First live birth in China after cryopreserved ovarian tissue transplantation to prevent premature ovarian insufficiency.

OBJECTIVE: This article reports the first live birth after cryopreserved ovarian tissue transplantation to prevent premature ovarian insufficiency in China. METHODS: A patient with myelodysplastic syndrome received ovarian tissue cryopreservation before hematopoietic stem cell transplantation, and six ovarian cortex strips were thawed and transplanted into her peritoneal pocket 2 years later. RESULTS: Pregnancy occurred spontaneously 27 months after grafting, and a healthy girl was born at 38 weeks gestation. Until now, the child has developed normally without any major diseases. CONCLUSIONS: We report the first live birth resulting from ovarian tissue cryopreservation and transplantation in China.

First pregnancy in China after ovarian tissue transplantation to prevent premature ovarian insufficiency.

OBJECTIVE: This article reports the first case of pregnancy after frozen-thawed ovarian tissue transplantation to prevent iatrogenic premature ovarian insufficiency in China. METHODS: Ovarian tissue cryopreservation was performed in a patient with myelodysplastic syndrome (MDS) before multi-agent chemotherapy and hematopoietic stem cell transplantation. Two years later, she showed complete remission from MDS, and six frozen-thawed ovarian tissue strips were transplanted into the peritoneal pocket. RESULTS: The patient's ovarian activity was restored 3 months after transplantation, and pregnancy occurred spontaneously 27 months after grafting. Until now, the pregnancy has progressed for 30 weeks, and the repeated ultrasound showed normal fetal development. CONCLUSION: This is the first pregnancy resulting from ovarian tissue cryopreservation and transplantation in China.

Ovarian tissue cryopreservation for patients with premature ovary insufficiency caused by cancer treatment: optimal protocol.

Objective: Premature ovary insufficiency is frequent after chemotherapy/radiotherapy in cancer patients. Ovarian tissue (OT) cryopreservation and later retransplantation, the routine method in Europe, has recently been implemented at the first center in China. We investigated the protective effect of the antioxidant N-acetyl-l-cysteine (NAC) during the decisive freezing-thawing steps. Methods: Fifteen OT samples were obtained from each of 13 cancer patients prospectively and randomly assigned to a control group and four groups with different NAC concentrations (Group 1, 0 mM NAC; Group 2, 0.5 mM NAC; Group 3, 1 mM NAC; Group 4, 5 mM NAC; Group 5, 25 mM NAC). After thawing, the follicle viability, DNA fragmentation, levels of reactive oxygen species (ROS), and total antioxidant capacity (TAC) were evaluated. Results: OT cryopreserved and thawed with 25 mM NAC (Group 5) has the lowest proportion of apoptotic stroma cells, but the worst follicle viability. The other four groups show similar anti-apoptosis and good follicle viability. Group 4 presented the lowest ROS and highest TAC levels. Conclusions: OT cryopreserved and thawed in medium supplemented with 5 mM NAC shows the highest antioxidant and lowest ROS capability, good apoptotic parameters, and follicle viability. Our results need to be confirmed in larger patient cohorts prior to being accepted as a standard protocol.

Artificial oocyte activation: evidence for clinical readiness.

Artificial oocyte activation using Ca(2+)ionophores or similar compounds is a widely applied technique in IVF laboratories. This is all the more interesting as most of the agents aiming for intracellular Ca(2+) increase do not result in physiological Ca(2+) oscillations but much rather cause a single Ca(2+) transient. Two observations from mammals may explain why a rather non-physiological single Ca(2+) peak caused by ionophores is sufficient to rescue cycles showing severe male factor infertility, deficient oocyte maturation, developmental problems in humans, or both. On the one hand, it has been shown that it is mainly the initial Ca(2+) rise that drives further downstream events, in particular calcium/calmodulin-dependent protein kinase II (CaMKII) action, and on the other, it is possible that this enzyme remains active even in the absence of Ca(2+). It therefore seems that mammalian oocytes can respond to a wide range of intracellular Ca(2+) signals and have a surprisingly high degree of tolerance for changes in cytosolic Ca(2+). As epigenetic consequences or differences in gene expression have not been studied to date, artificial oocyte activation has to be considered as experimental and should only be applied with a proper indication.

Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Efficacy of ovarian tissue cryopreservation in a major European center.

PURPOSE: To evaluate the effect of cryopreservation and thawing of ovarian tissue from oncological patients opting for fertility preservation on ovarian tissue viability. METHODS: In this prospective cohort study, the ovarian tissue viability before and after cryopreservation and thawing was measured for 25 newly diagnosed oncological patients who had their ovarian tissue cryopreserved. Outcome measures were follicle integrity (histology), follicle viability (Calcein viability assay), steroid hormone production (estradiol and progesterone production in vitro) and overall tissue viability (glucose uptake in vitro). This study was conducted at a Cryobank for storage of ovarian tissue in a university hospital. RESULTS: Cryopreserved/thawed ovarian tissue showed a decreased glucose uptake when compared to tissue that had not been cryopreserved. In addition, a diminished E2 and P4 production was observed after cryopreservation and thawing, despite the fact that numbers of viable follicles as determined by the Calcein viability assay were comparable. Histological examination revealed a higher percentage of degenerated follicles after cryopreservation and thawing. CONCLUSIONS: Ovarian tissue cryopreservation and thawing impairs the viability of ovarian tissue in oncological patients opting for fertility preservation.

[Preimplantation diagnosis--PID: preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)]. / Präimplantationsdiagnostik - PID : Präimplantative genetische Diagnostik (PGD) und präimplantatives genetisches Screening (PGS).

Preimplantation diagnosis (PID) comprises all the relevant diagnostic procedures for the investigation of genetic, structural, or numerical changes of the genetic information in spermatozoa and oocytes as well as in embryos after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). PID of oocytes is well established in Germany for the above-mentioned indications. PID at the embryonic level, i.e., trophectoderm biopsy of blastocysts, is possible in centers with proven expertise in reproductive medicine and human genetics. A high risk for genetic disease in the child or a high likelihood for stillbirth or miscarriage is a prerequisite for PID. A specialized ethics committee is required to look into each case before making a decision. While PID is still under development in Germany, it has been a well-established technology worldwide for 24 years. International experience in PID and the resulting implications are discussed in this article.

Procoagulant tissue factor-exposing vesicles in human seminal fluid.

Recent studies indicate that various types of vesicles, like microparticles (MP) and exosomes, are present in blood, saliva, bone marrow, urine and synovial fluid. These vesicles, which are released upon activation or shear stress, are thought to play a role in coagulation, neovascularisation, inflammation and intercellular signalling. Seminal fluid is a cell-, sperm- and protein-rich suspension. Although seminal fluid is known to contain vesicles like prostasomes, MP and exosomes have never been characterised. Therefore, the aim of our study was to analyse and characterise vesicles in seminal fluid in male partners of patients undergoing controlled ovarian stimulation for IVF/ICSI. MP from seminal fluid of patients during routine IVF/ICSI procedures were detected and analysed with flow cytometry (FACS) and transmission electron microscopy (TEM), using antibodies against tissue factor (TF), CD10, CD13, CD26 and annexin V. The coagulant properties of vesicles were studied using a fibrin generation test. MP were detected in human seminal fluid by both flow cytometry and TEM. Seminal fluid-derived MP expressed CD10, CD13, CD26 and TF, which was highly procoagulant and a powerful trigger of the extrinsic pathway of coagulation. The extent to which the procoagulant activity of MP in seminal fluid contributes to the implantation process itself and therefore affects human reproduction needs to be further elucidated.

Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter.

The objective of this study was to compare the toxicological effects of different source-related ambient PM10 samples in regard to their chemical composition. In this context we investigated airborne PM from different sites in Aachen, Germany. For the toxicological investigation human alveolar epithelial cells (A549) and murine macrophages (RAW264.7) were exposed from 0 to 96 h to increasing PM concentrations (0-100 µg/ml) followed by analyses of cell viability, pro-inflammatory and oxidative stress responses. The chemical analysis of these particles indicated the presence of 21 elements, water-soluble ions and PAHs. The toxicological investigations of the PM10 samples demonstrated a concentration- and time-dependent decrease in cell viability and an increase in pro-inflammatory and oxidative stress markers.

Temperature-induced sperm nuclear vacuolisation is dependent on sperm preparation.

The influence of temperature during incubation on the degree of sperm nuclear vacuolisation was assessed by two different experiments. In a first experiment, motile spermatozoa from 24 patients were prepared by the swim-up technique and incubated either at room temperature or at 37 °C for up to 4 h. The presence of sperm nuclear vacuoles was determined by contrast-enhanced high magnification microscopy. No statistically significant difference was found in the degree of sperm nuclear vacuoles in both groups (RT: 45.6 ± 17.6%; 37 °C: 48.4 ± 17.0%) following 4 h of incubation. In a second experiment, spermatozoa from six patients were either prepared by swim-up or washed and incubated at 37 °C. After 4 h of incubation, a significant increase in sperm nuclear vacuolisation was found in washed sperm (from 51.5 ± 15.4% to 68.6 ± 9.0%; P < 0.05) but not in swim-up sperm (from 51.5 ± 15.4% to 48.2 ± 17.1%; n.s.). Our data show that the mode of sperm preparation does influence sperm nuclear vacuolisation at 37 °C (Experiment II). However, sperm nuclear vacuolisation is unaffected by temperature in motile sperm after preparation and isolation by swim-up.

Cryopreservation of Ovarian Tissue: Detailed Description of Methods for Transport, Freezing and Thawing.

Purpose: In many cases cancer therapy leads to an irreversible reduction or even loss of ovarian reserve. Cryopreservation of ovarian tissue with subsequent thawing and re-transplantation of tissue after the cancer is in remission constitutes a promising method to preserve fertility in women. To date, more than 25 cases of live births after re-transplantation of cryopreserved ovarian tissue have been published worldwide. In Germany the first live birth after re-transplantation of cryopreserved tissue was in 2011. Material and Methods: After surgical removal of ovarian tissue in the Gynaecological Clinic of Dresden University, the tissue was sent to the Gynaecological Clinic of Bonn University in a special transport container at 5 °C and was frozen the next day using 1.5 M dimethyl sulfoxide cryosolution. In 2010 this ovarian tissue was thawed using a sucrose solution in the Gynaecological Clinic of Erlangen University Clinical Centre and was laparoscopically re-transplanted into the patient. Results: The patient became pregnant, the pregnancy was uneventful, and she gave birth to a healthy boy. Conclusion: Freezing of ovarian tissue with subsequent re-transplantation as described here is a viable method to preserve fertility in cancer patients.

Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential.

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.

ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:35-48.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHRE's recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos.

[Polar body diagnosis]. / Polkörperdiagnostik.

Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. The paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected in contrast to preimplantation genetic diagnosis by blastomere biopsy. The fast development in the field of molecular diagnostics will also influence PBD and probably allow a more general diagnosis in the future.

Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening.

Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle, which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. However, the paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected, in contrast to preimplantation genetic diagnosis (PGD) by blastomere biopsy. The rapid pace of developments in the field of molecular diagnostics will also influence the advantages of PBD, and probably allow more general diagnostic applications in the future.

[Complete anterolateral dislocation of the talus]. / Komplette anterolaterale Luxation des Talus.

A 22-year-old woman suffered an anterolateral dislocation of the talus in a horse-riding accident. Immediately after admission, closed reduction was performed, and then a low knee cast was used for 12 weeks of immobilisation with partial weight bearing (10 kg). Nine months later, the patient had achieved nearly free range of motion of the ankle without pain (dorsal-plantar 10-0-30 degrees ). There were no radiological signs of osteoarthritis or talus bone necrosis. Immediate closed reduction led to an excellent outcome.

Symposium: innovative techniques in human embryo viability assessment. Oocyte assessment and embryo viability prediction: birefringence imaging.

Embryo viability is a key element for success in assisted reproduction. Since the beginning of the era of assisted reproduction treatment, embryo viability was mostly considered to be a function of developmental progression during the preimplantation phase. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems in assisted reproduction has enabled the detection of structures within oocytes that possess a natural birefringence. Birefringence imaging of the meiotic spindle and the zona pellucida in living animal and human oocytes represents a new approach in the assessment of oocyte and embryo viability. The technique was applied in several studies to select oocytes in order to improve treatment success. This review will summarize the present knowledge of birefringence imaging. The various applications in basic and clinical research as well as in clinical treatment will be presented, especially with regard to their effect on assisted reproduction.

Simplified technique of human ovarian tissue freezing: quick cooling from -36 degree C.

Standard protocol of freezing of human ovarian tissue presupposes the very slow cooling (-0.3 C/min) from auto-seeding to -40 C, then slow cooling (-10 C/min) to -140 C and then direct plunging into liquid nitrogen. The aim of this investigation was to compare the -10 C/min cooling rate of human ovarian tissue from -40 C to -140 C with the -220 C/min cooling rate (direct plunging into liquid nitrogen) from -36 degree C. After post-thawing in vitro culture of tissue, hormonal activity as well as follicle viability was evaluated. After culture of fresh tissue pieces (Group 1), pieces after freezing and thawing with slow cooling (-10 C/min) from -40 C (Group 2) and pieces after freezing and thawing with direct plunging into liquid nitrogen (-220 C/min) from -36 C (Group 3), the supernatants showed estradiol 17-ss concentrations of 481, 441 and 459 pg per ml, respectively, and progesterone concentrations of 9.05, 5.06, 4.87 ng per ml, respectively. It is concluded that 94, 96, and 98 percent follicles for Groups 1, 2 and 3, respectively, were normal. Technique of human ovarian tissue cryopreservation with very slow cooling to -36 C and then direct plunging into liquid nitrogen with -220 C/min cooling rate is tolerated without apparent detriment.

[MR-colonography in hospitalized patients: feasibility and sensitivity]. / MR-Kolonografie bei stationären Patienten: Durchführbarkeit und Sensitivität.

INTRODUCTION: Besides conventional colonoscopy, CT and MR colonography offer alternate virtual imaging modalities of the colon. The sensitivity of CT colonography, which is associated with radiation exposure, has been published in prior, large studies. Regarding MR colonography, in particular dark lumen MR colonography with the rectal administration of a water enema as a contrast agent, only limited published data exist. The goal of this study was to compare MR colonography with conventional colonoscopy in the detection of colorectal polyps. In addition the feasibility and image quality in unselected hospitalised patients were assessed. PATIENTS/METHODS: Included were 103 hospitalised patients who had to undergo colonoscopy for various indications. Immediately prior to conventional colonoscopy, MR colonography with rectal water enema and additional intravenous administration of contrast material was performed. Detection rates for polyps and adenomas were documented with both imaging modalities. Image quality and completion rates (practicability) and other (incidental) findings were also recorded. RESULTS: In 15 of 103 patients the MR examination could not be done or was only partially completed. The detection rate of MR colonography for polyps (adenomas) was 2% (4%) for polyps (adenomas) up to 5 mm in diameter, 38% (56%) for polyps (adenomas) 6-10 mm in diameter and 89% (89%) for polyps (adenomas) up to 11 mm in diameter. One flat carcinoma seen with conventional coloscopy was missed on MR colonography. CONCLUSIONS: MR colonography offers the possibility of imaging the colon without exposure to radiation. Polyps and adenomas are detected, similar to the detection rate of CT colonography, with adequate sensitivity only if they are larger than 10 mm in diameter. Therefore this imaging technique is not (yet) suitable as a screening test. Additional limitations are the necessary cooperation of the patient which can reduce the practicability and image quality in selected patients. Further studies like the just started German multicentre trial are needed to assess the position of MR colonography.

Regional hippocampal atrophy in multiple sclerosis.

Gray matter brain structures, including deep nuclei and the cerebral cortex, are affected significantly and early in the course of multiple sclerosis and these changes may not be directly related to demyelinating white matter lesions. The hippocampus is an archicortical structure that is critical for memory functions and is especially sensitive to multiple insults including inflammation. We used high-resolution MR imaging at 3.0 T to measure hippocampal volumes in relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients and controls. We found that both groups of MS patients had hippocampal atrophy and that this volume loss was in excess of global brain atrophy. Subregional analysis revealed selective volume loss in the cornu ammonis (CA) 1 region of the hippocampus in RRMS with further worsening of CA1 loss and extension into other CA regions in SPMS. Hippocampal atrophy was not correlated with T2-lesion volumes, and right and left hippocampi were affected equally. Volume loss in the hippocampus and subregions was correlated with worsening performance on word-list learning, a task requiring memory encoding, but not with performance on the Paced Auditory Serial Addition Task (PASAT), a test of information processing speed. Our findings provide evidence for selective and progressive hippocampal atrophy in MS localized initially to the CA1 subregion that is associated with deficits in memory encoding and retrieval. The underlying histopathological substrate for this selective, symmetric and disproportionate regional hippocampal vulnerability remains speculative at this time. Further understanding of this process could provide targets for therapeutic interventions including neuroprotective treatments.