Preimplantation Genetic Testing within the Public Healthcare System in Slovenia.

Preimplantation genetic testing (PGT) is the earliest form of prenatal diagnosis that has become an established procedure for couples at risk of passing a severe genetic disease to their offspring. At UMC Ljubljana, we conducted a retrospective register-based study to present 15 years of PGT service within the public healthcare system in Slovenia. We collected the data of the PGT cycles from 2004 to 2019 and compared clinical outcomes for chromosomal and monogenic diseases using different embryo biopsy and testing approaches. In addition, we assessed the extent to which PGT has become the preferred option compared to classic prenatal diagnostics. We treated 211 couples, 110 with single gene disorder, 88 with structural chromosome rearrangement and 13 for numerical chromosome aberration. There were 375 PGT cycles with oocyte retrieval, while embryo transfer was possible in 263 cases resulting in 78 deliveries and 84 children. Altogether, the clinical pregnancy rate per embryo transfer was 31% in 2004-2016 (blastomere biopsy) and 43% in 2017-19 (blastocyst biopsy), respectively. We assessed that approximately a third of couples would opt for PGT, while the rest preferred natural conception with prenatal diagnosis. Our results show that providing a PGT service within the public healthcare system has become a considerable option in pregnancy planning for couples at risk of transmitting a severe genetic disease to their offspring. In Slovenia, approximately a third of couples would opt for PGT. Although the number of cycles is small, our clinical results are comparable to larger centres.

Proteomic analysis of peritoneal fluid identified COMP and TGFBI as new candidate biomarkers for endometriosis.

Endometriosis is a common non-malignant gynecological disease that significantly compromises fertility and quality of life of the majority of patients. The gold standard for diagnosis is visual inspection of the pelvic organs by surgical laparoscopy and there are no biomarkers that would allow non-invasive diagnosis. The pathogenesis of endometriosis is not completely understood, thus analysis of peritoneal fluid might contribute in this respect. Our prospective case-control study included 58 patients undergoing laparoscopy due to infertility, 32 patients with peritoneal endometriosis (cases) and 26 patients with unexplained primary infertility (controls). Discovery proteomics using antibody microarrays that covered 1360 proteins identified 16 proteins with different levels in cases versus the control patients. The validation using an ELISA approach confirmed significant differences in the levels of cartilage oligomeric matrix protein (COMP) and transforming growth factor-ß-induced protein ig-h3 (TGFBI) and nonsignificant differences in angiotensinogen (AGT). A classification model based on a linear support vector machine revealed AUC of > 0.83, sensitivity of 0.81 and specificity of 1.00. Differentially expressed proteins represent candidates for diagnostic and prognostic biomarkers or drug targets. Our findings have brought new knowledge that will be helpful in the understanding of the pathophysiology of endometriosis and warrant further studies in blood samples.

Is It Possible to Treat Infertility with Stem Cells?

Infertility is a major health problem, and despite improved treatments over the years, there are still some conditions that cannot be treated successfully using a conventional approach. Therefore, new options are being considered and one of them is cell therapy using stem cells. Stem cell treatments for infertility can be divided into two major groups, the first one being direct transplantation of stem cells or their paracrine factors into reproductive organs and the second one being in vitro differentiation into germ cells or gametes. In animal models, all of these approaches were able to improve the reproductive potential of tested animals, although in humans there is still too little evidence to suggest successful use. The reasons for lack of evidence are unavailability of proper material, the complexity of explored biological processes, and ethical considerations. Despite all of the above-mentioned hurdles, researchers were able to show that in women, it seems to be possible to improve some conditions, but in men, no similar clinically important improvement was achieved. To conclude, the data presented in this review suggest that the treatment of infertility with stem cells seems plausible, because some types of treatments have already been tested in humans, achieving live births, while others show great potential only in animal studies, for now.

Female Age Affects the Mesenchymal Stem Cell Characteristics of Aspirated Follicular Cells in the In Vitro Fertilization Programme.

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.

Vitamin D in human reproduction: the more, the better? An evidence-based critical appraisal.

OBJECTIVE: Vitamin D is a fat-soluble secosteroid hormone that regulates calcium, magnesium, and phosphate homeostasis and plays a pivotal role as antiproliferative and immunomodulatory mediator. Considering the different sources of synthesis and dietary intake as well as the pleiotropic actions in extremely diverse (micro)environments of the body, the supplementation of this Vitamin should be carefully evaluated taking into account the several pathways that it regulates. In the current brief review, we aimed to summarize the available evidence about the topic, in order to suggest the best evidence-based supplementation strategy for human reproduction, avoiding the unuseful (and sometimes hazardous) empiric supplementation. MATERIALS AND METHODS: Narrative overview, synthesizing the findings of literature retrieved from searches of computerized databases. RESULTS: Accumulating evidence from in vitro fertilization (IVF) trials suggests that fertilization rate decreases significantly with increasing levels of 25OH-D in follicular fluid; in addition, Vitamin D levels in the follicular fluid are negatively correlated to the quality of embryos and the higher values of Vitamin D are associated with lower possibility to achieve pregnancy. Both low and high Vitamin D serum concentrations decrease not only spermatozoa count, but their progressive motility as well as increase morphological abnormalities. Finally, studies in animal models found that severe hypervitaminosis D can reduce the total skeletal calcium store in embryos and may compromise the postnatal survival. CONCLUSIONS: Based on the retrieved data, we solicit to be extremely selective in deciding for Vitamin D supplementation, since its excess may play a detrimental role in fertility.

Embryo quality predictive models based on cumulus cells gene expression.

Since the introduction of in vitro fertilization (IVF) in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC) have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR)] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC) for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

Septate, subseptate and arcuate uterus decrease pregnancy and live birth rates in IVF/ICSI.

A retrospective matched-control study to evaluate the effect of uterine anomalies on pregnancy rates after 2481 embryo transfers in conventionally stimulated IVF/intracytoplasmic sperm injection (ICSI) cycles. The study group of 289 embryo transfers before and 538 embryo transfers following hysteroscopic resection of a uterine septum was compared with two consecutive embryo transfers in the control group. Groups were matched for age, body mass index, ovarian stimulation, embryo quality, IVF or ICSI and infertility aetiologies. Number of embryos transferred, embryo quality and absence of uterine anomalies significantly predicted the pregnancy rates in the study group: odds ratios (OR) 1.7, 2.6 and 2.5, respectively (P<0.001). Pregnancy rates after embryo transfer before hysteroscopic metroplasty were significantly lower, both in women with subseptate and septate uterus and in women with arcuate uterus compared with controls. If two or three embryos with at least one best-quality embryo were transferred, the differences were 9.6% versus 43.6%, OR 7.3 (P<0.001) and 20.9% versus 35.5%, OR 2.1 (P<0.03), respectively. Differences in terms of live birth rates were even more evident: 1.9% versus 38.6%, OR 32 (P<0.001) and 3.0% versus 30.4%, OR 14 (P<0.001). After surgery, the differences disappeared. This retrospective matched control study evaluated the influence of septate, subseptate and arcuate uterus on pregnancy and live birth rates after 2481 in conventionally stimulated IVF/intracytoplasmic sperm injection (ICSI) cycles. The study group included 827 embryo transfers (289 embryo transfers before and 538 embryo transfers following hysteroscopic resection of uterine septum ans was compared with two consecutive mebryo transfers in the control group. Both groups were matched by age, body mass index, stimulation protocol, quality of embryos, use of IVF or ICSI, and infertility aetiologies. Multivariate logistic regression analysis of the study group showed that the number of embryos, embryo quality and the absence of uterine anomalies significantly predicted the pregnancy rates: odds ratios (OR) 1.7, 2.6, and 2.5, respectively (P<0.001). The pregnancy and live birth rates before surgery were lower compared with controls, both in women with subseptate or septate uterus and in women with arcuate uterus. If two or three embryos with at least one best quality embryo were transferred, the differences in terms of pregnancy rates were 9.6% versus 43.6%, OR=7.3 (P<0.001) and 20.9% versus 35.5%, OR=2.1 (P<0.03), respectively. The differences in terms of live birth rates were even more evident: 1.9% versus 38.6%, OR=32 (P<0.001) and 3.0% versus 30.4%, OR=14 (P<0.001). After surgery, the differences disappeared. Negative impact of uterine anomalies on pregnancy and on live birth rates are two important arguments for treating uterine anomalies in infertile women.

Pre-vaccination distribution of human papillomavirus (HPV) genotypes in women with cervical intraepithelial neoplasia grade 3 (CIN 3) lesions in Slovenia.

BACKGROUND: High-risk human papillomaviruses (HPV) are the main etiological factor of cervical cancer. Cervical intraepithelial neoplasia grade 3 (CIN 3) is the latest pre-invasive stage of cervical cancer, with an approximately 20% progression rate to invasive cervical carcinoma. OBJECTIVE: To establish the pre-vaccination distribution of HPV genotypes in Slovenian women with CIN 3 lesions, in order to assess the potential benefit of prophylactic HPV vaccination in Slovenia, and to provide baseline data for monitoring the potential replacement of HPV genotypes under selective pressure of HPV vaccines. METHODS AND RESULTS: A total of 261 cervical swabs collected from women with histologically confirmed CIN 3 lesions were analyzed using four genotyping methods: the Abbott RealTime High Risk HPV Assay, the Innogenetics INNO-LiPA HPV Genotyping Extra Test, and the in-house PGMY09/11, and CPI/CPIIg polymerase chain reaction (PCR) and sequencing. Of 261 samples, 253 (96.9%) were HPV positive. The most common HPV genotype in CIN 3 lesions in the Slovenian samples was HPV-16 (59.0%), followed by HPV-31 (7.5%), HPV-33 (7.1%), HPV-58 (5.0%), and HPV-51 (4.0%). The presence of more than one HPV genotype was detected in 49/253 (19.4%) samples. Together, HPV-16 and HPV- 18 accounted for 67.4% of CIN 3 lesions in this Slovenian population. CONCLUSION: The high proportion of CIN 3 lesions caused by HPV-16 and HPV-18 should further support the recent decision to include the prophylactic vaccination against HPV in the national vaccination program in Slovenia.

Efficiency of three surgical procedures in eliminating high-risk human papillomavirus infection in women with precancerous cervical lesions.

PURPOSE OF INVESTIGATION: To establish the efficiency of laser vaporization (LV), large loop excision of the transformation zone (LLETZ) and cold knife conization, done for precancerous cervical lesions, in eliminating high-risk human papillomavirus (HPV) infection. Additionally, we determined whether the same HPV genotype persisted after surgery. METHODS: A total of 214 women were tested for HPV infection by the Hybrid Capture II (HCII) test prior to surgery. HPV-positive women were followed by HCII test ten months after surgery. In persistently HPV-positive women, HPV genotypes were determined by PCR - PGMY09/PGMY11. RESULTS: The HCII test showed elimination of HPV infection after LV, LLETZ and cold knife conization in 67.6%, 86.3%, and 100% (p < 0.05) of women, respectively. In seven (38.9%) women a different HPV genotype was found to be present after surgery, the corrected efficiency thus being 79.4%, 92.7% and 100% (p = NS), respectively. CONCLUSIONS: The three analyzed surgical procedures are effective in eliminating high-risk HPV infection. HPV testing is useful at follow-up, since it can identify a small proportion of women requiring close surveillance and potential treatment.

Role of human papillomavirus testing in reducing the number of surgical treatments for precancerous cervical lesions.

PURPOSE OF INVESTIGATION: To determine whether the addition of the Hybrid Capture II (HC II) test (Digene Corp., Gaithersburg, MD, USA) to cytological, colposcopical and histological results could reduce the number of surgical treatment procedures for precancerous cervical lesions. METHODS: Surgical treatment of precancerous cervical lesions was performed in 181 women. Priorly, the women were tested for high-risk human papillomavirus (HPV). Sensitivity, specificity, positive and negative predictive value were calculated to assess the performance characteristics of HC II in the detection of cervical intraepithelial neoplasia grade 2 or worse (CIN 2+) and grade 3 or worse (CIN 3+). RESULTS: Eighty (44.2%) women had a histological result < CIN 2; 117 (64.6%) women had < CIN 3. Fifty-three (29.3%) women with < CIN 2 tested HPV negative; 69 (38.1%) women with < CIN 3 tested HPV negative (p < 0.05). The sensitivity of HC II for detecting CIN 2+ and CIN 3+ was 76.2% and 87.5%, respectively. CONCLUSION: A high proportion of women were overtreated probably due to cytological and histological overestimations. HPV testing would reduce the number of unnecessary surgical treatments and should be used as an additional screening tool.

Human papillomavirus infection in relation to mild dyskaryosis in conventional cervical cytology.

PURPOSE OF INVESTIGATION: To establish the prevalence and distribution of high-risk human papillomavirus (HPV) genotypes in Slovene women with repeat mild dyskaryosis, and to evaluate three molecular methods for the detection of HPV that could be used as a complementary method to cervical cytology. METHODS: In this prospective study 148 women with three subsequent cervical cytologic tests within two years showing mild dyskaryosis were enrolled. HPV infection was determined using three molecular tests: Hybrid Capture II and two variants of polymerase chain reaction (PCR-PGMY11/PGMY09 and PCR-CPI/CPIIG). RESULTS: HPV was detected in 17 of the 45 women aged < or =30 years and in 21 of the 103 women aged >30 years (37.8% vs 20.4%, p = 0.04). The most common genotype was HPV 16 detected in eight (21.1%) women, the next were HPV 53 and HPV 51, each detected in five (13.2 %) women. The three molecular methods matched in 92.9%. CONCLUSION: Low prevalence of HPV infections indicates that cervical screening programmes in Slovenia are overburdened with mild dyskaryosis. Repeat cytology is not reliable; HPV testing might be useful as a complementary method.

Retrospective and prospective evaluation of the Amplicor HPV test for detection of 13 high-risk human papillomavirus genotypes on 862 clinical samples.

BACKGROUND: Persistent infection with a subgroup of at least 15 high-risk human papillomavirus (HPV) genotypes is considered as a necessary although insufficient etiological factor in the development of cervical carcinoma. As a consequence, HPV testing has recently become an important part of the cervical carcinoma screening and detection algorithms. AIM OF THE STUDY: To evaluate the analytical performance of a recently developed polymerase chain reaction (PCR)-based Amplicor HPV test (Roche Molecular Systems) in comparison with the Hybrid Capture 2 HPV DNA test (hc2) (Digene Corporation) for the detection of 13 high-risk HPV genotypes. Inhouse consensus PGMY09/PGMY11 and CPI/IIg PCRs targeting two different HPV genes coupled with HPV genotyping were used as an HPV internal reference standard. MATERIALS AND METHODS: In the retrospective evaluation, 550 cervical scrape specimens with previously established HPV status were included. Additionally, 312 cervical scrape specimens were tested prospectively for the presence of 13 high-risk HPV genotypes by both hc2 and Amplicor HPV test. RESULTS: In the retrospective evaluation, the Amplicor HPV test results agreed almost completely with the HPV internal reference standard results. In the prospective evaluation performed on 312 samples, the concordant Amplicor and hc2 results were obtained in 85.9% of samples tested. CONCLUSION: In our hands, the Amplicor HPV test demonstrated high analytical sensitivity and specificity. The higher analytical specificity of Amplicor in comparison to that of hc2 can be considered clinically useful. Prospective studies with clinical endpoints are urgently needed to assess the clinical utility of the higher analytical sensitivity of the Amplicor HPV test for primary HPV screening and triaging patients with ASC-US.

Utero-ovarian arterial blood flow and hormonal profile in patients with polycystic ovary syndrome.

Utero-ovarian arterial blood flow and hormonal profile were evaluated in 20 patients with polycystic ovary syndrome (PCOS) and in 22 patients with normal menstrual cycle (NMC) undergoing in-vitro fertilization (IVF) treatment. In this controlled prospective clinical study the vascular impedance in the utero-ovarian arteries was measured in both groups on days 4, 13, 22 [introduction of gonadotrophin releasing hormone agonist (GnRHa) administration], 36 (after the 14 day GnRHa administration) and on the day of human chorionic gonadotrophin (HCG) administration. Simultaneously, serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol, androstendione and sex hormone-binding globulin (SHBG) were determined. In the PCOS group significantly (P < 0.05) higher concentrations of FSH and LH were found on day 22, whereas the excess of androstendione production and lower SHBG values were registered throughout the studied cycles. Also, no effect of the 14 day GnRHa administration on the androstendione discharge was registered. In both groups the serum LH concentration decreased significantly (P < 0.05); however, the value remained significantly (P < 0.05) higher in the PCOS group. In the PCOS group no changes regarding the vascular impedance in the uterine artery were registered in the phase of the cycle preceding the GnRHa administration. The resistance index (RI) on day 22 of the cycle was 0.86 in the PCOS and 0.82 in the NMC group. In the NMC group the active ovary showed dramatic changes during the cycle, with vascular impedance almost constant throughout the cycle in the inactive ovary. In the PCOS group the vascular impedance to ovarian arterial blood flow in both ovaries was similar to that in inactive ovaries in the NMC group. After the 14 day GnRHa administration the utero-ovarian vascular impedance was high in both groups with changes registered only in the NMC group, as the PCOS patients exhibited maximal vascular impedance throughout the cycle. On the day of HCG administration strong negative correlation between the vascular impedance in the uterine artery and the serum oestradiol concentration in stimulated cycles was found in both groups, but not in anovulatory PCOS patients. As in PCOS patients the utero-ovarian vascular impedance was raised throughout the cycle we concluded that the 14 day GnRHa administration neither affected the utero-ovarian arterial blood flow nor suppressed the androstendione discharge.

Short-term GnRH-analogue administration in patients with PCOS and its impact on hemodynamics and hormonal profile.

PURPOSE: The ovarian response to 14 days of GnRH-analogue (GnRHa) application in patients with polycystic ovary syndrome (PCOS) was evaluated by measuring hemodynamic parameters and hormonal profile. METHODS: Twenty patients with PCOS represented the study group, and 22 patients with normal menstrual cycle (NMC) the reference group. The resistance index (RI) of uteroovarian arterial blood flow and serum level of LH and androstendione were measured on days 4, 13, and 22 of the cycle and after 14 days of GnRHa administration. RESULTS: On day 22 of the cycle the RI in the uterine artery was 0.86 in the PCOS and 0.82 in the NMC group. Throughout the cycle the ovarian arterial blood flow in both ovaries in the PCOS group was similar to that in inactive ovaries in the NMC group. After the 14-day GnRHa administration the RI of uteroovarian blood flow and the serum level of androstenedione remained unchanged in the PCOS group. CONCLUSIONS: RI in the uteroovarian blood flow and serum androstendione level in the PCOS group are not affected by 14-day GnRHa administration.

[Comparison of prostaglandin E2 and oxytocin infusions for induction of labor in primiparas at term with an unripe cervix and premature rupture of fetal membranes]. / Usporedba prostaglandina E2 i oksitocina u infuziji za indukciju porodaja u prvorotkinja u terminu sa nezrelim cerviksom i preranim prsnucem vodenjaka.

The study aimed at examining the effectiveness of labor induction in term pregnant nulliparas with the premature rupture of the membranes (PRM) and unriped cervix (Bishop less than 6). Each group contained 17 nulliparas. In the first group the labor was induced with the prostaglandin E2 (PgE2) infusion, in the second group the oxytocin infusion was used. The results of both groups were compared. PgE2 was found to be effective in labor induction; the rate of caesarean sections was 18.75% in the first group and 29.41% in the second group. The PgE2 drug was found to be safe for the fetus and also well tolerated by pregnant women. The effect of the drug on the ripening of the cervix has not been noticed. The time interval from the induction of labor to the delivery is equal in both groups. However, in the first group significantly less work was done by the uterus at the same interval. No incidence of uterine hyperactivity was recorded. All newborns in the first group were in good condition: in the second group two cases of fetal distress were recorded. The pregnancy outcome shows that the PgE2 drug is superior to oxytocin for labor induction in term pregnant nulliparas with the PRM and unriped cervix.