Determinants of transplantation success with cryopreserved ovarian tissue: data from 196 women of the FertiPROTEKT network.

STUDY QUESTION: What are the pregnancy and live birth rates for ovarian tissue transplantation and which factors are associated with the success rate? SUMMARY ANSWER: Pregnancy and live birth rates per transplanted woman are 32.7% and 26.5% and success rate is associated with female age and first versus repeated transplantation. WHAT IS KNOWN ALREADY: Live birth rates after ovarian tissue transplantations have been reported to be between around 24% and 41% per patient. Success rates seem to be negatively associated with increasing female age at the time of tissue cryopreservation and with pelvic radiation. Success rates are apparently not reduced after overnight transportation of ovarian tissue before freezing. STUDY DESIGN, SIZE, DURATION: Registry analysis of 244 transplantations in 196 women, performed by 26 FertiPROTEKT network centres from 2007 to 2019 with follow-up till December 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Orthotopic ovarian tissue transplantations were performed in 196 women, 191 with previous malignant and 5 with previous non-malignant diseases. Size of transplanting centres varied between 1 and 100 transplantations per centre (median: 2). Factors possibly associated with success rate such as female age, first and repeated transplantation, experience of the transplanting centre and overnight transportation of the ovarian tissue before freezing were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Average age of all 196 transplanted women was 31.3 years (SD 5.2; range 17-44) at the time of cryopreservation of tissue and 35.9 years (SD 4.8; range 23-47) at the time of transplantation. Pregnancy rate was 30.6% (95% CI, 24.2-37.6%) per first transplantation and 32.7% (95% CI, 26.1-39.7%) per patient. Pregnancy rate was higher after first transplantation (30.6% (95% CI, 24.2-37.6%)) compared to second and subsequent transplantations (11.8% (95% CI, 3.3-27.5%)). Live birth rate per first transplantation was 25.0% (95% CI, 19.1-31.7%) and per patient 26.5% (95% CI, 20.5-33.3%). Success rate decreased with increasing age at the time of ovarian tissue freezing. Live birth rate was 28.2% (95% CI, 20.9-36.3%) in women <35 years and 16.7% (95% CI, 7.9-29.3%) in women >35 years. Pregnancy rates after first transplantation were higher in centres who had performed ≥10 transplantations (35.1%) compared to centres with <10 transplantation (25.4%) (P = 0.12). Corresponding live birth rates were 27.0% and 18.6%. Success rates were not different in women with and without overnight transportation of tissue before cryopreservation. LIMITATIONS, REASONS FOR CAUTION: The data were drawn from a registry analysis. Data such as ovarian reserve and premature ovarian insufficiency were not available for all women. Data might be influenced by different follow-up policies of the centres. WIDER IMPLICATIONS OF THE FINDINGS: The study reveals the high potential of ovarian tissue freezing and transplantation, but only if freezing is performed in younger women. The study suggests focus should be placed on the first and not on repeated transplantations. It also opens the discussion of whether transplantation should rather be performed by experienced centres. STUDY FUNDING/COMPETING INTEREST(S): No funding. No competing interests. TRIAL REGISTRATION NUMBER: N/A.

Refinement of detecting atrial fibrillation in stroke patients: results from the TRACK-AF Study.

BACKGROUND AND PURPOSE: Detection of occult atrial fibrillation (AF) is crucial for optimal secondary prevention in stroke patients. The AF detection rate was determined by implantable cardiac monitor (ICM) and compared to the prediction rate of the probability of incident AF by software based analysis of a continuously monitored electrocardiogram at follow-up (stroke risk analysis, SRA); an optimized AF detection algorithm is proposed by combining both tools. METHODS: In a monocentric prospective study 105 out of 389 patients with cryptogenic stroke despite extensive diagnostic workup were investigated with two additional cardiac monitoring tools: (a) 20 months' monitoring by ICM and (b) SRA during hospitalization at the stroke unit. RESULTS: The detection rate of occult AF was 18% by ICM (n = 19) (range 6-575 days) and 62% (n = 65) had an increased risk for AF predicted by SRA. When comparing the predictive accuracy of SRA to ICM, the sensitivity was 95%, specificity 35%, positive predictive value 27% and negative predictive value 96%. In 18 patients with AF detected by ICM, SRA also showed a medium risk for AF. Only one patient with a very low risk predicted by SRA developed AF revealed by ICM after 417 days. CONCLUSIONS: A combination of SRA and ICM is a promising strategy to detect occult AF. SRA is reliable in predicting incident AF with a high negative predictive value. Thus, SRA may serve as a cost-effective pre-selection tool identifying patients at risk for AF who may benefit from further cardiac monitoring by ICM.

Twenty-five-year-old Woman with Bilateral Borderline Ovarian Tumour Desiring to Preserve Fertility - Case Report and Literature Review on the Current State of Fertility Preservation in Women with Borderline Ovarian Tumours.

Borderline ovarian tumours are semimalignant tumours occurring unilaterally or bilaterally with a peak incidence among women of reproductive age. Since the affected women often wish to preserve fertility, particular precautions must be taken when counselling the patient and obtaining consent prior to planning an individual treatment. Options for preserving fertility include an organ-sparing surgical procedure and cryopreservation of oocytes and/or ovarian tissue. In this article, we report on a 25-year-old patient with a bilateral seromucinous borderline tumour who desired all fertility-preserving options. In order to perform the procedure without delay, we opted to perform luteal phase stimulation prior to oocyte retrieval. We conclude by discussing the current literature on the state of fertility preservation in the treatment of borderline ovarian tumours.

Surgical Aspects of Ovarian Tissue Removal and Ovarian Tissue Transplantation for Fertility Preservation.

Introduction: The removal of ovarian tissue prior to starting oncologic treatment and the subsequent transplantation of this tissue after completing therapy have become increasingly important surgical fertility-preserving techniques. The aim of this review was to investigate the different surgical techniques used for this method reported in the literature to date and to discuss the advantages and disadvantages of the respective techniques. Review: A search was done in MEDLINE using a defined algorithm to find studies published between January 2004 and December 2015. All study designs were included in our review if they contained statements on the surgical technique used. We found 16 publications (8 retrospective cohort studies, 6 case reports and 2 systematic reviews) with a total of 1898 female patients which reported on the surgical technique used for ovarian biopsy and 15 publications (7 retrospective cohort studies, 6 case reports and 2 systematic reviews) with a total of 455 women which mentioned the surgical technique used for ovarian transplantation. Different surgical techniques can be used both for ovarian biopsy and for the transplantation of ovarian tissue. A number of different surgical routes have been used, and the amount of tissue extracted, the instruments used, the treatment of the ovary, the transplantation site, the blood supply to the transplanted ovarian tissue and the procedure used for simultaneous surgical interventions vary. Conclusion: In future, one of the tasks will be to establish a standard surgical method for ovarian extraction and transplantation which will have a low rate of complications and a high pregnancy and birth rate while ensuring that the transplanted tissue is fully functional.

Ninety-five orthotopic transplantations in 74 women of ovarian tissue after cytotoxic treatment in a fertility preservation network: tissue activity, pregnancy and delivery rates.

STUDY QUESTION: What is the success rate in terms of ovarian activity (menstrual cycles) as well as pregnancy and delivery rates 1 year after orthotopic ovarian transplantations conducted in a three-country network? SUMMARY ANSWER: In 49 women with a follow-up >1 year after transplantation, the ovaries were active in 67% of cases and the pregnancy and delivery rates were 33 and 25%, respectively. WHAT IS KNOWN ALREADY: Cryopreservation of ovarian tissue in advance of cytotoxic therapies and later transplantation of the tissue is being performed increasingly often, and the total success rates in terms of pregnancy and delivery have been described in case series. However, published case series have not allowed either a more detailed analysis of patients with premature ovarian insufficiency (POI) or calculation of success rates based on the parameter 'tissue activity'. STUDY DESIGN, SIZE, DURATION: Retrospective analysis of 95 orthotopic transplantations in 74 patients who had been treated for cancer, performed in the FertiPROTEKT network from 2008 to June 2015. Of those 95 transplantations, a first subgroup (Subgroup 1) was defined for further analysis, including 49 women with a follow-up period >1 year after transplantation. Of those 49 women, a second subgroup (Subgroup 5) was further analysed, including 40 women who were transplanted for the first time and who were diagnosed with POI before transplantation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transplantation was performed in 16 centres and data were transferred to the FertiPROTEKT registry. The transplantations were carried out after oncological treatment had been completed and after a remission period of at least 2 years. Tissue was transplanted orthotopically, either into or onto the residual ovaries or into a pelvic peritoneal pocket. The success rates were defined as tissue activity (menstrual cycles) after 1 year (primary outcome) and as pregnancies and deliveries achieved. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of all transplanted 74 women was 31 ± 5.9 years at the time of cryopreservation and 35 ± 5.2 at the time of transplantation. Twenty-one pregnancies and 17 deliveries were recorded. In Subgroup 1, tissue was cryopreserved at the age of 30 ± 5.6 and transplanted at 34 ± 4.9 years. Ovaries remained active 1 year after transplantation in 67% of cases (n = 33/49), the pregnancy rate was 33% (n = 16/49) and the delivery rate was 25% (n = 12/49). In Subgroup 5, tissue was cryopreserved at the age 30 ± 5.9 years and transplanted at 34 ± 5.2 years. Ovaries remained active 1 year after transplantation in 63% of cases (n = 25/40), the pregnancy rate was 28% (n = 11/40) and the delivery rate was 23% (n = 9/40). The success rates were age dependant with higher success in women who cryopreserved at a younger age. In Subgroup 5, tissue was exclusively transplanted into the ovary in 10% (n = 4/40) of women and into a peritoneal pocket in 75% (n = 30/40), resulting in spontaneous conceptions in 91% of patients (n = 10/11). LIMITATIONS, REASONS FOR CAUTION: The data were drawn from a retrospective analysis. The cryopreservation and transplantation techniques used have changed during the study period. The tissue was stored in many tissue banks and many surgeons were involved, leading to heterogeneity of the procedures. However, this does reflect the realistic situation in many countries. Although patients with POI were evaluated before transplantation to allow specific analysis of the transplanted tissue itself, the possibility cannot be excluded that residual ovarian tissue was also reactivated. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest case series worldwide to date and it confirms that cryopreservation and transplantation of ovarian tissue can be a successful option for preserving fertility. Persistent tissue activity 12 months after transplantation suggests that the pregnancy and delivery rates may increase further in the future. As transplantation into the peritoneum results in a high success rate, this approach may be an alternative to transplantation into the ovary. However, in order to establish the best transplantation site, a randomized study is required. STUDY FUNDING/COMPETING INTEREST: This study was in part funded from the Deutsche Forschungsgemeinschaft (# DI 1525) and the Wilhelm Sander Foundation (2012.127.1) and did not receive any funding from a commercial company. No competing interests. TRIAL REGISTRATION NUMBER: None.

Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil.

The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.

Gestational and Non-gestational Trophoblastic Disease. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Registry No. 032/049, December 2015).

Purpose: The aim was to establish an official interdisciplinary guideline, published and coordinated by the German Society of Gynecology and Obstetrics (DGGG). The guideline was developed for use in German-speaking countries. In addition to the Germany Society of Gynecology and Obstetrics, the guideline has also been approved by the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). The aim was to standardize diagnostic procedures and the management of gestational and non-gestational trophoblastic disease in accordance with the principles of evidence-based medicine, drawing on the current literature and the experience of the colleagues involved in compiling the guideline. Methods: This s2k guideline represents the consensus of a representative panel of experts with a range of different professional backgrounds commissioned by the DGGG. Following a review of the international literature and international guidelines on trophoblastic tumors, a structural consensus was achieved in a formalized, multi-step procedure. This was done using uniform definitions, objective assessments, and standardized management protocols. Recommendations: The recommendations of the guideline cover the epidemiology, classification and staging of trophoblastic tumors; the measurement of human chorionic gonadotropin (hCG) levels in serum, and the diagnosis, management, and follow-up of villous trophoblastic tumors (e.g., partial mole, hydatidiform mole, invasive mole) and non-villous trophoblastic tumors (placental site nodule, exaggerated placental site, placental site tumor, epitheloid trophoblastic tumor, and choriocarcinoma).

Comparison of Whole Ovary Cryotreatments for Fertility Preservation.

The goal of this study was to compare a traditional slow-freeze method (TF) with an open unidirectional slow freeze cooling system (UF) for whole ovary cryopreservation. Therefore, whole pig ovaries were randomly assigned to (A) fresh control, (B) traditional slow freeze (TF) or (C) unidirectional slow freeze (UF). Ovaries were perfused with 10% DMSO in Krebs-Ringer. For TF, whole ovaries were placed in specimen jars containing 10% DMSO and placed into a specialized container for freezing filled with propan-2-ol. For UF, whole ovaries were placed within a specially designed container containing 10% DMSO and transferred to a specialized freezing machine (CTE 920). Histological evaluation demonstrated intact morphology of follicles in all groups; however, an overall decrease of follicle numbers in TF (46%) and UF (50%) compared to fresh control. Live/dead assay indicated significantly lower populations of live cells in both TF (60%) and UF (58%) compared to fresh tissue (74%). TUNEL assay confirmed a difference in percentage of apoptotic follicles between fresh and TF, but there was no significant difference between fresh and UF. To improve the structural and functional integrity of whole ovaries, further investigation, especially into directional freezing, is needed. Whole ovary cryopreservation could provide opportunities for women facing fertility loss due to chemo- or radiotherapy treatment.

Histopathological hoof laminar changes in horses with pituitary pars Intermedia adenoma: cases report / Alterações histopatológicas do tecido laminar do casco em cavalos com adenoma da pars intermedia da hipófise: relato de casos

Laminitis in horses is often associated with endocrine disorders, especially the pituitary pars intermedia dysfunction (PPID) in older animals. Morphologic exams of the laminar tissue of the hoof were performed in two horses with suspected PPID, with no clinical signs of laminitis. Changes compatible with laminitis of endocrine origin were observed, such as rounding of the nuclei of the basal cells, thinning and stretching of the secondary epidermal laminae and tissue proliferation. PPID horses with no clinical signs of laminitis may be affected by lesions of the laminar tissue of the hoof that compromise the integrity of the dermal-epidermal junction and may develop clinical symptoms of the disease. It has been suggested that the development stage of endocrine laminitis is longer, but further studies should be conducted to confirm it.

A laminite em cavalos está frequentemente associada a distúrbios endócrinos, como a disfunção da pars intermedia da pituitária (PPID) em animais mais velhos. Exames morfológicos do tecido laminar do casco foram realizados em dois cavalos com suspeita de PPID após o óbito, os quais não apresentaram sinais clínicos de laminite. Alterações compatíveis com a laminite de origem endócrina foram observadas, como arredondamento da núcleo das células basais, alongamento e afilamento das lâminas epidérmicas secundárias e proliferação tecidual. Cavalos com PPID sem sinais clínicos de laminite podem estar acometidos por lesões do tecido laminar do casco que comprometam a integridade das interdigitações dérmico-epidérmicas e podem desenvolver a sintomatologia clínica da doença. Sugere-se que o período de desenvolvimento da laminite endócrina seja mais longo, porém estudos adicionais devem ser realizados para confirmar essa hipótese.

Fertility Protection in Female Oncology Patients: How Should Patients Be Counseled?

Protecting the fertility of patients with oncologic disease is becoming more and more important, as fulfilling the wish to have children is increasingly occurring at a later stage in life and long-term survival rates after cancer are continuing to improve. A number of fertility-preserving options exist. In addition to techniques which have been around for some time such as medical ovarian suppression, ovarian transposition, and organ-preserving surgery, there are other, more recent, innovative methods which have developed over the last few years such as cryopreservation of oocytes or ovarian tissue transplantation after completing cancer therapy. As every procedure has its specific advantages and disadvantages, informed patient consent is essential. The physician's aim must be to select the optimal procedure for each patient. The extent of patients' information about the options to preserve fertility in women with oncologic disease remains limited. One of the main reasons for this is that clinicians are not sure how to inform patients about existing procedures and methods. The aim of this review article is to provide help in clinical practice.

Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine.

On August 1, 2013, the German Patent and Trademark Office issued a patent for the "Non-embryo-destructive extraction of pluripotent embryonic stem cells, stem cells obtained by this process and their uses" (DE 10 2004 062 184 B4). The patent document describes a non-embryo-destructive process to harvest embryonic stem cells from the inner cell mass (ICM) during the blastocyst development stage. The patent application was filed with the German Patent Office in Munich on December 23, 2004 and the patent claim was published in 2006. The patent was granted on August 1, 2013. Processing the patent application was a lengthy affair due to the fact that, for a long time, the prevailing opinion in Germany was that genetic screening of embryos (preimplantation genetic diagnosis) was prohibited under the German Embryo Protection Act (ESchG). A ruling by the German Federal Court in 2010 proved this opinion to be false. Animal studies have provided the evidence that the described procedure is technically feasible; healthy offspring were born after stem cells were harvested from the blastocyst and stored. We report here on a technique for the non-embryo-destructive extraction of pluripotent embryonic stem cells together with potential future applications for stem cells harvested in this manner.

Risk of recurrent cervical artery dissection during pregnancy, childbirth and puerperium.

BACKGROUND AND PURPOSE: Hormonal and mechanical factors might increase the risk for cervical artery dissection (CAD) during pregnancy and the puerperium. There is uncertainty how to counsel women with a previous CAD regarding the risk of CAD recurrence during pregnancy and the puerperium. METHODS: In an observational study of four stroke centers, all women aged 16-45 years with primary CAD in the previous decade were asked to participate in a standardized assessment on long-term follow-up with a special focus on pregnancies and recurrent CAD. RESULTS: Ninety-two women were identified and 53 of them were included in the analysis (60%). Eleven women declined to participate, 28 were untraceable. The 39 non-participants did not differ from participants regarding key baseline characteristics. Average follow-up time was 72 months. Nine women (17%) had recurrent CAD after a median of 14 days (range 2 days to 117 months). Eleven women (20%) had a total of 13 completed pregnancies at a median of 44 months (range 12-84 months) after index CAD. Two of the pregnant women (18%) had suffered recurrent CAD ≥18 months prior to the pregnancy. All 13 pregnancies and puerperia went without recurrent CADs or cerebrovascular events. This includes giving birth by vaginal delivery (n = 6) and caesarean section (n = 7). None of the five women with typical connective tissue disease became pregnant. CONCLUSIONS: Our observation suggests that the risk of recurrent CAD may not be greatly increased with pregnancies starting at least 12 months after CAD in women without typical connective tissue disease.

Synthesis and physicochemical, in vitro and in vivo evaluation of an anisotropic, nanocrystalline hydroxyapatite bisque scaffold with parallel-aligned pores mimicking the microstructure of cortical bone.

Scaffolds for bone regeneration are mostly prepared with an isotropic, sponge-like structure mimicking the architecture of trabecular bone. We have developed an anisotropic bioceramic with parallel aligned pores resembling the honeycomb arrangement of Haversian canals of cortical bone and investigated its potential as a scaffold for tissue engineering. Parallel channel-like pores were generated by ionotropic gelation of an alginate-hydroxyapatite (HA) slurry, followed by ceramic processing. Organic components were thermally removed at 650 °C, whereas the pore system was preserved in the obtained HA bioceramic in the processing stage of a bisque. Even without further sintering at higher temperatures, the anisotropic HA bisque (AHAB) became mechanically stable with a compressive strength (4.3 MPa) comparable to that of native trabecular bone. Owing to the low-temperature treatment, a nanocrystalline microstructure with high porosity (82%) and surface area (24.9 m(2)/g) was achieved that kept the material dissolvable in acidic conditions, similar to osteoclastic degradation of bone. Human mesenchymal stem cells (hMSCs) adhered, proliferated and differentiated into osteoblasts when osteogenically induced, indicating the cytocompatibility of the bisque scaffold. Furthermore, we demonstrated fusion of human monocytes to osteoclast-like cells in vitro on this substrate, similar to the natural pathway. Biocompatibility was demonstrated in vivo by implantation of the bisque ceramic into cortical rabbit femur defects, followed by histological analysis, where new bone formation inside the channel-like pores and generation of an osteon-like tissue morphology was observed.

Culture of Individually Required Number of 2-Pronuclei-Stage Oocytes - Patient Participation in Decision-Making is in Accordance with the Aim of Avoiding Surplus Embryo Freezing.

Background: The aim of this study was to evaluate how many embryos will develop if more than 3 2-pronuclei-stage oocytes (2-PNOs) are cultured at the patient's request and in accordance with the Germany Embryo Protection Law. Methods: A total of 106 cycles of patients undergoing their 1st, 2nd or 3rd cycle of IVF or ICSI treatment in 2010 were prospectively included in the study. In each individual case, a decision was taken prior to treatment about the number of 2-PNOs to be cultured after each cycle. Results: Ninety female patients were treated for a total of 106 cycles. A mean of two to six 2-PNOs were cultivated for a period of between 3 and 6 days for each patient. After culture, no viable embryo was identified for 5 patients (4.7 %), a single viable embryo was identified for 37 cycles (34.7 %), and 2 viable embryos were identified for 52 cycles (48.8 %). Eleven patients (10.3 %) had 3 viable embryos after a further 11 cycles and 1 patient had 4 viable embryos in a single cycle. Ten of the patients with 3 embryos each opted to have all 3 embryos transferred in the same cycle. This meant that a single embryo from one patient with 3 viable embryos and a single embryo of the patient with 4 viable embryos were cryopreserved after culture. The pregnancy rate was 19 % per embryo transfer and 25 % per blastocyst transfer (20 pregnancies in total). All cryopreserved embryos were transferred in a subsequent cycle. Discussion: Based on this study it is possible to make a statement about the number of viable embryos which should be cultivated to obtain, at best, two embryos for transfer without running an unacceptably high risk of producing too many embryos which would then need to be cryopreserved. Only 12 patients (13.3 %) had more than 2 viable embryos. The number of supernumerary pre-implantation-stage embryos was acceptably low (only 2 patients had additional viable embryos, 2.2 %). This means that it is possible to fulfil the wishes of individual patients while complying with the German Embryo Protection Law.

Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor.

Sera from 112 mares from 5 horse-breeding farms was examined for the presence of antibodies to Neospora caninum and Toxoplasma gondii by an indirect fluorescent antibody test (IFAT), as well as from dogs and cattle present on these properties for the presence of antibodies to N. caninum. Among the 112 mares, 35 had a history of reproductive problems in the last breeding season and 77 had no reproductive problems. The rates of seroprevalence of N. caninum in mares with and without a history of reproductive problems were 25.71% and 6.49% and from T. gondii 2.85% and 1.29%, respectively. In dogs and cattle, the rates of seroprevalence of N. caninum were 10.52% and 15.55%, respectively. A positive correlation was found between the presence of antibodies against N. caninum (p=0.010) in mares and the occurrence of reproductive problems using the Fisher's exact test. Significantly higher seroprevalence for N. caninum in mares was observed on the farm that had seropositive dogs (p=0.018). Cattle on this farm were also seropositive. No significant differences in seropositivity were found on farms where dogs were seronegative, or absent. This result suggests, for the first time, the presence of seropositive dogs as a risk factor for N. caninum in mares and the necessity for further investigation of the epidemiology of this parasite in horse-breeding farms with reproductive problems and the presence of cattle and dogs. This is the first report on the occurrence of antibodies against N. caninum in horses from the state of Santa Catarina, Brazil.

Isolamento e caracterização de células mesenquimais do tecido adiposo de cães / Isolation and characterization of canine adipose-derived mesenchymal stem cells

As células-tronco mesenquimais (CTMs) diferenciam-se em várias linhagens e têm potencial de utilização na medicina regenerativa. As CTMs podem ser isoladas de vários tecidos de animais adultos. O objetivo deste estudo foi o isolamento das CTMs do tecido adiposo de cães, seu cultivo e diferenciação. Foram coletadas amostras de tecido adiposo subcutâneo de cinco cães. As CTMs foram isoladas, obtendo-se 146.803 (±49.533) células/g, cultivadas e diferenciadas em osteoblastos, adipócitos e condrócitos. Avaliaram-se a cinética do crescimento, a morfologia e a viabilidade celular. A caracterização citoquímica comprovou a natureza mesenquimal das células isoladas. O cultivo foi iniciado com 20.000 células/mL, verificando-se crescimento rápido até 72 horas (220.000 células/mL), fase exponencial entre 72 e 192 horas (455.000 células/mL), seguida de platô por saturação da densidade com 240 horas (355.000 células/mL). A viabilidade celular variou entre 96 e 100%. As CTMs em cultivo são fibroblásticas, fusiformes, com citoplasma basofílico e núcleo esférico. O comprimento médio das células variou entre 80,85 e 98,36µm, a largura média entre 17,40 e 28,79µm e o diâmetro médio do núcleo entre 15,46 e 17,74µm.

The applications of mesenchymal stem cells (MSCs) are becoming increasingly more promising for regenerative medicine and tissue engineering fields. MSCs can be isolated from adult animals from a variety of tissues, such as the adipose. This study focused on the isolation, culture and differentiation of MSCs from canine adipose tissue. Samples of subcutaneous adipose tissue from five dogs were collected. These cells were isolated, cultured and differentiated into osteoblasts, adipocytes and chondrocytes. We obtained 146,803 (±49,533) cells/g. Growth kinetics and viability studies were conducted during cell culture and the evaluation of cell differentiation was successfully performed by cytochemistry. The cell cultures were initiated with 20,000 MSCs/ml. Rapid growth was observed at 72 hours (220,000 cells/ml), the exponential phase between 72 and 192 hours (455,000 cells/ml) and saturation at 240 hours (355,000 cells/ml). The cellular viability ranged from 96 to 100%. MSCs in culture are fibroblastic cells, fusiform with basophilic cytoplasm and spherical nucleus. The length and width means of the cells and nuclear diameter ranged from 80.85-98.36µm, 17.40-28.79µm and 15.46-17.74µm respectively.

Nanocrystalline spherical hydroxyapatite granules for bone repair: in vitro evaluation with osteoblast-like cells and osteoclasts.

Conventionally sintered hydroxyapatite-based materials for bone repair show poor resorbability due to the loss of nanocrystallinity. The present study describes a method to establish nanocrystalline hydroxyapatite granules. The material was prepared by ionotropic gelation of an alginate sol containing hydroxyapatite (HA) powder. Subsequent thermal elimination of alginate at 650 °C yielded non-sintered, but unexpectedly stable hydroxyapatite granules. By adding stearic acid as an organic filler to the alginate/HA suspension, the granules exhibited macropores after thermal treatment. A third type of material was achieved by additional coating of the granules with silica particles. Microstructure and specific surface area of the different materials were characterized in comparison to the already established granular calcium phosphate material Cerasorb M(®). Cytocompatibility and potential for bone regeneration of the materials was evaluated by in vitro examinations with osteosarcoma cells and osteoclasts. Osteoblast-like SaOS-2 cells proliferated on all examined materials and showed the typical increase of alkaline phosphatase (ALP) activity during cultivation. Expression of bone-related genes coding for ALP, osteonectin, osteopontin, osteocalcin and bone sialoprotein II on the materials was proven by RT-PCR. Human monocytes were seeded onto the different granules and osteoclastogenesis was examined by activity measurement of tartrate-specific acid phosphatase (TRAP). Gene expression analysis after 23 days of cultivation revealed an increased expression of osteoclast-related genes TRAP, vitronectin receptor and cathepsin K, which was on the same level for all examined materials. These results indicate, that the nanocrystalline granular materials are of clinical interest, especially for bone regeneration.

Vascularization of carotid plaque in recently symptomatic patients is associated with the occurrence of transcranial microembolic signals.

BACKGROUND AND PURPOSE: Microembolic signals (MES) are detectable in the middle cerebral artery by transcranial ultrasound downstream to atherosclerotic lesions of the internal carotid artery (ICA) in patients with stroke or transient ischaemic attack. The occurrence of MES predicts future risk of stroke in patients with symptomatic and asymptomatic carotid stenosis. The detection of intra-plaque neo-vascularization by contrast-enhanced ultrasound (CEUS) in atherosclerotic plaques of the ICA is associated with future cardiovascular/cerebrovascular events. We investigated whether there is an association between both surrogate markers of future vascular events. METHODS: Forty-one patients with symptomatic atherosclerotic plaques underwent ipsilateral transcranial ultrasound MES detection for 30 min followed by a CEUS investigation of the plaque. The occurrence and number of MES was documented, and the degree of intra-plaque neo-vascularization was measured semi-quantitatively. RESULTS: During the 30 min investigation, 17 patients had MES and nine of them showed neo-vascularization of the atherosclerotic plaque. The remaining 24 patients did not have MES, and only in four patients of this group could plaque neo-vascularization be demonstrated (P = 0.020). CONCLUSIONS: We found an association between the occurrence of MES and the presence of neo-vascularization in patients with symptomatic atherosclerotic carotid plaque. Therefore, plaque neo-vascularization might also be a surrogate marker of future stroke risk.