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Background In vitro maturation (IVM) of oocytes is a laboratory method that allows the maturation of immature (GV) oocytes retrieved from patients enrolled in the in vitro fertilization (IVF) programme. However, this method is still sparsely researched and used in clinical practice, leading to suboptimal clinical results. Anti-Müllerian hormone (AMH) is an important hormone with known effects on human ovaries, especially on follicles (follicular cells) during folliculogenesis. In contrast, the effect of AMH on the human oocyte itself is unknown. Therefore, we wanted to determine whether human oocytes express AMH receptor 2 (AMHR2) for this hormone. Recombinant AMH was added to the IVM medium to determine whether it affected oocyte maturation. Methods In total, 247 human oocytes (171 immature and 76 mature) were collected from patients enrolled in the intracytoplasmic sperm injection (ICSI) programme who were aged 20 to 43 years and underwent a short antagonist protocol of ovarian stimulation. The expression of AMHR2 protein and AMHR2 gene was analysed in immature and mature oocytes. Additionally, maturation of GV oocytes was performed in vitro in different maturation media with or without added AMH to evaluate the effect of AMH on the oocyte maturation rate. Results Immunocytochemistry and confocal microscopy revealed that AMHR2 protein is expressed in both immature and mature human oocytes. AMHR2 was expressed in a spotted pattern throughout the whole oocyte. The IVM procedure revealed that AMH in maturation medium improved GV oocyte maturation in vitro, as all oocytes were successfully matured in maturation medium containing recombinant AMH only. Furthermore, antagonism between AMH and follicle-stimulating hormone (FSH) during the maturation process was observed, with fewer oocytes maturing when both AMH and FSH were added to the maturation medium. Finally, AMHR2 gene expression was found in immature and in vitro matured oocytes but absent in mature oocytes. Conclusions The positive AMHR2 protein and AMHR2 gene expression in human oocytes shows that AMH could directly act on human oocytes. This was further functionally confirmed by the IVM procedure. These findings suggest the potential clinical application of recombinant AMH to improve IVM of human oocytes in the future.

The ICSI procedure was invented to treat severe male infertility but is often used even if the sperm quality parameters are normal. This practice has recently been called into question, but there is still no clear answer especially in terms of sperm morphology, regarding when it is necessary to perform ICSI and when conventional IVF is indeed more beneficial. In borderline cases it seems logical to fertilize oocytes using ICSI and conventional IVF at the same time. Since we also use this approach we performed a retrospective analysis of such cycles to elucidate, which procedure results in a better clinical outcome in terms of fertilization rate, the quality of day 3 and day 5 embryos, and the pregnancy rate. The data from fifty-one couples who were treated with ART and whose male factor of infertility was defined as teratozoospermia were included. The fertilization rates were similar between ICSI and conventional IVF groups (per COCs: 54.5% vs. 58.2%, P = 0.322; per MII oocytes: 63.9% vs. 67.2%; P = 0.399), but more oocytes degenerated after ICSI (11.7% vs. 4.3%; P = 0.0003). The quality of cleaved embryos was similar between the groups, but more embryos reached the blastocyst stage after conventional IVF (43.7% vs. 55.0%; P = 0.032) and furthermore, more of them were of good quality (19.8% vs. 29.2%; P = 0.037). The pregnancy rate did not significantly differ between the groups (21.4% vs. 45.5%; P = 0.175), although there was a trend in favor of conventional IVF. This retrospective analysis suggests that when sperm morphology is not severely impaired and sperm concentration and motility are normal, it is better to use conventional IVF to fertilize oocytes and not ICSI. The main advantage of conventional IVF is reflected in improved blastocyst rate and quality.

The present study aimed to determine clinical IVF parameters and gene expression in cumulus cells (CCs) in obese and normal weighting women after administration of 150 mcg of corifollitropin alfa for controlled ovarian hyperstimulation (COH). 150 mcg of corifollitropin alfa and gonadotropin releasing hormone antagonist were used for COH. Analysis of CC gene expression was performed using quantitative real-time PCR. We did not find significant differences in biochemical and clinical pregnancy rates between obese and normal weighting women. Obese women required twice as much of additional gonadotropins for ovarian stimulation and had a significantly lower proportion of good quality embryos on day 5 of IVF cycle. Expression of PGR and PTX3 was significantly higher in CCs of obese women. Obese women require significantly larger amounts of gonadotropins to achieve similar IVF success rates as normal weighting women. Differences in CC gene expression and smaller proportion of good quality embryos may imply that oocytes derived from obese women are of lower quality. Further studies are needed to evaluate whether obesity itself or the higher amount of gonadotropins used in obese women causes this effect.

Objective Embryo culture media are important factors in IVF, which can significantly influence clinical outcome of IVF/ICSI cycles. Despite this, it is still not completely clear which formulation is most optimal and whether sequential or continuous media should be favored. Materials and methods This study retrospectively analyzed the outcome of IVF/ICSI cycles with regard to different types of culture media used to culture embryos, namely sequential and two types of single step continuous embryo culture media. Results If the data were combined for both types of single step continuous embryo culture media the only significant difference we observed was the proportion of poor quality embryos on day 3, which was significantly higher (16.9% vs. 22.5%; P = 0.017) in the sequential media. The pregnancy (55.1% vs. 40.5%; P = 0.113) and live birth rates (42.9% vs. 33.8%; P = 0.308) were lower in continuous media, although the difference was not statistically significant. Furthermore, the blastocyst rate (sequential vs. continuous; 47.4% vs. 47.3%; P = 1), and birthweight (3280 ± 630g vs. 3272 ± 575g; P = 0.96) did not significantly differ regardless of the medium used to culture embryos. Additional comparison of each type of continuous medium to sequential media revealed that the difference in the quality of cleavage stage embryos for combined data of both continuous culture media may be derived from the group of cycles were SAGE 1-Step was used to culture embryos. Conclusion These results therefore indicate that continuous media can be equivalent to sequential media and could help lower the workload in busy IVF labs without impairing the clinical results. Although, caution is needed because this study is limited by its retrospective design. To confirm the results, especially in terms of live birth rates and perinatal outcome, a prospective study is needed with a higher number of included couples.

Research question Does the site of semen collection influence IVF/intracytoplasmic sperm injection (ICSI) cycle outcome? Design Retrospective study performed at the University Medical Centre Ljubljana, including all stimulated and modified natural IVF/ICSI cycles (with at least one oocyte retrieved) performed in 2019 with fresh ejaculated semen samples. IVF/ICSI cycle outcomes, in terms of oocytes, embryos and pregnancy rates according to site of semen sample collection (at home or at clinic) were evaluated. Results Samples collected at clinic had significantly lower sperm concentration (median [interquartile range, IQR], 50 [20–100] million/ml versus 70 [30–100] million/ml, adjusted odds ratio [OR] 0.001, 95% confidence interval [CI] 1.574 × 10–6 to 0.196, P = 0.012) and motility (60 [50–70]% versus 70 [50–70]%, adjusted OR 0.034, 95% CI 0.002 to 0.563, P = 0.018, adjusted for age). There was no difference in total sperm count, semen volume or sperm morphology, or women's age (36 [32–39] versus 36 [33–39] years) and men's age (37 [34–41] versus 38 [34–42] years), between semen samples collected at clinic versus at home. When all IVF/ICSI cycles were analysed together using generalized estimating equation analysis, no significant difference in cycle outcomes attributed to site of semen sample collection was observed. There were also no significant differences in cycle outcomes when only first cycles were analysed. Conclusions Collecting semen samples at home has a positive effect on sperm quality (sperm concentration and motility were higher), but no significant differences in cycle outcomes are observed when these samples are used in IVF/ICSI cycles. Therefore, it is suggested that collecting semen samples at home for IVF/ICSI procedures is safe and has no negative effect on treatment outcomes.

Background Acute myeloid leukemia (AML) in monozygotic twins is a rare event and, until now, only a few cases have been reported. Due to improved oncological treatment and cancer survival rates, quality of life considerations post-treatment have become an important aspect in the treatment regime. The ability to have their own biological children is considered one of the most important indicators of quality of life by cancer survivors. As fertility following oncological treatment is often impaired, fertility preservation methods should be offered to these patients prior to the treatment. Here, we present a very rare case in which we can in vivo observe the impact of oncological treatment on female fertility when applied before and after puberty. Case presentation This is a very rare case of concordant AML in monozygotic twin sisters. Twin A became sick at the age of 21 months. She was treated with cytostatic medications and then underwent bone marrow transplantation (BMT), the donor being her twin sister B. After 27 years, she is disease free and has regular periods. After trying to conceive for 4 years, she was seen by an infertility specialist. She underwent hysteroscopic uterine septum removal and laparoscopic enucleation of bilateral paraovarian cysts. Following those procedures, she immediately conceived naturally. Twin B became sick at 15 years of age. She was treated with chemotherapy and cranial radiation and relapsed after 10 years. She then received chemotherapy and had a BMT. Until relapse, she had normal menstrual cycles. After the second treatment she became amenorrhoeic and is now part of an oocyte donation program. Conclusions This is a case of AML in monozygotic twins who, after treatment, have completely different reproductive potential. They both received oncological treatment, and one of them conceived conceived naturally while the other suffered premature ovarian failure and is not able to have a biological child. Based on the outcome of this case, it appears that the pre-pubertal state truly serves as protection against ovarian failure.

With the development of IVF procedures, the role of reproductive surgery in the management of infertile couples has been questioned. Pregnancy rates (PR) after IVF procedures are well known, but recent data on spontaneous PR after reproductive surgery are scarce. This study aimed to prospectively evaluate how often fertility is restored by reproductive surgery and to identify which independent factors influence spontaneous pregnancy after reproductive surgery. Eight hundred eighty-eight infertile women who underwent surgery for infertility were prospectively included. Women who were referred to IVF after surgery, ceased to plan pregnancy and were lost to follow-up were excluded. Spontaneous PR was analysed for 519 women. A total of 252 (48.6%) women, including 30 treated with clomiphene citrate, conceived spontaneously in the 12–18 months observation period following surgery. Multivariate logistic regression showed that woman's age (OR 0.95, 95% CI 0.90–0.99) and duration of infertility (OR 0.86, 95% CI 0.74–0.99) significantly influence spontaneous PR. Each year of infertility lowers spontaneous PR following surgery by 14% and each year of woman's age by 5%. The study shows a relatively high percentage of women conceived spontaneously after reproductive surgery. The role of reproductive surgery in the management of infertility should be re-evaluated.

Aspirated follicular cells (AFCs) from the in vitro fertilization program can express various stem cell markers and are even able to differentiate into different types of cells in vitro. The female reproductive potential decreases with increasing age due to lowered ovarian reserve and oocyte quality, but data on the effect of female age on stem cell characteristics of AFCs are scarce. Therefore, the aim of this study was to elucidate whether female age affects the mesenchymal stem cell (MSC) characteristics of AFCs. Follicular aspirates were collected from 12 patients included in the in vitro fertilization programme with a normal ovarian reserve. Patients were divided into four age groups: Group A ≤ 30 years, Group B 31–35 years, Group C 36–39 years and Group D ≥ 40 years. After removal of the oocytes, AFCs were collected from follicular aspirates using hypo-osmotic technique and cultured in vitro, and their stemness was compared according to female age. The cultured AFCs were analysed for gene expression using the Human Mesenchymal Stem Cell RT2 Profiler™ PCR Array, for their potential for differentiation into adipogenic and osteogenic lineage, and for their expression of MSC-related markers using immunocytochemistry. We found that female age can significantly influence their stemness: expression of pluripotency and MSC-related genes, and their differentiation potential. Despite the relatively high expression of MSC-related genes, the AFCs of the oldest patients had the lowest potential to differentiate into osteogenic and adipogenic lineages in vitro, which may be related to their age and the changed ovarian function.

The aim of our study was to explore the risk factors for very preterm (gestation under 32 weeks) and moderate preterm birth (gestation weeks 32-36 6/7) in singleton pregnancies in a national retrospective cohort study. We also wanted to establish whether IVF/ICSI is an independent risk factor for preterm birth after adjusting for already known confounders. We used data for 267 718 singleton births from 2002-2015 from the National Perinatal Information System of Slovenia, containing data on woman, pregnancy, birth, the postpartum period, and the neonate for each mother-infant pair. Mode of conception, maternal age, education, BMI, parity, smoking, history of cervical excision procedure, history of hysteroscopic resection of uterine septum, presence of other congenital uterine malformations, bleeding in pregnancy, preeclampsia or HELLP and maternal heart, and pulmonary or renal illness were included in the analyses. Unadjusted OR for very preterm birth after IVF-ICSI [corrected] was 2.8 and for moderate preterm birth was 1.7. After adjusting for known confounders, the OR was still significantly elevated (1.6 and 1.3, respectively). Risk factors for very preterm birth with OR higher than 2.4 were history of cervical excision procedure, resection of uterine septum, operation or having other congenital uterine malformations, and bleeding in pregnancy. Risk factors for very preterm birth with OR between 1.4 and 2.1 were age35 years, being underweight or obese, not having professional education, smoking, first birth, preeclampsia/HELLP, and IVF/ICSI. Risk factors for moderate preterm birth with OR higher than 2.4 were history of cold knife conization and other congenital uterine malformations. We found that even after adjustment, IVF/ICSI represents a single risk factor for early and late preterm birth even after adjustment with other risks such as maternal age, smoking, or a history of invasive procedures for either cervical intraepithelial neoplasia or infertility treatment.

Objectives Our aim was to explore the association between cold-knife conisation and large loop excision of transformation zone (LLETZ) with spontaneous preterm birth in a large 10-year national sample. We wanted to explore further the association of these procedures with preterm birth according to gestation. Study design We conducted a population based retrospective cohort study, using data from national Medical Birth Registry. The study population consisted of all women giving birth to singletons in the period 2003–2012 in Slovenia, excluding all induced labors and elective cesarean sections before 37 weeks of gestation ( N = 192 730). We compared the prevalence of spontaneous preterm births (before 28 weeks, before 32 weeks, before 34 weeks and before 37 weeks of gestation) in women with cold-knife conisation or LLETZ compared to women without history of conisation, calculating odds ratios (OR), adjusted for potential confounders. Chi-square test was used for descriptive analysis. Logistic regression analyses were performed to estimate crude odds ratio (OR) and adjusted odds ratio (aOR) and their 95% confidence intervals (95% CI) with two-sided probability ( p ) values. Results A total of 8420 (4.4%) women had a preterm birth before 37 weeks of gestation, 2250 (1.2%) before 34 weeks of gestation, 1333 (0.7%) before 32 weeks of gestation and 603 (0.3%) before 28 weeks of gestation. A total of 4580 (2.4%) women had some type of conisation in their medical history: 2083 (1.1%) had cold-knife conisation and 2498 (1.3%) had LLETZ. In women with history of cold-knife conisation, the adjusted OR for preterm birth before 37 weeks of gestation was 3.13 (95% CI; 2.74–3.57) and for preterm birth before 28 weeks of gestation 5.96 (95% CI; 4.3–8.3). In women with history of LLETZ, the adjusted OR was 1.95 (95% CI; 1.68–2.25) and 2.88 (95% CI; 1.87–4.43), respectively. Conclusions Women with cervical excision procedure of any kind have significantly increased odds for preterm birth, especially for preterm birth before 28 weeks and before 32 weeks of gestation.

Earlier data suggest a relationship between PIBF concentrations and the outcome of pregnancy. The aim of the study was to compare serum and urine concentrations of PIBF in women with successful pregnancy after IVF with those of women without pregnancy after IVF procedure, and to evaluate the potential relation between PIBF and the outcome of pregnancy. Urine and serum were collected from 120 women, undergoing IVF. 87.5% of patients had primary infertility. 69.2% faced female causes of infertility: 10.8% tubal cause, 11.7% ovulation disorder, and 46.7% other causes of infertility. 30.8% of patients had male factor of infertility. Among non-pregnant women (42) mean concentrations of PIBF in urine and serum were significantly lower (15.8 ng/mL; 148.4 ng/mL) than in women with positive beta HCG value (78) (19.1 ng/mL; 225.9 ng/mL). In 49 patients pregnancy terminated with a term delivery, in 10 patients with pretem delivery, while in 19 patients the pregnancy terminated with a miscarriage. PIBF concentrations in urine (13.9 ± 2.8 ng/mL) and serum (124.6 ± 46.7 ng/mL) samples of women with miscarriage were significantly lower of those with preterm delivery (180.6 ± 54.4 ng/mL; 18.1 ± 4.4 ng/mL) and of those with term delivery (20.4 ± 8.5 ng/mL; 208.7 ± 114.3 ng/mL). Successful pregnancy after IVF procedure is predictable by measuring of urine and serum PIBF concentrations and could be important for predicting of early implantation and pregnancy outcome after IVF procedure and maybe to protect the risk pregnancy.

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.

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. J. Med. Virol. 79:431–438, 2007. © 2007 Wiley-Liss, Inc.

Background Testing cervical smears for the presence of high-risk human papillomaviruses (hrHPV) increases the sensitivity for detecting women with underlying high-grade cervical intraepithelial neoplasia (CIN) and provides better and longer protection against invasive cervical cancer compared to cytology testing alone. The Abbott RealTime High Risk HPV test (RealTime) is a hrHPV DNA test with concurrent partial genotyping for HPV16 and HPV18 and aggregate detection of 12 other hrHPV types that have been extensively analytically and clinically evaluated over the last 6 years. Objectives and study design To provide the first 3-year longitudinal data regarding the clinical performance of RealTime, the risk of CIN2+ according to various negative baseline characteristics, and baseline and future risk for CIN2+ at 3 years for women with baseline infection with various hrHPV types were assessed in a cohort of 3,920 Slovenian women that had hrHPV DNA and/or cytology in 36- to 48-month follow-up results after a baseline screening round in 2009/2010. Results A total of 36 CIN2+ cases were identified in the second screening round. Of these, 17 CIN2+ cases were identified passively through questionnaires/data registries and 19 cases actively as the result of actions triggered by second-round cytology and/or HPV test results. Accumulation of CIN2+ cases during follow-up occurred predominantly in woman with normal cytology at baseline. Among women >30 years old, significantly better protection against CIN2+ at 3 years was associated with a negative hrHPV DNA result at baseline (risk for CIN2+ 0.04% [95 CI, 0.00–0.22%]) than by normal cytology at baseline (risk for CIN2+ 0.68% [95 CI, 0.40–1.08%]). Women with baseline HPV16 infection had a significantly higher risk of CIN2+ at baseline (21.9% [95 CI, 15.2–30.4%]) and baseline plus future risk at 3 years for CIN2+ (33.3% [95 CI, 24.7–44.0%]) in comparison to women with baseline non-HPV16/18 hrHPV infection (7.0% [95 CI, 4.6–10.2%]) or those that were hrHPV-positive (11.7% [95 CI, 9.1–14.9%]). Conclusions 3-year longitudinal data reinforce evidence from previous studies that RealTime can be safely used in primary HPV-based cervical cancer screening. Concurrent partial genotyping for HPV16/18 should be strongly considered as a triage method for HPV screen-positive women.

High-risk HPV, particularly HPV-16, is etiologically associated with the development of cervical cancer and its precursor lesions - cervical intraepithelial neoplasia (CIN). However, most precancerous lesions will not progress to cancer. Numerous studies have shown that HPV-16 consists of several genomic variants, which differ in their association with cervical cancer, viral persistence and the frequency of recurrence of cervical disease. Recently, a novel, presumably less pathogenic, HPV-16 E6-T350G genomic variant has been identified, carrying a 63-bp in-frame insertion in the E1 gene. No data from Slovenian patients have so far been reported for this specific HPV-16 variant. In the present study, therefore, a total of 390 HPV-16 positive samples obtained from the same number of women with normal cytology, CIN I, CIN II, CIN III or cervical cancer, were analyzed. The HPV-16 E1 insert variant was detected using real-time PCR-amplification of a 146-210-bp fragment of the E1 gene and PCR-sequencing of a 169-bp fragment of the E6 gene. The HPV-16 E1 insert variant was identified in 7/48 (14.6%), 1/21 (4.8%), 2/20 (10.0%), 9/131 (6.9%) and 12/170 (7.1%) of women with normal cytology, CIN I, CIN II, CIN III and cervical cancer, respectively. All HPV-16 E1 insert variants with an amplifiable E6 gene belonged to the European HPV-16 E6-350G variant group. No statistically significant differences in the prevalence of HPV-16 E1 insert genomic variant in women presenting with normal cytology and those with the different stages of HPV-16-induced disease were found.

Our aim was to analyze genomic variants of the three most common human papillomavirus (HPV) genotypes found in Slovenian women with cervical cancer (CC).A total of 40 isolates of HPV 16, 20 isolates of HPV 18 and 11 isolates of HPV 33 were included in the study. The genomic diversity of HPV 16, HPV 18 and HPV 33 isolates was investigated within the long control region (LCR), and E6 and E7 genomic regions using direct polymerase chain reaction-sequencing.A total of 26 genomic variants of HPV 16, consisting of 22 LCR, 10 E6 and 5 E7 variants were identified. Thirty-eight (95%) HPV 16 isolates belonged to the European branch, one (2.5%) to the African 2 branch and one (2.5%) to the Asian-American branch. A total of 18 genomic variants of HPV 18 consisting of 18 LCR, two E6 and four E7 variants were identified: 19 (95%) HPV 18 isolates belonged to the European branch and one (5%) to the African branch. A total of seven genomic variants of HPV 33 consisting of seven LCR, two E6 and three E7 variants were identified: five (45.5%) HPV 33 isolates belonged to prototypic and six (54.5%) to non-prototypic HPV 33 genomic variants.The majority of HPV 16 and HPV 18 isolates from Slovenian patients with CC analyzed in this study belonged to European branches. Prototypic and non-prototypic HPV 33 genomic variants were equally distributed among Slovenian patients with CC. Several novel mutations were identified in all three genotypes examined.

This study compared serum and follicular anti-mullerian hormone, LH, FSH, E 2 , and P concentrations in modified natural cycles and controlled ovarian hyperstimulation cycles to analyze their effect on oocyte and embryo quality and implantation. Follicular anti-mullerian hormone, FSH, E 2 , and P concentrations had no effect on embryo quality, whereas high serum and follicular LH concentrations in natural cycles modified only with hCG did not compromise oocyte and embryo quality but might have a negative effect on implantation.

Objective: The purpose of the present study was to establish the distribution of human papillomavirus (HPV) genotypes in a representative population of women with cervical cancer in Slovenia in order to contribute to the lacking data on HPV in cervical cancer and to assess the potential local benefit of future prophylactic HPV vaccination. Study design: A total of 284 samples of cervical cancer were analyzed including archival samples, cervical scrapes and fresh tissue samples. Polymerase chain reaction with GP5+/GP6+ primers was performed in all samples for HPV deoxyribonucleic acid (DNA) detection. All GP5+/GP6+ negative samples were additionally tested using CPI/CPIIg primers and INNO-LiPA HPV genotyping assay. Results: After exclusion of 6 samples with unsuccessful amplification of beta-globin gene, 262 of 278 cervical cancer samples (94.2%) were HPV DNA positive. HPV genotypes found in the decreasing order of frequency were: HPV 16 (64.9%), HPV 18 (12.2%), HPV 33 (4.7%), HPV 45 (4.1%), followed by HPV 31, 51, 58, 59, 35, 52, 73 and 82 (3.5–0.2%). HPV positive samples were more frequent among squamous cell carcinomas than among adenocarcinomas/adenosquamous carcinomas (95.8% versus 85.5%; P = 0.003). HPV 16 was more frequently found in squamous cell carcinomas than in adenocarcinomas/ adenosquamous carcinomas (69.9% versus 37.5%; P < 0.001), while the opposite was true for HPV 18 (6% versus 41.7%; P < 0.001). Conclusion: Prophylactic HPV vaccination with currently available vaccines could prevent up to 77.1% of cervical cancer in Slovenia, which is caused by HPV 16 or HPV 18.

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, P0.001). 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.

The aim was to establish the impact of human granulosa cell apoptosis and reactive oxygen species (ROS) production on fertilization competence of the oocyte, embryo developmental stage and implantation rate.Thirty women undergoing IVF-ET for tubal factor infertility were included; GnRH antagonists and gonadotrophins were used for ovarian stimulation. Granulosa cells were isolated from each aspirated follicle using gradient centrifugation. Apoptosis was studied by flow cytometry using annexin V and propidium iodide. ROS production was studied with hydroethidine staining and analyzed by flow cytometry.There were no differences in characteristics of granulosa cells between the follicles with fertilized and non-fertilized oocytes. The analyzed characteristics of granulosa cells in corresponding follicles had no effect on embryo developmental stage on day 5. The percentage of ROS producing granulosa cells was lower in the follicles giving rise to blastocysts that resulted in implantation compared to those that did not (39.9% versus 69.9%, P = 0.031).Apoptosis and ROS production in granulosa cells have no significant impact on fertilization and do not correlate with the development of blastocysts. An increased percentage of ROS producing granulosa cells results in fewer oocytes retrieved and diminishes implantation rate.