Publications (14)32.01 Total impact
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Article: Small SSEA-4-positive cells from human ovarian cell cultures: related to embryonic stem cells and germinal lineage?
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ABSTRACT: BACKGROUND: It has already been found that very small embyronic-like stem cells (VSELs) are present in adult human tissues and organs. The aim of this study was to find if there exists any similar population of cells in cell cultures of reproductive tissues and embryonic stem cells, and if these cells have any relation to pluripotency and germinal lineage.Methods and results: Here we report that a population of small SSEA-4-positive cells with diameters of up to 4 mum was isolated by fluorescence-activated cell sorting (FACS) from the human ovarian cell cultures after enzymatic degradation of adult cortex tissues. These small cells -- putative ovarian stem cells -- were also observed during cell culturing of up to 6 months and more. In general, small putative ovarian stem cells, isolated by FACS, showed a relatively low gene expression profile when compared to human embryonic stem cells (hESCs) and human adult fibroblasts; this may reflect the quiescent state of these cells. In spite of that, small putative ovarian stem cells expressed several genes related to primordial germ cells (PGCs), pluripotency and germinal lineage, including VASA. The PGC-related gene PRDM1 was strongly expressed in small putative ovarian stem cells; in both hESCs and fibroblasts it was significantly down-regulated. In addition, putative ovarian stem cells expressed other PGC-related genes, such as PRDM14 and DPPA3. Most of the pluripotency and germinal lineage-related genes were up-regulated in hESCs (except VASA). When compared to fibroblasts, there were several pluripotency-related genes, which were up-regulated in small putative ovarian stem cells. Similar populations of small cells were also isolated by FACS from human testicular and hESC cultures. CONCLUSIONS: Our results confirm the potential embryonic-like character of small putative stem cells isolated from human adult ovaries and their possible relation to germinal lineage.Journal of Ovarian Research 04/2013; 6(1):24. · 2.57 Impact Factor -
Article: Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients.
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ABSTRACT: This prospective randomized study investigated whether intracytoplasmic sperm injection (ICSI) outcome can be improved with sperm preselection under ×6000 magnification and intracytoplasmic morphologically selected sperm injection (IMSI) in patients with teratozoospermia and characterized embryo development and quality regarding sperm morphology and presence of head vacuoles. Couples with isolated teratozoospermia were divided into two groups: IMSI group (n=52) and ICSI group (n=70) and fertilization, blastocyst and clinical pregnancy rates were compared. Oocytes from 30 randomly chosen patients from the IMSI group were injected with spermatozoa that had been previously classified under ×6000 magnification into four classes according to the number and size of vacuoles in the head and then cultured separately. Pronuclear morphology, embryo development and blastomere viability were estimated to investigate the influence of sperm morphology, especially vacuoles, on embryo developmental capacity. A significantly higher clinical pregnancy rate was achieved in the IMSI group compared with the ICSI group (48% versus 24%, P<0.05). Fertilization with spermatozoa without head vacuoles yielded higher number of morphologically normal zygotes, higher blastocyst rate and smaller proportion of arrested embryos than spermatozoa with vacuoles and other head defects. IMSI is a method of choice in patients with teratozoospermia.Reproductive biomedicine online 04/2012; 25(2):168-79. · 2.04 Impact Factor -
Article: The IMSI procedure improves poor embryo development in the same infertile couples with poor semen quality: a comparative prospective randomized study.
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ABSTRACT: Sperm of poor quality can negatively affect embryo development to the blastocyst stage. The aim of this comparative prospective randomized study was to evaluate the role of an intracytoplasmic morphologically selected sperm injection (IMSI) in the same infertile couples included in the programme of intracytoplasmic sperm injection (ICSI) due to their indications of male infertility which had resulted in all arrested embryos following a prolonged 5-day culture in previous ICSI cycles. Couples exhibiting poor semen quality and with all arrested embryos following a prolonged 5-day culture in previous ICSI cycles were divided into two groups: Group 1: IMSI group (n = 20) with IMSI performed in a current attempt and Group 2: ICSI group (n = 37) with a conventional ICSI procedure performed in a current attempt of in vitro fertilization. Fertilization rate, embryo development, implantation, pregnancy and abortion rates were compared between current IMSI and conventional ICSI procedures, and with previous ICSI attempts. The IMSI group was characterized by a higher number of blastocysts per cycle than the ICSI group (0.80 vs. 0.65) after a prolonged 5-day embryo culture. There was a significantly lower number of cycles with all arrested embryos and cycles with no embryo transfer in the IMSI group versus the ICSI group (0% vs. 27.0%, p = 0.048). After the transfer of embryos at the blastocyst or morula stage (on luteal day 5) a tendency toward higher implantation and pregnancy rates per cycle was achieved in the IMSI group compared to the ICSI group (17.1% vs. 6.8%; 25.0% vs. 8.1%, respectively), although not statistically significant. After IMSI, all pregnancies achieved by the blastocyst transfer were normally on-going, whereas after ICSI, two of three pregnancies ended in spontaneous abortion. After IMSI, two pregnancies were also achieved by the morula stage embryos, whereas after the conventional ICSI procedure, embryos at the morula stage did not implant. The IMSI procedure improved embryo development and the laboratory and clinical outcomes of sperm microinjection in the same infertile couples with male infertility and poor embryo development over the previous ICSI attempts.Reproductive Biology and Endocrinology 08/2011; 9:123. · 2.05 Impact Factor -
Article: Comparison of clinical and analytical performance of the Abbott Realtime High Risk HPV test to the performance of hybrid capture 2 in population-based cervical cancer screening.
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ABSTRACT: The clinical performance of the Abbott RealTime High Risk HPV (human papillomavirus) test (RealTime) and that of the Hybrid Capture 2 HPV DNA test (hc2) were prospectively compared in the population-based cervical cancer screening setting. In women >30 years old (n = 3,129), the clinical sensitivity of RealTime for detection of cervical intraepithelial neoplasia of grade 2 (CIN2) or worse (38 cases) and its clinical specificity for lesions of less than CIN2 (3,091 controls) were 100% and 93.3%, respectively, and those of hc2 were 97.4% and 91.8%, respectively. A noninferiority score test showed that the clinical specificity (P < 0.0001) and clinical sensitivity (P = 0.011) of RealTime were noninferior to those of hc2 at the recommended thresholds of 98% and 90%. In the total study population (women 20 to 64 years old; n = 4,432; 57 cases, 4,375 controls), the clinical sensitivity and specificity of RealTime were 98.2% and 89.5%, and those of hc2 were 94.7% and 87.7%, respectively. The analytical sensitivity and analytical specificity of RealTime in detecting targeted HPV types evaluated with the largest sample collection to date (4,479 samples) were 94.8% and 99.8%, and those of hc2 were 93.4% and 97.8%, respectively. Excellent analytical agreement between the two assays was obtained (kappa value, 0.84), while the analytical accuracy of RealTime was significantly higher than that of hc2. RealTime demonstrated high intralaboratory reproducibility and interlaboratory agreement with 500 samples retested 61 to 226 days after initial testing in two different laboratories. RealTime can be considered to be a reliable and robust HPV assay clinically comparable to hc2 for the detection of CIN2+ lesions in a population-based cervical cancer screening setting.Journal of clinical microbiology 03/2011; 49(5):1721-9. · 4.16 Impact Factor -
Article: Slow oocyte freezing and thawing in couples with no sperm or an insufficient number of sperm on the day of in vitro fertilization.
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ABSTRACT: The clinical results of in vitro fertilization of slowly frozen-thawed oocytes are known to be significantly worse than those obtained by fresh oocytes. Little is known about the factors affecting the clinical outcome of frozen-thawed oocytes. The aim of this retrospective study was to explore the role of oocyte cryopreservation in the group of patients with no available sperm on the day of in vitro fertilization. Additionally, the effects of the female serum FSH level and sperm quality on the clinical outcome of frozen-thawed oocytes were evaluated. Oocytes were slowly frozen and thawed in 22 infertile couples with no sperm or insufficient number of sperm on the day of in vitro fertilization (IVF). In 9 couples with severe azoospermia or oligoasthenoteratozoospermia frozen-thawed oocytes were fertilized by autologous sperm of bad quality when available (Group 1). In 13 couples with non-ejaculation due to psychological stress on the day of classical IVF or severe azoospermia frozen-thawed oocytes were fertilized by autologous or donated sperm of normal quality (Group 2). Oocytes were thawed in 23 cycles and microinjected by the autologous or donated sperm, when available. The clinical outcome of intracytoplasmic sperm injection--ICSI (fertilization, blastocyst, and pregnancy rates) was compared to the outcome of fresh oocytes of the same group of patients; additionally, the female serum FSH level and the sperm quality were compared. In all couples, 70.5% of oocytes survived the freeze-thaw procedure. After ICSI, 61.5% of thawed oocytes were fertilized. Twenty one% of embryos developed to the blastocyst stage. The pregnancy rates per embryo transfer and freeze-thaw cycle were 33.3% and 17.4%, respectively. All pregnancies ended in the birth of a baby without congenital anomalies. In patients with severe azoospermia or oligoasthenoteratozoospermia there was no statistically significant difference in pregnancy rates per cycle obtained by thawed oocytes vs. fresh oocytes in previous ICSI cycles (14.2% vs. 13.6%) but there was a higher proportion of abnormal, non-cleaved or triploid zygotes when frozen-thawed oocytes were microinjected (33.3% vs. 11.8%; P < 0.01). The female serum FSH levels did not affect the survival and fertilization of frozen-thawed oocytes, but in patients with increased serum FSH level no pregnancies were achieved. After the complete freeze-thaw cycle, there was a significantly higher fertilization rate and tendency to higher pregnancy rates per thawing cycle after the microinjection of autologous or donated sperm of normal quality than autologous sperm of poor quality. The slow oocyte freezing and thawing is a valuable method when no or insufficient number of sperm are available on the day of in vitro fertilization. The quality of sperm is an important factor affecting the clinical outcome achieved by frozen-thawed oocytes.Reproductive Biology and Endocrinology 01/2011; 9:19. · 2.05 Impact Factor -
Article: The Abbott RealTime High Risk HPV test: comparative evaluation of analytical specificity and clinical sensitivity for cervical carcinoma and CIN 3 lesions with the Hybrid Capture 2 HPV DNA test.
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ABSTRACT: The Abbott RealTime High Risk HPV test (RealTime) is a novel assay designed to detect 14 high-risk human papillomavirus genotypes (hr-HPV) and concurrently distinguish HPV-16 and HPV-18 from other hr-HPV within a single test. To evaluate analytical specificity and clinical sensitivity for cervical carcinoma and cervical intraepithelial neoplasia grade 3 (CIN3) of the RealTime test in comparison with the Digene Hybrid Capture II Test (hc2). Materials and methods: Analytical specificity of the RealTime assay was evaluated on 37 samples with previously determined hc2 false-positive results due to cross-reactivity of the hc2 high-risk probe cocktail with untargeted low-risk HPV genotypes. All 37 samples were negative for 14 hr-HPV using the RealTime test. Clinical sensitivity of RealTime was evaluated in comparison to hc2 on 95 and 267 archived routine cervical specimens collected from women with histologically confirmed cervical carcinoma and CIN3 lesions, respectively. Archived specimens were selected for the present study after linkage with the Slovenian national registry of CIN3 and cervical cancer to obtain histology data. Concordant results between RealTime and hc2 were obtained in 90/95 cervical cancer samples (94.7% agreement) and in 250/267 CIN3 samples (93.6% agreement). Clinical sensitivity of RealTime and hc2 for cervical cancer in the total study cohort was 88.4% (95% confidence interval (CI): 80.3-93.6%) and 87.3% (95% CI: 79.0-92.8%), respectively, and analytical sensitivity for samples containing at least one targeted hr-HPV was 98.8% (95% CI: 93.0-100.0%) and 95.3% (95% CI: 88.2-98.5%), respectively. Clinical sensitivity of RealTime and hc2 for CIN3 lesions of the total study cohort was 91.8% (95% CI: 87.8-94.5%) and 89.1% (95% CI: 84.8-92.3%), respectively, and analytical sensitivity for samples containing at least one targeted hr-HPV was 96.4% (95% CI: 93.3-98.2%) and 92.5% (95% CI: 88.5-95.2%), respectively. The RealTime test showed excellent analytical specificity and no cross-reactivity with low risk HPV genotypes that tested positive with hc2. Clinical sensitivity of the RealTime assay using archived routine cervical specimens was comparable to hc2. The RealTime test is an important new method applicable to cervical carcinoma screening and management of cervical precancerous lesions.Acta dermatovenerologica Alpina, Panonica, et Adriatica 09/2009; 18(3):94-103. -
Article: Follicular oestradiol and VEGF after GnRH antagonists or GnRH agonists in women with PCOS.
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ABSTRACT: The aim of this study was to determine whether follicular oestradiol and vascular endothelial growth factor (VEGF) concentrations in women with polycystic ovarian syndrome (PCOS) differ according to the use of gonadotrophin-releasing hormone (GnRH) antagonists or GnRH agonists. Furthermore, the effect of follicular oestradiol and VEGF concentrations on oocyte and embryo quality was investigated. In this prospective clinical study, 20 women with PCOS undergoing intracytoplasmic sperm injection for male factor infertility were included using a GnRH antagonist or a GnRH agonist protocol. In each follicle, oestradiol and VEGF concentrations were determined. In the GnRH antagonist group 254 follicles and in the GnRH agonist group 245 follicles, were aspirated. Fewer metaphase II (MII) and more immature and degenerative oocytes were registered in the GnRH antagonist group. Follicular oestradiol and VEGF were lower in the GnRH antagonist group (P = 0.014 and P < 0.001, respectively). Moreover, higher oestradiol concentrations were related to embryos of higher quality (P = 0.037). It is concluded that GnRH antagonists decrease follicular oestradiol and VEGF concentrations and the number of retrieved MII oocytes in women with PCOS.Reproductive biomedicine online 01/2009; 18(1):21-8. · 2.04 Impact Factor -
Article: Parthenogenetic embryo-like structures in the human ovarian surface epithelium cell culture in postmenopausal women with no naturally present follicles and oocytes.
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ABSTRACT: Little is known about parthenogenesis in the human ovary. What is known is related to patients with teratoma in their medical history. Ovarian surface epithelium (OSE) was often proposed as a source of ovarian stem cells with an embryonic character in the past, and was also termed "germinal epithelium." The aim of this study was to isolate putative stem cells from OSE scrapings, to set up an OSE cell culture, to follow the in vitro oogenesis and possible formation of parthenogenetic embryos in 21 postmenopausal women with no naturally present follicles and oocytes. Small round cells with a bubble-like structure and with a diameter from 2 to 4 microm were isolated from the material obtained by OSE scrapings in all women. They expressed early embryonic developmental markers such as stage-specific embryonic antigen-4 (SSEA-4) surface antigen and Oct-4, Nanog, Sox-2, and c-kit transcription factors. These cells were separated by density gradient centrifugation and grown in vitro, where they proliferated and formed embryoid body-like structures. Their markers of pluripotency such as telomerase activity were decreased during in vitro culture and they did not form teratoma after the injection into SCID mice. Some of them grew intensively and reached a diameter of approximately 20 microm after 5-7 days of culture. In the OSE cell culture, oocyte-like cells developed among them, which reached a diameter up to 95 mum, and expressed Oct-4, c-kit, VASA, and ZP2 transcription markers after 20 days of culture. Some of them expressed a zona pellucida-like structure and rarely germinal vesicle- and polar body-like structures. At the same time, parthenogenetic blastocyst-like structures developed, which expressed transcription markers Oct-4, Sox-2, and Nanog and were normal for chromosomes X, Y, 13, 16, 18, 21, and 22. In conclusion, the discovered cells expressed embryonic stem cell markers, gave rise to embryoid body-, oocyte-, and blastocyst-like structures, and might be involved in the human reproduction and formation of ovarian tumors.Stem cells and development 07/2008; 18(1):137-49. · 4.15 Impact Factor -
Article: Apoptosis, reactive oxygen species and follicular anti-Müllerian hormone in natural versus stimulated cycles.
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ABSTRACT: The aim of this study was to compare granulosa cell apoptosis, reactive oxygen species (ROS) and anti-Müllerian hormone (AMH) production in natural and stimulated cycles, and to determine their effect on IVF outcome. Of 60 women with tubal factor infertility, 30 underwent natural cycle IVF and 30 underwent ovarian stimulation with gonadotrophin-releasing hormone antagonist and gonadotrophin. Apoptosis and ROS production was measured in isolated granulosa cells from each aspirated follicle using flow cytometry. Follicular AMH concentration was measured using enzyme immunoassay. A total of 188 follicles were analysed. A lower incidence of apoptotic granulosa cells and higher mean follicular AMH concentration was observed in the natural cycle than in the ovarian stimulation group (20.2 +/- 15.6% versus 35.6 +/- 27.7%, P = 0.007; 6.9 +/- 7.1 ng/ml versus 2.5 +/- 1.7 ng/ml, P < 0.001). Ovarian stimulation increases the incidence of apoptotic granulosa cells, but has no effect on their ROS production. Ovarian stimulation reduces follicular AMH concentrations, presumably due to a negative effect of exogenous FSH on AMH production. This study was unable to explain the low pregnancy rate in natural cycle IVF using the data on apoptosis, ROS and AMH production in granulosa cells.Reproductive biomedicine online 06/2008; 16(5):640-8. · 2.04 Impact Factor -
Article: Genomic diversity of human papillomavirus genotype 53 in an ethnogeographically closed cohort of white European women.
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ABSTRACT: Human papillomavirus (HPV) genotype 53 is classified taxonomically in alpha HPV genus-species 6, together with HPV-30, HPV-56, and HPV-66 and is considered to be one of three "probable high-risk" HPV genotypes. Recent worldwide comparison of 44 isolates of HPV-53 showed the existence of nine long control region (LCR) genomic variants, which formed a phylogenetic tree with two deep dichotomic branches. In order to investigate further the genomic diversity of HPV-53, a total of 94 isolates of HPV-53 obtained from an ethnogeographically closed cohort of 70 white European women was analyzed. The identification and characterization of HPV-53 genomic variants was based on analysis of three different HPV genomic regions: LCR, E6 and E7. A higher genomic diversity of HPV-53 was identified in the ethnogeographically closed cohort of white European women than has been reported previously on isolates collected worldwide. Altogether, 19 HPV-53 genomic variants, composed of 13 LCR, 13 E6, and 5 E7 genomic variants, were identified. Eleven out of 13 LCR, all E6, and four out of five E7 genomic variants were described for the first time. The present study confirmed dichotomic phylogeny of HPV-53 described previously and, in addition, showed for the first time that after a dichotomic split, both groups of HPV-53 genomic variants formed star-like phylogenetic clusters. In women with persistent HPV-53 infection, HPV-53 genomic variants remained unchanged for up to 51 months. In rare cases, infection with multiple HPV-53 genomic variants is possible. Taking into account the results of this and previous studies, at least 26 different HPV-53 genomic variants exist today.Journal of Medical Virology 05/2007; 79(4):431-8. · 2.82 Impact Factor -
Article: Increased ammonium in culture medium reduces the development of human embryos to the blastocyst stage.
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ABSTRACT: The objective of this prospective study was to evaluate the effect of ammonium accumulated in sequential media and determined by enzymatic spectrophotometric method on the blastocyst development in 281 human embryos from 100 stimulated and natural in vitro fertilization (IVF) cycles. Ammonium concentration was increased in 62% of cycles and was correlated negatively with the blastocyst development after classical IVF, but not after intracytoplasmic sperm injection (ICSI).Fertility and sterility 03/2006; 85(2):526-8. · 3.97 Impact Factor -
Article: Uterine arterial blood flow and the substances of ovarian renin-angiotensin system in women with polycystic ovaries undergoing in vitro fertilization.
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ABSTRACT: To find whether plasma and follicular prorenin concentrations have any effect on the uterine arterial blood flow in women with polycystic ovarian syndrome (PCOS) compared to those with normal menstrual cycles (NMC). Controlled prospective clinical study involved 55 women undergoing in vitro fertilization: 24 with PCOS and 31 with NMC. In both groups transvaginal colour Doppler assessment of uterine arterial blood flow was analysed on day 22 of the cycle, on the day of human chorionic gonadotrophin (HCG) administration and 36 h after HCG. Plasma and follicular (in the dominant follicle containing mature oocyte, and in the pooled follicles) prorenin and active renin, and serum estradiol and androstenedione concentrations were measured at these time-points. The Student's t-test and Pearson correlation were used for the statistical analysis. The resistance index (RI) in the NMC group decreased from 0.84+/-0.05 on the day of HCG to 0.78+/-0.08 36 h after HCG (P < 0.05); in the PCOS group the RI did not decrease. Follicular prorenin concentrations in the dominant follicle and in the pooled follicles were lower in the PCOS than in the NMC group (20,210+/-10,831 microU/l, 16,753+/-8634 microU/l versus 42,637+/-35,400 microU/l, 33,067+/-26,200 microU/l; P < 0.05). Plasma prorenin concentrations do not affect vascular impedance to the uterine artery, but follicular prorenin do by newly formed low resistant vessels in the follicles.European Journal of Obstetrics & Gynecology and Reproductive Biology 02/2006; 124(1):77-81. · 1.97 Impact Factor -
Article: [Prevalence of human papillomavirus infection in Slovenian women with repeated Pap II smears].
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ABSTRACT: In the detection of precancerous lesions the cervical Papanicolaou smear screening is used in Slovenia and worldwide. Management of patients with repeat abnormal smears (Pap II) represents a great and complex clinical and public health problem. Repeated cytologic examinations are routine procedure in many countries, also in Slovenia, although the sensitivity of Pap smear testing in the detection of cervical intraepithelial neoplasia (CIN) II and III is relatively low. In cases of abnormal squamous cells and mildly dyskaryotic cells the presence of infections with high-risk HPV genotypes is being increasingly used as a complementary method to Pap smear testing. In the study we enrolled 148 cervical samples of women who within two years had three subsequent Pap II smears (abnormal squamous cells or mildly dyskaryotic cells). The prevalence of HPV infections was determined using three molecular tests: Hybrid Capture 2 (hc2) test and two variants of polymerase chain reaction (PCR-PGMY11/PGMY09 and PCR-CPI/CPIIG). HPV infection was detected in 25.7% of women. In women aged < or = 30 years a statistically significant higher prevalence of HPV infections was found (37.8%) than in women aged > 30 years (20.4%). Our findings show that repeat Pap smear as the method of follow-up and detection of precancerous lesions of the observed population do not provide relevant results due to low prevalence of HPV infections in Slovenia, which indirectly indicates low sensitivity and specificity of Pap smear testing. In the detection of HPV infections, molecular methods are thus sensitive screening tests to be used complementary to cytologic tests in women with abnormal squamous cells and mildly dyskaryotic cells.Medical Archives 01/2005; 59(1):47-51. -
Article: Follicular fluid renin concentration in patients with polycystic ovaries treated with gonadotrophins in an in vitro fertilisation programme.
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ABSTRACT: The initial hypothesis was that the follicular fluid prorenin concentration was higher in women with polycystic ovaries (PCO) compared to normally cycling women stimulated by gonadotrophins. In a controlled prospective study, 47 women undergoing in vitro fertilisation (IVF) were enrolled: 20 women with PCO represented the study group and 27 normally cycling women the control group. Plasma prorenin, active renin, serum oestradiol, and androstenedione concentrations were measured on the day of human chorionic gonadotrophin (HCG) administration and 36 hours after HCG administration. Follicular fluid prorenin, active renin, oestradiol, and androstenedione concentrations from the dominant follicle and pooled follicles were measured 36 hours after HCG administration. Plasma prorenin concentration 36 hours after HCG administration was significantly higher in the PCO group (1867 microU/l; range 1137-3162 microU/l) than in controls (860 microU/l; range 433-1763 microU/l) (p = 0.007). The follicular fluid prorenin concentration in the dominant follicle and in the pooled follicles was lower in the PCO group (20190 microU/l; range 11130-25955 microU/l) than in controls (46930 microU/l; range 20671-66171 microU/l) (p = 0.003). We conclude that plasma prorenin concentrations in gonadotrophin-stimulated PCO cycles are augmented due to numerous follicles. Lower follicular fluid prorenin concentrations in women with PCO show that the synthesis of prorenin in theca cells starts later after HCG administration than in normally cycling women.Clinical Chemistry and Laboratory Medicine 06/2003; 41(5):663-7. · 2.15 Impact Factor
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Institutions
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2009
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University of Ljubljana
- Institute of Microbiology and Immunology
Ljubljana, Ljubljana, Slovenia
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2003–2009
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Ljubljana University Medical Centre
Ljubljana, Ljubljana, Slovenia
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