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Study question How does significant increase of the proportion of single embryo transfer over 10 years period affect pregnancy, live birth and twin rate. Summary answer Increase in single embryo transfer doesn’t change pregnancy and live birth rate, although it significantly lowers twin rate. What is known already Due to widely used approach in IVF of transferring multiple embryos to improve the pregnancy and birth rate, multiple pregnancies, mostly twin, are quite common. But because they are more often associated with adverse neonatal and perinatal outcomes as singleton pregnancies, they are not desirable. Therefore, more and more often the transfer of single embryo is encouraged. Furthermore, in the last years with improvements in cryopreservation techniques leading to effective cryopreservation of supernumerary embryos, there is more options for performing repeated single embryo transfers. Study design, size, duration We retrospectively collected the data of all fresh embryo transfers in couples treated in our centre from January 2010 to December 2019. We excluded embryo transfer where embryos were derived from cryopreserved oocytes and analysed the outcome of fresh embryo transfer regarding to the number of transferred embryos. Participants/materials, setting, methods In our analysis we included 10583 fresh embryo transfers. We tried to evaluate how the proportion of single embryo transfer has changed through analysed period of time and if this led to any differences in pregnancy, live birth, and twin rate. To determine the differences between the groups, the data were analysed with one-way ANOVA and Pearson’s chi-square, as appropriate. Statistical significance was set at P Main results and the role of chance The analysis revealed that the proportion of single embryo transfers significantly increased from year 2010 to year 2019 (from 28% to 73%; P Limitations, reasons for caution The limitation of the study is retrospective design, and not evaluating the influence of elective single embryo transfer. Also, the IVF laboratory methods and IVF culture media improved over the years meaning they could be partly responsible for observed differences. Wider implications of the findings: Single embryo transfer could probably be performed in even higher proportion without lowering the chances for pregnancy. Trial registration number not applicable’

Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged < 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

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 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 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 age >35 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.

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.

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.

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.

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.

We report a case of ureteral injury with delayed hematuria after transvaginal oocyte retrieval. A 28-year-old infertile patient with a history of previous laparoscopic resection of endometriotic nodes of both sacrouterine ligaments presented with abdominal pain one day after oocyte retrieval. Four days after oocyte retrieval, she presented with massive hematuria that reappeared 6 days after oocyte retrieval. Monopolar coagulation with wire electrode and insertion of a double-J-stent was performed during operative cystoscopy. The patient recovered completely after transfusion and had no signs of renal impairment after ureteric stent removal. This is the first report of ureteral injury after oocyte retrieval presenting itself with delayed massive hematuria and no signs of renal dysfunction or urinary leakage into retroperitoneal space.

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.

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.

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.