Laparoscopic ovarian tissue harvesting for cryopreservation: an effective and safe procedure for fertility preservation

Objective

To critically review our experience with the method of laparoscopic ovarian tissue harvesting for ovarian cryopreservation.

Study design

In a retrospective cohort study 85 patients undergoing surgery for cryopreservation of ovarian tissue were included. One random ovarian cortical slice was histologically examined in order to determine the presence of primordial follicles and to detect possible malignant cells. Selective microbiological culture techniques from swabs were taken from all slices immediately after excision. Intra- and postoperative courses, histological and microbiological findings were evaluated.

Results

Eighty-five patients underwent cryopreservation of ovarian tissue, mostly for malignant diseases (78/85, 91.8%). Sixty patients (70.6%) underwent laparoscopy for ovarian tissue harvesting only, without any additional surgical procedure. The median operating time was 30 min (range 10-75 min). The intraoperative course was uneventful in these patients. In two patients slight postoperative increases in C-reactive protein levels were found. Microbiological examination revealed no contamination apart from one case revealing sporadic Propionibacterium acnes. Histological examination revealed intact ovarian tissue with primordial follicles in 81/85 patients (95.3%).

Conclusion

Laparoscopy is a safe and effective procedure for ovarian tissue harvesting. We suggest microbiological and histological testing of ovarian tissue as mandatory tools to guarantee safety regarding ovarian tissue transplantation.     

Accidental poisoning with autumn crocus

We describe a case of a 43-yr-old female with severe multiorgan injury after accidental poisoning with Colchicum autumnale, which was mistaken for wild garlic (Allium ursinum). Both plants grow on damp meadows and can be confused in the spring when both plants have leaves but no blossoms. The autumn crocus contains colchicine, which inhibits cellular division. Treatment consisted of supportive care, antibiotic therapy, and granulocyte-directed growth factor. The patient was discharged from the hospital after three weeks. Three years after recovery from the acute poisoning, the patient continued to complain of muscle weakness and intermittent episodes of hair loss.     

Development of a Clinically Relevant Dissolution Method for Metaxalone Immediate Release Formulations Based on an IVIVC Model

Purpose

The aim of the present work was to classify metaxalone according to the Biopharmaceutics Classification System (BCS), to develop a clinically relevant dissolution method that can be used to predict the oral absorption of metaxalone and to establish an in vitro-in vivo correlation (IVIVC).

Methods

Solubility of the drug was studied in different pH media and permeability studies were performed using a Caco-2 cell model. The in vitro dissolution and in vivo disposition of metaxalone from 3 different immediate release (IR) tablet formulations were investigated using USP 2 apparatus and a single dose, four-way, crossover bioequivalence study in healthy humans along with an oral solution of the drug, respectively. An IVIVC was established by using a direct, differential based method.

Results

Metaxalone has been confirmed as a Class II drug according to BCS. Bioavailability studies performed in humans demonstrated that dissolution was the rate limiting step for bioavailability of the drug and one of the test products had significantly improved bioavailability compared to the marketed product Skelaxin®. An IVIVC model was developed that demonstrated an acceptable internal predictability.

Conclusion

The IVIVC demonstrated that formulation factors play a significant role in dissolution and absorption of metaxalone. A pH 4.5 dissolution medium containing 0.5% NaCl with 0.2% SLS (USP apparatus 2 at 50 rpm) is clinically relevant to predict bioavailability of the drug and is superior to the USP method in terms of the Quality by Design (QbD) concept.

     

White matter disruptions in patients with bipolar disorder

Bipolar disorder (BD) patients show aberrant white matter microstructure compared to healthy controls but little is known about the relation with clinical characteristics. We therefore investigated the relation of white matter microstructure with the main pharmacological treatments as well its relation with IQ. Patients with BD (N = 257) and controls (N = 167) underwent diffusion tensor imaging (DTI) and comprehensive clinically assessments including IQ estimates. DTI images were analyzed using tract-based spatial statistics. Fractional anisotropy (FA) and Mean Diffusivity (MD) were determined. Patients had significantly lower FA and higher MD values throughout the white matter skeleton compared to controls. Within the BD patients, lithium use was associated with higher FA and lower MD. Antipsychotic medication use in the BD patients was not associated with FA but, in contrast to lithium, was associated with higher MD. IQ was significantly positively correlated with FA and negatively with MD in patients as well as in controls. In this large DTI study we found evidence for marked differences in FA and MD particularly in (but not restricted to) corpus callosum, between BD patients and controls. This effect was most pronounced in lithium-free patients, implicating that lithium affects white matter microstructure and attenuates differences associated with bipolar disorder. Effects of antipsychotic medication intake were absent in FA and only subtle in MD relative to those of lithium. The abnormal white matter microstructure was associated with IQ but not specifically for either group.     

Expression of adipokine ghrelin and ghrelin receptor in human colorectal adenoma and correlation with the grade of dysplasia

BACKGROUNDGhrelin is an adipokine that plays an important role in energy balance. Expression of ghrelin and ghrelin receptor has been investigated in different tissues and tumors. Studies regarding expression of ghrelin and ghrelin receptor in colorectal tumors are scarce and no data on expression of ghrelin and its receptor in colorectal adenomas has been published. Ghrelin and ghrelin receptor were highly expressed in colon carcinoma cells while expression was decreased in less differentiated tumors, presuming that ghrelin might be important in early phases of tumorigenesis.AIMTo investigate the expression of ghrelin and ghrelin receptor in human colorectal adenomas and adjacent colorectal tissue.METHODSIn this prospective study (conducted from June 2015 until May 2019) we included 92 patients (64 male and 28 female) who underwent polypectomy for colorectal adenomas in the Department of Gastroenterology and Hepatology, “Sestre milosrdnice” Clinical Hospital Center in Zagreb, Croatia. After endoscopic removal of colorectal adenoma, an additional sample of colon mucosa in the proximity of the adenoma was collected for pathohistological analysis. Adenomas were graded according to the stage of dysplasia, and ghrelin and ghrelin receptor expression were determined immunohistochemically in both adenoma and adjacent colon tissue using the polyclonal antibody for ghrelin (ab150514, ABCAM Inc, Cambridge, United States) and ghrelin receptor (ab48285, ABCAM Inc, Cambridge, United States). Categorical and nominal variables were described through frequencies and proportions and the difference between specific groups were analyzed with Fisher’s and Fisher-Freeman-Halton’s method respectively. Spearman''s rank correlation coefficient was determined for correlation of expression of ghrelin and ghrelin receptor in adenoma and adjacent colon tissue with the grade of adenoma dysplasia.RESULTSAmong 92 patients with colorectal adenoma 43 had adenomas with high-grade dysplasia (46.7%). High expression of ghrelin was 7 times more common in high-grade adenoma compared to low-grade adenomas (13.95% to 2.04%, P = 0.048), while the expression of ghrelin in adjacent colon tissue was low. We found no correlation between ghrelin receptor expression in adenoma and adjacent colon tissue and the grade of colorectal adenoma dysplasia. The most significant correlation was found between ghrelin and ghrelin receptor expression in adenomas with high-grade dysplasia (rho = 0.519, P < 0.001).CONCLUSIONGhrelin and ghrelin receptor are expressed in colorectal adenoma and adjacent tissue with ghrelin expression being more pronounced in high grade dysplasia as a possible consequence of increased local synthesis.     

Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow

AimTo evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples.MethodsThree long-range high-fidelity DNA polymerases (Platinum^TM PCR SuperMix High Fidelity, LA Taq® Hot Start, and PrimeSTAR® GXL) were tested for their performance in the amplification of mtDNA fragments. Sequencing results of ten samples, as well as negative controls, which underwent library preparation with 12 and 15 cycles in limited-cycle PCR were compared. Additionally, two library normalization methods were compared: bead-based normalization vs quantification and individual normalization.ResultsPrimeSTAR® GXL performed best for mitochondrial DNA enrichment. Increment of amplification cycles to 15 in limited-cycle PCR step did not affect either the sequencing process or variant calling. Library quantification combined with individual library-by-library dilution outperformed bead-based normalization.ConclusionOptimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar massively parallel sequencing workflows (eg, small genomes, PCR amplicons, and plasmids).

Library preparation in massively parallel sequencing (MPS) protocols is a sensitive process, usually consisting of multiple elaborate steps. To ensure high quality of sequencing results, it is important to optimize library preparation according to characteristics of a particular target molecule. There are several critical aspects of Illumina® Human mtDNA Genome (1) assay for analysis of whole mitochondrial DNA (mtDNA) on MiSeq® instrument: initial enrichment of the target molecule (achieved, in this case, by long-range PCR); PCR step wherein index-adapter oligonucleotides are added and libraries are amplified (termed “limited-cycle PCR” step); and normalization of libraries prior to their pooling for sequencing.In this study, we aimed to test three long-range high-fidelity DNA polymerases for their performance in amplification of mtDNA fragments, in order to determine the one best suited for Illumina® assay, in which mitochondrial genomes are amplified in two large fragments (sizes 9.1 kb and 11.2 kb) (Supplementary Figure 1(Supplementary Figure 1)).Various optimizations and evaluations have been previously published (2-6), but, to our knowledge, none of them assessed the impact of increasing the number of amplification cycles in limited-cycle PCR. Therefore, we also aimed to test and observe how sequencing results were affected by this step and whether there were any adverse effects that would impact variant calling.Lastly, we aimed to compare two library normalization methods: bead-based normalization vs quantification and individual normalization. Nextera® XT Library Prep Kit (Illumina, San Diego, CA, USA) has been known to produce uneven read depth profiles (2-4,6-8). Therefore, a great risk in this protocol is potential loss of sequence information in regions that achieve very low read depth (ie, too low for analysis and subsequent variant calling), or possibly receive no reads at all. The choice of library normalization method may affect this through distribution of reads among the sequenced libraries. So, a method that provides more uniform distribution of reads would be preferable, which we hypothesized would be the method of quantification and individual normalization rather than bead-based normalization.     

Pro‐inflammatory effects of extracellular Hsp70 on NCI‐H292 human bronchial epithelial cell line

Extracellular Hsp70 (eHsp70) exerts its biological actions via Toll‐like receptors 2 and 4, and is increased in sera of chronic obstructive pulmonary disease (COPD) patients. The aim of this study was to explore the pro‐inflammatory effects and cytotoxicity of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on NCI‐H292 airway epithelial cells. NCI‐H292 cells were treated with recombinant human Hsp70 protein (rhHsp70), LPS, LTA and their combinations for 4, 12, 24 and 48 hours. IL‐6, IL‐8 and TNF‐α levels were measured by an ELISA method. Cell viability was determined by the MTS method, and caspase‐3/7, caspase‐8 and caspase‐9 assays. rhHsp70 induced secretion of IL‐6 and IL‐8 in a concentration‐ and time‐dependent manner, with the highest secretion at 24 hours. rhHsp70 combined with LTA had antagonistic and with LPS synergistic effect on IL‐6 secretion, while the interactions between rhHsp70 and LPS or LTA on IL‐8 were synergistic. TNF‐α was not detected in the applied conditions. rhHsp70, LPS or LTA did not affect cell viability, and rhHsp70 even suppressed caspase‐3/7 activities. We suggest that pro‐inflammatory effects of eHsp70, together with other damaging molecules and/or COPD risk factors, might contribute to the aggravation of chronic inflammation in human bronchial epithelium.     

Drug-induced gingival overgrowth in cardiovascular patients

Drug-induced gingival overgrowth(DIGO) is a pathological growth of gingival tissue, primarily associated with calcium channel blockers and immunosuppressants. Consequently, it is mainly seen in cardiovascular and transplanted patients. Nifedipine remains the main calcium channel blocker related to the development of this unpleasant side-effect. As for immunosuppress-ants, cyclosporin is the leading causative agent, whereas other drugs from this druggroup, including tacrolimus, have better safety profiles. Accumulated collagen with inflammatory infiltrates is the histological hallmark of this condition. Several factors are involved in the pathogenesis and can increase the risk, such as male gender, younger age, pre-existing periodontal inflammation, and concomitant use of other DIGO-inducing medications. Patients with DIGO may experience severe discomfort, trouble with speech and mastication, pain, and teeth loss, aside from cosmetic implications. Furthermore, these patients also have an increased risk for cardiovascular diseases. The interdisciplinary approach and cooperation with dental care experts are necessary for patient management. Treatment includes discontinuing the drug and switching to one with a better profile, improving oral hygiene, and surgical removal of enlarged tissue. Recognizing the potential of commonly used medications to cause DIGO and its effect on patients' health is necessary for early detection and adequate management of this complication.     

Hepatotropic viruses:Is Roma population at risk?

Roma people make up a significant ethnic minority in many European countries,with the vast majority living in Central and Eastern Europe.Roma are a vulnerable population group in social,economic,and political terms.Frequent migrations,life in segregated communities,substandard housing,poverty,and limited access to quality health care,including low immunization coverage,affect their health status and predispose them to various diseases,including viral hepatitis.Hepatitis A,B,and E are highly prevalent among Roma and mainly associated with low socioeconomic status.In contrast,hepatitis C does not seem to be more frequent in the Roma population.Enhanced efforts should be directed towards the implementation of screening programs,preventive measures,and treatment of viral hepatitis in Roma communities throughout Europe.