± 3089.1) in pretreatment and 2.3 (± 6.4) in post-treatment samples (p < 0.05). The copy number of ccfHPV-DNA increased with higher FIGO stages (p < 0.05), which are commonly used for clinical staging/assessment. Furthermore, we compared the distribution of copy numbers between T-stage 1 versus T-stage 2/3. We could show higher copy number level of ccfHPV-DNA in T-stage 2/3 (p < 0.05). CONCLUSIONS: Therapy monitoring with determination of ccfHPV-DNA by ddPCR with a small amount of plasma reflects response to therapy and appears feasible for patients in advanced cancer stages of cervical and vulvar cancer. This promising tool should be examined as marker of therapy monitoring in particular in novel HPV-directed therapies.

Impact of Advanced Maternal Age on Maternal and Neonatal Outcomes.

BACKGROUND/AIM: Due to better career opportunities for women and a shift in sex roles, as well as improved reproductive medicine, the age of women who conceive children is rising. A variety of maternal risks and complications that may occur during pregnancy or childbirth in women with advanced maternal age has been examined and reported controversial results. The present study focused on controversial and debatable conclusions regarding the impact of advanced maternal age on maternal and neonatal outcomes. PATIENTS AND METHODS: Data from 8,523 patients, who gave singleton birth at the Women's University Hospital Cologne between 2014 and 2018, were subdivided into two groups: those with maternal age ≥40 years and those <40, and analyzed. RESULTS: A significantly higher rate of C-section, more preterm births, more low birth weight, and higher incidence of retained placenta were observed in women older than or equal to 40. There were no significant differences regarding postpartum hemorrhage and fetal position. Younger patients tend to have more birth injuries and use more epidural administration. The evaluation of neonatal outcomes using fetal base-excess, birth pH, and Apgar score showed no significant clinical differences. CONCLUSION: More antenatal complications could be identified in patients with advanced maternal age. Nonetheless, the neonatal outcomes were comparable and no severe complications in women with advanced maternal age were observed. These findings are due to a well standardized management system for women with risk pregnancies. This encourages better monitoring and care of pregnant women with risk factors.

FeTPPS, a Peroxynitrite Decomposition Catalyst, Ameliorates Nitrosative Stress in Human Spermatozoa.

Excessive levels of reactive nitrogen species (RNS), such as peroxynitrite, promote nitrosative stress, which is an important cause of impaired sperm function. The metalloporphyrin FeTPPS is highly effective in catalyzing the decomposition of peroxynitrite, reducing its toxic effects in vivo and in vitro. FeTPPS has significant therapeutic potential in peroxynitrite-related diseases; however, its effects on human spermatozoa under nitrosative stress have not been described. This work aimed to evaluate the in vitro effect of FeTPPS against peroxynitrite-mediated nitrosative stress in human spermatozoa. For this purpose, spermatozoa from normozoospermic donors were exposed to 3-morpholinosydnonimine, a molecule that generates peroxynitrite. First, the FeTPPS-mediated peroxynitrite decomposition catalysis was analyzed. Then, its individual effect on sperm quality parameters was evaluated. Finally, the effect of FeTPPS on ATP levels, motility, mitochondrial membrane potential, thiol oxidation, viability, and DNA fragmentation was analyzed in spermatozoa under nitrosative stress conditions. The results showed that FeTPPS effectively catalyzes the decomposition of peroxynitrite without affecting sperm viability at concentrations up to 50 µmol/L. Furthermore, FeTPPS mitigates the deleterious effects of nitrosative stress on all sperm parameters analyzed. These results highlight the therapeutic potential of FeTPPS in reducing the negative impact of nitrosative stress in semen samples with high RNS levels.

RNA Transcripts in Human Ovarian Cells: Two-Time Cryopreservation Does Not Affect Developmental Potential.

Sometimes, for medical reasons, when a frozen tissue has already thawed, an operation by re-transplantation may be cancelled, and ovarian tissues should be re-frozen for transplantation next time. Research about the repeated cryopreservation of ovarian cells is rarely reported. It has been published that there is no difference in the follicle densities, proportions of proliferation of early preantral follicles, appearance of atretic follicles, or ultrastructural quality of frozen-thawed and re-frozen-rethawed tissue. However, the molecular mechanisms of a repeated cryopreservation effect on the developmental potential of ovarian cells are unknown. The aim of our experiments was to investigate the effect of re-freezing and re-thawing ovarian tissue on gene expression, gene function annotation, and protein-protein interactions. The morphological and biological activity of primordial, primary, and secondary follicles, aimed at using these follicles for the formation of artificial ovaries, was also detected. Second-generation mRNA sequencing technology with a high throughput and accuracy was adopted to determine the different transcriptome profiles in the cells of four groups: one-time cryopreserved (frozen and thawed) cells (Group 1), two-time cryopreserved (re-frozen and re-thawed after first cryopreservation) cells (Group 2), one-time cryopreserved (frozen and thawed) and in vitro cultured cells (Group 3), and two times cryopreserved (re-frozen and re-thawed after first cryopreservation) and in vitro cultured cells (Group 4). Some minor changes in the primordial, primary, and secondary follicles in terms of the morphology and biological activity were detected, and finally, the availability of these follicles for the formation of artificial ovaries was explored. It was established that during cryopreservation, the CEBPB/CYP19A1 pathway may be involved in regulating estrogen activity and CD44 is crucial for the development of ovarian cells. An analysis of gene expression in cryopreserved ovarian cells indicates that two-time (repeated) cryopreservation does not significantly affect the developmental potential of these cells. For medical reasons, when ovarian tissue is thawed but cannot be transplanted, it can be immediately re-frozen again.

Anatomical and functional outcomes after bilateral sacrospinous colposuspension (BSC) for the treatment of female genital prolapse.

BACKGROUND: Pelvic organ prolapse is a bothersome condition affecting many women at advanced age, but also frequently observed in young women with certain risk factors. Various surgical techniques have been developed with the aim of providing effective surgical treatment for apical prolapse. The vaginal bilateral sacrospinous colposuspension surgery (BSC) with ultralight mesh and utilization of the i- stich is a relatively new minimal invasive technique with very promising outcomes. The technique offers apical suspension, in the presence or absence of the uterus. The objective of this study is to evaluate the anatomical and functional outcomes of bilateral sacrospinous colposuspension with ultralight mesh in 30 Patients treated with the vaginal single incision standardized technique. METHODS: In this retrospective study, 30 patients were treated by BSC for significant vaginal, uterovaginal or cervical prolapse. A simultaneous anterior colporrhaphy, posterior colporrhaphy or both were performed when indicated. Anatomical and functional outcomes were assessed 1 year postoperatively using the Pelvic Organ Prolapse Quantification system (POP-Q) and the standardised Prolapse Quality of Life (P-QOL) questionnair. RESULTS: The POP-Q parameters were significantly improved at twelve months after surgery compared to baseline. The total score and all four subdomains of the P-QOL-questionnaire showed positive trends and improvement at twelve months after surgery when compared to preoperative values. All patients were asymptomatic and expressed high satisfaction one year after surgery. No intraoperative adverse events were recorded for all patients. Only minimal postoperative complications were recorded and they all resolved completely with conservative management. CONCLUSION: This study highlights the functional and anatomical outcomes of the minimally invasive vaginal bilateral sacrospinal colposuspension with ultralight mesh for the management of apical prolapse. The one year postoperative results of the proposed procedure reflect excellent outcomes with minimal complications. The data published here are very promising and warrant further investigations and more studies to evaluate the long-term outcomes of BSC in the surgical management of apical defects. TRIAL REGISTRATION: The study protocol was approved by the Ethics Committee at the University Hospital of Cologne, Germany (Date of registration: 08.02.2022) (Registration number: 21-1494-retro) (retrospectively registered).

Incidental FOXL2 mutated adult granulosa cell tumour of the ovary with thecoma-like foci.

We report on the incidental finding of a FOXL2 mutated adult granulosa cell tumour of the ovary with thecoma-like foci, a rare entity recently described by Jennifer N. Stall and Robert H. Young in a series of sixteen cases in 2019, displaying features differing from conventional adult granulosa cell tumour. Our aim is to specify the morphologic and molecular particularities of this presumably underrecognized finding, with a short presentation of the typical clinical context. Awareness of this rare and challenging neoplasm with indeterminate clinical course is crucial in routine diagnostics.

Statement of the Uterus Commission of the Gynecological Oncology Working Group (AGO) on the Use of Primary Chemoimmunotherapy to Treat Patients with Locally Advanced or Recurrent Endometrial Cancer.

The publication of two large randomized studies - the ENGOT-EN-6-NSGO/GOG-3031/RUBY trial and the NRG-GY018 trial - which investigated combining chemotherapy with immunotherapy to treat patients with primary advanced or recurrent endometrial cancer (EC) has transformed the clinical study landscape in terms of first-line therapy for affected patients and has set a new standard of therapy. In the ENGOT-EN-6-NSGO/GOG-3031/RUBY trial, the addition of dostarlimab to standard chemotherapy with carboplatin and paclitaxel resulted in a significant and clinically relevant improvement of progression-free survival and overall survival in the overall population, a significant and clinically relevant improvement of progression-free survival and overall survival in the subgroup with dMMR/MSI-high tumors, and a significant and clinically relevant improvement of progression-free survival in the subgroup with pMMR/MSI-low tumors. In the NRG-GY018 trial, the addition of pembrolizumab to standard chemotherapy with carboplatin and paclitaxel resulted in a significant and clinically relevant improvement of progression-free survival in the group with dMMR tumors, and a significant and clinically relevant improvement of progression-free survival in the group with pMMR tumors. As expected, the effect in both trials was much more pronounced in the group of patients with dMMR/MSI-high tumors. According to the assessment of the Uterus Organ Commission of the AGO, all patients with dMMR/MSI-high tumors should receive chemoimmunotherapy and all patients with pMMR/MSI-low tumors who meet the inclusion criteria of the two trials discussed here may have chemoimmunotherapy. For dostarlimab this means: patients with EC recurrence who will not undergo surgery or radiotherapy, patients with stage IIIA, IIIB or IIIC1 disease and a measurable lesion postoperatively, patients with stage IIIA, IIIB or IIIC1 disease with histological findings of serous EC, clear-cell EC or carcinosarcoma with or without a measurable lesion postoperatively, and patients with stage IIIC2 or IV disease with or without a measurable lesion postoperatively. For pembrolizumab this means: patients with EC recurrence (except carcinosarcoma) who will not undergo surgery or radiotherapy, and patients with stage III or IVA disease (except carcinosarcoma) and a measurable lesion postoperatively or with stage IVB disease with or without a measurable lesion.

Sarcoma of the Uterus. Guideline of the DGGG, OEGGG and SGGG (S2k-Level, AWMF Registry No. 015/074, April 2021).

Purpose This is an official guideline, published and coordinated by the Germany Society for Gynecology and Obstetrics (Deutsche Gesellschaft für Gynäkologie und Geburtshilfe, DGGG). Because of their rarity and heterogeneous histopathology, uterine sarcomas are challenging in terms of their clinical management and therefore require a multidisciplinary approach. To our knowledge, there are currently no binding evidence-based recommendations for the appropriate management of this heterogeneous group of tumors. Methods This S2k guideline was first published in 2015. The update published here is once again the result of the consensus of a representative interdisciplinary committee of experts who were commissioned by the Guidelines Committee of the DGGG to carry out a systematic search of the literature on uterine sarcomas. Members of the participating professional societies achieved a formal consensus after a structured consensus process. Recommendations 1.1 Epidemiology, classification, staging of uterine sarcomas. 1.2 Symptoms, general diagnostic workup, general pathology or genetic predisposition to uterine sarcomas. 2. Management of leiomyosarcomas. 3. Management of low-grade endometrial stromal sarcomas. 4. Management of high-grade endometrial stromal sarcoma and undifferentiated uterine sarcomas. 5. Management of adenosarcomas. 6. Rhabdomyosarcomas of the uterus in children and adolescents. 7. Follow-up of uterine sarcomas. 8. Management of morcellated uterine sarcomas. 9. Information provided to patients.

Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells.

Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-ß. TGF-ß plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-ß within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-ß, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-ß and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-ß.

Automatic Evaluation for Bioengineering of Human Artificial Ovary: A Model for Fertility Preservation for Prepubertal Female Patients with a Malignant Tumor.

INTRODUCTION: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. METHODS: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. RESULTS: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. CONCLUSIONS: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects.

BACKGROUND: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. METHODS: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. RESULTS: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5' splice sites (A5SS), alternative 3' splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). CONCLUSIONS: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer.

BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors. RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

Oral Misoprostol for the Induction of Labor: Comparison of Different Dosage Schemes With Respect to Maternal and Fetal Outcome in Patients Beyond 34 Weeks of Pregnancy.

BACKGROUND/AIM: Labor is induced in 1 out of 5 pregnancies. This is why we aimed to compare two different protocols of orally administered misoprostol for the induction of labor (IOL), with special regard to maternal and fetal outcome, delivery mode and duration. PATIENTS AND METHODS: One hundred and twenty four patients with a medical indication for IOL were divided into two groups: Group A (n=63), which initially received 50 µg misoprostol escalated to 100 and, subsequently, to 200 µg every 4 h with a daily maximum of 600µg, between 11/2007 and 01/2008; and Group B (n=61), which initially received 25 µg misoprostol followed by 100 µg every 4 h with a daily maximum of 300 µg, between 12/2009 and 04/2010. RESULTS: The mean administration-delivery interval was significantly lower in Group A (19.0 h) compared to Group B (27.1 h, p<0.05). Overall caesarean section rate, average birth weight, APGAR score, umbilical cord pH and meconium-stained fluid rates were similar between both groups. CONCLUSION: A higher dosage protocol of orally administered misoprostol significantly reduces the mean induction-delivery interval without increasing the risk for an adverse maternal or fetal outcome.

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling.

INTRODUCTION: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. METHODS: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. RESULTS: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. CONCLUSION: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.