Krzysztof Szymanowski

An Efficient 3D Cell Culture Method on Biomimetic Nanostructured Grids

Current techniques of in vitro cell cultures are able to mimic the in vivo environment only to a limited extent, as they enable cells to grow only in two dimensions. Therefore cell culture approaches should rely on scaffolds that provide support comparable to the extracellular matrix. Here we demonstrate the advantages of novel nanostructured three-dimensional grids fabricated using electro-spinning technique, as scaffolds for cultures of neoplastic cells. The results of the study show that the fibers allow for a dynamic growth of HeLa cells, which form multi-layer structures of symmetrical and spherical character. This indicates that the applied scaffolds are nontoxic and allow proper flow of oxygen, nutrients, and growth factors. In addition, grids have been proven to be useful in in situ examination of cells ultrastructure.

Endometriosis prophylaxis and treatment with the newly developed xenogenic immunomodulator RESAN in an animal model

OBJECTIVE The objective was to assess the effectiveness of the newly developed immunomodulator RESAN in the prophylaxis and treatment of endometriosis induced in rats. STUDY DESIGN The study was performed on 58 Wistar rats. Twelve weeks before endometriosis induction, the RESAN vaccine was administered to 24 rats (100 mg i.m. and 100 mg s.c.). Endometriosis induction was performed in 48 rats, which were divided into two groups: group I, the prophylaxis group, consisting of 24 previously vaccinated rats; and group II, the treatment group, comprising the other 24 rats, which had not been vaccinated. The graft (4 mm x 4 mm) of endometrium was attached to the parietal peritoneum. A sham operation was performed in 10 rats (group III). After 3 months, a second laparotomy was performed in all animals, and endometriotic foci were excised when present. RESAN was administered to the group II animals. After an additional 3 months, a third laparotomy was performed in all animals of the three groups. RESULTS Positive, histologically confirmed endometriosis was found in 4.3% of the animals in group I and in 69.6% of group II rats (p<0.0001). Macroscopic assessment revealed endometriosis in 21.7% and 91.3% of animals in groups I and II, respectively (p<0.0001). At final laparotomy, 3 months after excision of the previously suspected foci, no signs of endometriosis were found according to both macroscopic assessment and histological examination. During the second laparotomy intraperitoneal adhesions were present in 13.0% of the animals in group I and in 61.0% of those in group II. No adhesions were present in group III. At the final laparotomy, the adhesions were present in only three of the animals in group II (p<0.0009). CONCLUSIONS RESAN seems to be effective in both the prophylaxis and treatment of endometriosis, as well as in the prophylaxis of adhesions. Histological confirmation of endometriosis should be mandatory.

Photodiagnosis and photodynamic therapy of endometriotic epithelial cells using 5-aminolevulinic acid and steroids

BACKGROUND The photodynamic diagnosis and therapy represent relatively new methods used, i.a., in the detection of some preneoplastic and neoplastic conditions. They are based on selective accumulation of photosensitizers in the altered cells, which can be identified by fluorescence of the sensitizers and, using light of an appropriate wavelength, can be eliminated. Currently, investigations continue on application of the methods in diagnosis and therapy of endometriosis, one of the most prevalent causes of a reduced fertility in women. METHODS In this study protoporphyrin IX, a photosensitizer derived from 5-aminolevulinic acid, was used to locate and destroy endometrial epithelium. Material for the investigations involved primary epithelial cells, isolated from 15 normal endometria and 15 ovarian endometriotic epithelia. Taking into account the cyclical hormonal alterations, which affect endometrial cells in individual phases of the menstrual cycle, experiments were conducted on accumulation of the photosensitizer and photodestruction of the cells preceded by their hormonal stimulation (17β-estradiol and progesterone). RESULTS AND CONCLUSION It was found that following 48 h stimulation with 17β-estradiol and/or progesterone a significantly augmented synthesis of protoporphyrin IX can be obtained in cells of endometrial epithelium as compared to the normal epithelium. Moreover, the endometriotic epithelial cells were most effectively eliminated following 48 h prestimulation with progesteron alone. The obtained result permits to assume that photodynamic diagnosis and photodynamic therapy of endometrial epithelium should be performed in the secretory phase of endometrium in order to optimise their results.

Matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of matrix metalloproteinases (TIMP-1) and transforming growth factor-β2 (TGF-β2) expression in eutopic endometrium of women with peritoneal endometriosis

INTRODUCTION The prevalence of endometriosis among reproductive age women is 7-17%; however, these figures reach 20-50% in patients suffering from infertility. Matrix metalloproteinases (MMPs) activity is thought to be particularly essential in the early phases of endometriosis development. Any changes in the equilibrium between MMPs activity and their tissue inhibitors (TIMPs) could be potentially harmful, promoting endometriosis development. The aim of this study was to investigate whether the MMP-2, MMP-9, TIMP-1 or TGF-B2 expression in eutopic endometrium from women with early endometriosis differ when compared with healthy subjects. The results were referred to the serum progesterone levels. MATERIALS AND METHOD Endometrial biopsy was taken from 42 patients (18 in the study group, 22 in thecontrol group) at the time of hysteroscopy for routine histology and for RT-PCR procedures. Comparison of the quantity of gene products was performed with a programme for densitometry and compared to GADPH product, which was a reference value. RESULTS The obtained results did not reveal any statistical difference in endometrial expression of MMP-2, MMp-9, TIMP-1, and TGF-β2 or serum progesterone level between women with endometriosis and without visible signs of this illness. CONCLUSION Despite the lack of statistical differences, it was observed that both examined metalloproteinases expressed a tendency to higher gene expression in the eutopic endometrium of women with endometriosis. However, both TIMP-1 and TGF-β2 expressions had the same tendency - higher values in endometriosis patients.

Threatened preterm labour – analysis of the cytokine profile and progesterone treatment efficiency

Abstract Objective: Preterm labour is defined as the onset of labour between weeks 23 and before 37 of gestation. The purpose of this study was to compare the cytokine profile in the blood serum of patients experiencing threatened premature labour, along with the time of delivery and the foetus body mass at birth. Methods: The study included 89 women hospitalised at the Obstetrics and Gynaecology Clinical Hospital in the period 2011–2013. Group 1 comprised 31 patients diagnosed with threatened preterm labour, prior to tocolytic treatment. Group 2 comprised 32 patients with tocolytic treatment. Group 3 constituted the control group. Results: Groups 1, 2 and 3 differed considerably in terms of the week of delivery of pregnancy (p = 0.006). Analogous results were obtained by analysing the body mass at birth, where a statistically significant difference in body mass at birth was found between Groups 1, 2 and 3 (p  < 0.001). Our analysis focussed on the cytokine profile of the women included in the study, but no considerable changes in cytokine concentration were observed. Conclusions: No statistically significant differences were found in the cytokine profile among those patients in normally progressing pregnancy or in threatened preterm labour. No suitability of progesterone treatment in threatened preterm labour was observed.

Costs of the use of carbetocin in the prevention of uterine atony following delivery of the infant by Caesarean section — retrospective multicenter study

OBJECTIVES The aim of this study was to compare the costs of using carbetocin in the prevention of uterine atony following delivery of the infant by Cesarean section (C-section) under epidural or spinal anesthesia with standard methods of prevention (SMP). MATERIAL AND METHODS This retrospective multicenter study was based on data from three medical centers. A questionnaire was developed to gather patient records on consumption and costs of resources related to C-section, prevention of uterine atony and postpartum hemorrhage (PPH) treatment. Six subpopulations were considered, depending on patient characteristics. The analysis covered two perspectives: that of the hospital and of the public payer. RESULTS The subpopulations were homogenous, which was a premise for pooling the data. The use of carbetocin in the prevention of uterine atony following Cesarean section generates savings for hospital in comparison with SMP (oxytocin) in 5 of 6 subpopulations. The biggest savings were observed amongst patients who experienced severe PPH and reached 2.6-6.2 thousand PLN per patient. Costs of services related to C-section borne by the hospitals were higher than the refund received from a public payer. The greatest underestimation reached 12.1 thousand PLN per patient. Nevertheless, loss generated by this underfunding was lower in carbetocin versus oxytocin group. CONCLUSIONS The use of carbetocin instead of SMP gives hospitals an opportunity to make savings as well as to reduce losses resulting from the underfunding of the services provided by the National Health Fund.

Trichrome Mallory's stain may indicate differential rates of RNA synthesis in eutopic and ectopic endometrium.

Mallorys triple staining is a histochemical technique used mainly for analysing connective tissues and glands and other tissues. We have described the differences in the nuclear staining between eutopic and ectopic endometrium as well as endometrial hyperplasia and adenocarcinoma using the Mallorys method. The ultrastructural differences between eutopic and ectopic endometrium have been detected. In normal and hyperplastic endometrium the presence of stromal cell nuclei with an increased affinity to aniline blue has been observed. The affinity has disappeared after digestion of tissues with RNase. In cases of endometriosis, independently of cell types, the nuclei have shown affinity to orange G. Similar effects in adenocarcinoma have been noted. The ultrastructural studies have shown that in normal endometrium the stroma contained cells with euchromatic and low electron density cell nuclei. In endometriosis heterochromatic cell nuclei present both in the stroma and within glands have been detected. The results indicate that the Mallorys technique may be a useful tool for recognizing the differences between eutopic and ectopic endometrium. The affinity for aniline blue in normal and hyperplastic endometrium occurs most likely due to increased RNA synthesis. Based on Mallorys staining a similarity between hyperplasia and unchanged endometrium in contrast to similar results of the staining obtained in cases of adenocarcinoma and endometriosis may be suggested.

Recurrent fetal complex ovarian cysts with rupture followed by simple cyst in the neonatal period with no adverse sequelae

Abstract Fetal ovarian cysts are the most frequent type of abdominal tumors in female fetuses with prenatal detection rate of more than 30%. The etiology of fetal ovarian cysts is unclear, but hormonal stimulation as well as presence of maternal diabetes, hypothyroidism, Rh iso-immune hemolytic disease and toxemia has been generally considered responsible for the disease. Complications of fetal ovarian cysts include compression of other viscera, cyst rupture, hemorrhage and, most frequently, ovarian torsion with consequent loss of the ovary. Management is controversial with several options described in the literature, including watchful expectancy, antenatal aspiration of simple cysts to prevent torsion and ovarian loss and finally, resection of all complex cysts in the neonatal period. To date, no case report has described recurrent complex cysts with rupture in the fetal period and recurrence of simple cyst in neonatal period. By presenting this case, we wanted to show that surgical intervention in case of prenatally diagnosed fetal ovarian cyst should be considered postnatally and only in symptomatic or complicated cases.

Ectopic pregnancy: which treatment method least affects fertility?

We would like to present our data on important topic which is the influence of treatment of ectopic pregnancy (EP) on future fertility of the woman. Since women with EP often want to continue reproducing, it is crucial to offer patients a treatment method that least affects fertility. The aim of our study was to elucidate which of the three main treatments for tubal EP: medical treatment with methotrexate (MTX), conservative surgical treatment with salpingostomy or radical surgical treatment with salpingectomy serves this purpose best. We also aimed to investigate which therapy was associated with the greatest risk of future EP. We analyzed the group of 126 patients treated from EP in our hospital. The cut-off was spontaneous pregnancy occurring 24 months after treatment. Fertility was confirmed in patients with clinical IUP. Subsequent EP was considered a failure with regard to fertility. The patients in our study had undergone one of three treatment methods: MTX, conservative surgical treatment or radical surgical treatment. Qualification of patients to MTX was based on the guidelines of American Society of Reproductive Medicine (ASRM) from 2013. The characteristic of the study group is presented in Table 1. We found no statistically significant between-group difference in the IUP rates (p = 0.938). There was no significant betweengroup difference in average time to pregnancy (p > 0.05). We neither found significant between-group difference regarding the cumulative pregnancy rate. We compared the incidence of consequent EP, according to the treatment method and found again no significant between-group difference (p = 0.638). Since the groups were relatively small, we summed the patients who had conservative treatment (MTX + salpingostomy) and compared the incidence of second EP with the group of women who underwent salpingectomy. Neither this comparison showed significant

Studies on function of P-glycoprotein in photodynamic therapy of endometriosis.

OBJECTIVE The aim of the present study was to examine whether the effects of endometriosis-targeted photodynamic therapy (PDT), dependent on 5-aminolevulinic acid (ALA), rely on the presence of P-glycoprotein (P-gp), which is regarded as constituting one of the causes of multidrug resistance phenomenon. BACKGROUND The significance of the undertaken studies reflects the complete absence of reports related to the modulation of P-gp activity and efficacy of PDT in patients with endometriosis. MATERIALS AND METHODS Tissue samples of normal endometria were obtained from eight women after hysterectomy who were diagnosed with cervical intra-epithelial neoplasia. Fragments of ovarian endometriosis were obtained from 15 women. Epithelial cells were isolated from the material and in in vitro conditions were preincubated with P-gp blocker-verapamil-before ALA-PDT. The cytotoxicity was evaluated using the XTT test, allowing us to estimate cell growth inhibition. Statistical analysis of the results involved the nonparametric Wilcoxon paired rank test and the Mann-Whitney U-test using the Statistica v5 software (p < 0.05). In parallel, P-gp presence in the analyzed material was evaluated using immunohistochemistry. RESULTS In normal endometrial epithelium, verapamil was shown to intensify phototoxic effects at 2 and 4 mmol/L ALA (p < 0.05). In endometriotic epithelium, such intensification was noted in all examined concentrations of ALA (p < 0.001). Moreover, the ectopic epithelial cells were more sensitive than eutopic epithelial cells to PDT upon ALA alone, as well as after preincubation with verapamil. Immunohistohemical analysis allowed us to demonstrate the absence of glycoprotein P in normal endometrium. In endometriosis, P-gp was localised in both the epithelium and the stroma of the examined material. CONCLUSION Phototoxic effects could be amplified in epithelial cells of endometriotic foci by appropriate action of verapamil and 5-aminolevulinic acid.