Prenatal and postnatal risk factors for developing bronchopulmonary dysplasia

Aim. To determine prenatal and postnatal risk factors for developing bronchopulmonary dysplasia in infants < 30 weeks of gestational age. Methods. Over a 22-month period, 115 newborns were enrolled in the study. Details including gestational age, sex, birth weight, prenatal steroids, surfactant treatment, ventilatory support, days of postnatal oxygen requirement, late onset sepsis/ pneumonia, air leaks, patency of ductus arteriosus, and fluid intake were collected. The presence of chorioamnionitis was diagnosed by histological examination. Commercial ELISA kits were used for the determination of the IL-6 and IL-8 levels. Results. Twenty-five infants developed BPD and 90 were enrolled in the non BPD group. Lower gestational age and male sex increased the risk for BPD. There was no difference in the presence of chorioamnitis and the level of IL-6 and IL-8 measured in cord blood and gastric aspirate between the groups. Intubation in the delivery room (resuscitation), need for surfactant treatment, mechanical ventilation, late onset sepsis/pneumonia, increased oxygenation index increased the risk for BPD after adjustment for GA and gender. Conclusion. In our cohort of infants with GA < 30 weeks exposure to prenatal inflammation did not increase the risk for BPD. However, low gestational age, male sex, need for resuscitation, mechanical ventilation and late onset sepsis were major risk factors for BPD development

OVARIAN INFERTILITY, STEM CELLS AND OOGENSIS IN VITRO

Background: In different human adult tissues and organs embryonic-like stem cells have already been found. A question is whether they are present also in the adult human ovary. In this study we tried to find whether embryonic-like stem cells are present in the ovarian surface epithe- lium of women with premature ovarian failure, and whether these cells can differentiate into oocytes by in vitro culture. Methods: In 10 women with premature ovarian failure the ovarian surface epithelium was laparo- scopically scraped. We tried to immunomagnetically isolate embryonic-like stem cells from the suspension of scraped cells and to confirm their existence immunocytochemically by the expression of the markers c-kit and surface antigen SSEA-4, characteristic of pluripotent stem cells. In the lab, we tried to differentiate stem cells into oocytes in a cell culture medium with added heterologous follicular fluid from the in vitro fertilization programme, which is rich in substances, and important for oocyte growth and maturation. The differentiation of oocyte-like cells was screened. Oocyte-like cells were isolated from the cell culture and im- munocytochemically stained for oocyte-specific markers (c-kit, VASA, ZP2-zona pellucida). Oocyte-like cells were analyzed for the expression of oocyte-specific genes (Oct-4A, ZP3, c-kit, SCP-3) by single-cell PCR. Results: From the ovarian surface epithelium of all patients small yellow coloured cells with diam- eters of 2 to 4 μm and positive for markers of pluripotent stem cells were isolated. In the lab, these cells were cultured into oocyte-like cells with diameter ranging from 60 to 80 μm. Some of oocyte-like cells were positive for oocyte-specific markers and expressed the genes, characteristic of oocytes, including meiotic genes. Conclusions: In patients with premature ovarian failure it is possible to culture oocyte-like cells from ovarian surface epithelium stem cells. Intense research is needed to evaluate the quality of oocyte-like cells developed in vitro and to estimate their potential use for in vitro fertilization in the far future

DISTRIBUTION OF HUMAN PAPILLOMA VIRUS GENOTYPES IN WOMEN WITH CERVICAL CANCER IN SLOVENIA AND GENOMIC VARIANTS OF HPV 16, HPV 18 AND HPV 33

Background. To establish the distribution of human papillomavirus (HPV) genotypes in representative population of women with cervical cancer (CC) in Slovenia in order to contribute the lacking data on HPV in CC and to assess the potential local benefit of future prophylactic HPV vaccination. Furthermore, we wanted to determine genomic variants of the most common HPV genotypes. Methods. Polymerase chain reaction with GP5+/GP6+ primers was performed in all 278 CC samples for HPV DNA detection and genotyping. Negative samples were additionally tested using CPI/CPIIg primers and INNO-LiPA HPV genotyping assay. Genomic variants of HPV 16, HPV 18 and HPV 33 were determined by sequencing of LCR, E6 and E7 genetic regions. Results. A total of 262/278 CC samples (94.2 %) were HPV DNA positive. HPV genotypes in Slovenian women with CC, in decreasing order of frequency, were: 16, 18, 33, 45, 31, 51, 58, 59, 35, 52, 73 and 82. Detailed genomic analysis was carried out on 40/178 isolates of HPV 16, 20/34 isolates of HPV 18 and 11/13 isolates of HPV 33. A total of 26 genomic variants of HPV 16 were identified. Thirty-eight isolates (95 %) belonged to the European branch; one isolate (2.5 %) belonged to the Asian-American branch and one (2.5 %) to African branch.1, 2 A total of 18 genomic variants of HPV 18 were identified. Nineteen isolates (95 %) belonged to the European branch and one isolate (5 %) belonged to the African branch.2, 3 Seven genomic variants of HPV 33 were identified. Five isolates (45.5 %) belonged to prototypic variants and 6 (54.5 %) belonged to non-prototypic variants.4 Conclusions. Distribution of all HPV genotypes in Slovenian women with CC was in the present study established for the first time and represents baseline distribution before mandatory HPV vaccination. Prophylactic HPV vaccination with currently available vaccines could prevent up to 77.1 % of CC in Slovenia caused by HPV 16 or HPV 18. Almost all isolates of HPV 16 and HPV 18 belonged to European branches; prototypic and non-prototypic HPV 33 variants were almost equally distributed among Slovenian patients with CC

Ovarian surface epithelium stem cells: oogenesis in vitro?

Background: The dogma that the total number of follicles and oocytes available for reproduction are determined at birth, and that one follicle and one oocyte are recruited from the existing pool to mature in each menstrual cycle still persists. There is an increasing experimental evidence that this might not be true. In the ovarian surface epithelium there might be nondifferentiated stem cells that might differentiate into oocytes also in adult life. In this research we aimed at isolating putative stem cells from the ovarian surface epithelium and at evaluating in vitro oogenesis in the ovarian cell culture in vitro in women without naturally present follicles and oocytes in their ovarian cortex – postmenopausal women and women with premature ovarian failure.Methods: Ovarian surface epithelium was scraped from the ovaries of 20 postmenopausal women and 5 women with premature ovarian failure. We tried to find putative stem cells in ovarian scraping and to confirm them by transcription markers Oct-4, Sox-2, and Nanog, and by surface antigen SSEA-4. Cell culture was set up by scraped cells in DMEM/F-12 medium with phenol red, which shows a weak estrogenic activity. Ovarian cell culture was cultured for 20 days in a CO2-incubator at 37 °C and 5 % CO2. Development of cells in the culture was followed and the presence of oocyte-like cells was evaluated by using different methods – evaluation of morphology, transcription markers, surface antigen markers, oocyte immunohistochemical markers, and flow cytometry after propidium iodide staining.Results: In all postmenopausal women and in 4 out of the 5 women with premature ovarian failure putative stem cells were isolated from the ovarian surface epithelium, which were positive for transcription markers of embryonic stem cells Oct-4, Sox-2, Nanog, and for surface antigen SSEA-4. In all these women ovarian cell culture was successfully established. In all cultures oocyte-like cells developed approximately on day 5 of culture, which were positive for transcription marker Oct-4 and immunohistological germ cell markers c-kit, Oct-4, ZP2, DAZL, and even meiotic marker SCP3. Marker SCP3 was expressed in oocyte-like cells developed in ovarian cell culture of postmenopausal women, but not in patients with premature ovarian failure. Flow cytometry after propidium iodide staining revealed a population of potentially haploid oocyte like cells in postmenopausal women. Stem cells developed also into some somatic cell types, including fibroblasts, neuron-, and mioblast-like cells. In patients with premature ovarian failure oocyte-like cells were developed only at the presence of autologous patient’s serum and mostly at the presence of heterologous follicular fluid serum, whereas they did not develop at the presence of fetal calf serum.Conclusions: First results are promising as they indicate the new insight into the physiology of the adult human ovary. In the ovarian surface epithelium putative stem cells with embryonic character are present, which can develop into the oocyte-like cells and other types of cells in vitro. Further research is required to understand better these new findings.</p