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The aim is to present a single-center case series of patients with symptomatic hyperpronated feet treated with arthroereisis by using a second generation extra-osseous talo-tarsal stabilization device. This case series enrolled 123 feet in 87 patients (20 [6-75] years) treated with arthroereisis, either isolated (76 cases) or combined procedure (47 cases). At their final follow-up, a patient reported questionnaire (overall satisfaction, foot stability and shape, activities of daily living, pain level, and analgesics usage) was distributed. The average postoperative follow-up period was 30 (13-55) months. Nineteen (15%) cases required at least one revision surgery: the implant was manipulated in 5 (4%), while 14 cases (11%) required definitive implant removal. The predominant reason for implant removal was pain (50%), followed by implant migration (27%). The pediatric population with isolated procedure showed lowest revision rate (5%), while adults with combined ankle/hindfoot procedures demonstrated revision rate of 50%. The overall patient satisfaction after arthroereisis was 84%. The patients' perceived improvement in foot stability was 75%, foot shape 85%, and activities of daily living 64%. Eighty-two percent of cases reported no analgesics usage in the last month and mean visual analogue scale (0-10) pain level decreased from 5.5 to 2.2 (p.001). The subgroup analyses of patient-reported questionnaires revealed the best outcome in the pediatric-isolated cases, while adults with combined procedures reported the lowermost outcome. Extra-osseous talo-tarsal stabilization demonstrated a low rate of revisions surgery and a high satisfaction rate as an isolated procedure. Patients with conjoined procedures experienced more revisions and considerably lower satisfaction rates.

Study question Does the presence of mild adenomyosis, a common acquired uterine anomaly, affect the endometrial gene expression levels during window of receptivity? Summary answer Mild adenomyosis has no significant influence on gene expression signature in the window of implantation (WOI). What is known already The improvements in imaging techniques have led to frequent detection of adenomyosis in women undergoing investigations for infertility. Although the data are conflicting, some clinical studies have shown that the presence of adenomyosis may interfere with embryo implantation and lead to poor pregnancy outcomes. The knowledge of molecular background that would lead to the phenomenon of altered endometrial receptivity in women with adenomyosis is limited and mainly demonstrated by selected candidate genes. Next-generation sequencing platforms enable genome-wide transcriptomic profiling of desired tissue samples and present a powerful tool to identify differentially expressed genes (DEGs) between women with adenomyosis and controls. Study design, size, duration We designed a prospective case-control study comparing women with sonographic evidence of mild adenomyosis (n = 10) and women with normal uteri seeking assisted reproduction due to male factor infertility as the control group (n = 10). All eligible women underwent infertility treatment at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia between years 2018 and 2020. For the present study, they were scheduled for cycle monitoring by urinary luteinizing hormone (LH) tests. Participants/materials, setting, methods Each endometrial biopsy was obtained in the presumed window of implantation (WOI) on days LH + 7 to LH + 9 after LH surge (LH + 0). Isolated total RNA was applied for mRNA + lncRNA sequencing (RNA-seq) by Illumina Novaseq 6000. An aliquot of RNA samples was used to verify the WOI by the endometrial receptivity test “beREADY” (CCHT, Estonia). Gene Ontology and Reactome pathway enrichment analyses were conducted in ClueGO bioinformatics tool to study biological role behind obtained DEGs. Main results and the role of chance The R program language and Bioconductor packages were used to align generated RNA-seq reads on the human reference genome assembly (hg19) and to calculate gene expression differences between study groups using normalized counts per million (CPM)>10 in at least 10 samples. A total 233 DEGs (p Limitations, reasons for caution Only mild adenomyosis was considered in this study, which is most commonly detected in women. The results could differ in women in severe cases of adenomyosis. Multicellular whole-tissue endometrial samples that were used for RNA isolation could mask gene expression differences of specific cell types between study groups. Wider implications of the findings: According to our results of transcriptome analysis, the presence of mild adenomyosis has no significant influence on the gene expression signature during endometrial receptivity in natural menstrual cycle. Women being investigated for infertility can be reassured that this common acquired anomaly does not significantly influence the chances of successful conception. Trial registration number 0120–259/2018/16

Study question Does laser-induced artificial blastocoel collapse result in better blastocyst cryopreservation survival and higher live birth rate (LBR) in comparison with intact counterparts? Summary answer Compared to vitrification of intact blastocysts, collapsed blastocysts resulted in higher survival and for 5% higher LBR. Neonatal outcomes were comparable in both groups. What is known already Blastocysts have long been considered a stage that is suboptimal for freezing-thawing procedures due to their high fluid content and different cell types. The development of a modified vitrification technique has enabled blastocysts to better survive cryopreservation compared to a slow freezing procedure. Many studies on the optimization of cryopreservation of blastocysts have mentioned the need for artificial collapsing of the blastocoel prior to cryopreservation, thereby reducing the risk of intracellular ice-crystals formation. However, the effectiveness of artificial collapsing on blastocyst survival rate, single vitrified-warmed blastocyst transfer (SVBT) outcome and on safety of such intervention remains to be confirmed. Study design, size, duration A retrospective matched case-control study of transfers of single blastocysts being artificially collapsed (case) or intact (control) before vitrification. A sample size of 306 cycles in both arms was needed to achieve 80% power to detect a difference between the groups of 10% with P Participants/materials, setting, methods Artificial collapsing was introduced into clinical practice gradually. In fresh IVF cycles (performed in university clinic from 2012 until 2014) with supernumerary blastocysts, half of the blastocysts were randomly selected before vitrification for laser-induced artificial collapsing. The other half was vitrified in intact form. Only the first transfers of a single vitrified-warmed blastocyst (n = 818) were included in the study. By matching, 309 pairs of collapsed (study) and intact (control) SVBTs were identified. Main results and the role of chance Both groups were comparable by their characteristics in indications, female age, type and length of ovarian hyperstimulation, insemination method in fresh cycle, protocol for warmed blastocyst transfer, blastocyst quality and day of blastocyst vitrification. Survival rates in case and control group ((309/316) 97.8% and (309/323) 95.7%; P = 0.13) were comparable, but optimal survival rates (100% survival and re-expansion after warming) was significantly higher in artificial collapse group ((247/316) 78.2% and (225/323) 69.7%; P = 0.01). Clinical pregnancy rates ((120/309) 38.8% and (110/309) 35.6%; P = 0.4), miscarriage rates ((15/120) 12.5% and (24/110) 21.8%; P = 0.06) and LBR per transfer ((100/309) 32.4% and (85/309) 27.5%; P = 0.19) or LBR per warmed blastocyst ((100/316) 31.6% and (85/323) 26.3%; P = 0.14) were not statistically different between case and control groups. Since the study was powered to detect a 10% difference, the possibility of type 2 error cannot be excluded. Perinatal outcomes were available for 175 live births. There were 10.5% (10/95) preterm births in the study group vs. 16.3% (13/80) in control group (P > 0.05). Birth weights (3,308 g (SD 592 g) vs 3,308 g (SD 738 g) and sex ratio (50.7% vs 49.2% boys) were also comparable between both groups (P > 0.05). There were no major malformations detected in the study population. Limitations, reasons for caution The research is retrospective, but the cycles from both groups were performed in the same time period. The groups were balanced according to all possible confounders. Blastocysts for vitrification were first categorized by quality groups and embryos from each category were randomized for collapsing or for remaining intact. Wider implications of the findings: No significant difference was found in live births by this sample size. Nevertheless, increasing the success by 5% with the introduction of artificial collapsing can be an important step towards optimizing of blastocyst cryopreservation. To confirm a 5% improvement in results, a sample size of > 2500 cases would be needed. Trial registration number The study has been approved by the National Ethics Committee of the Republic of Slovenia (0120–204/2016–2).

Background: Adenomyosis is a gynaecological condition with limited evidence of negative impact to endometrial receptivity. It is commonly associated with endometriosis, which has been shown to alter endometrial expression patterns. Therefore, the candidate genes identified in endometriosis could serve as a source to study endometrial function in adenomyosis. Methods: Transcripts/proteins associated with endometrial receptivity in women with adenomyosis or endometriosis and healthy women were obtained from publications and their nomenclature was adopted according to the HUGO Gene Nomenclature Committee (HGNC). Retrieved genes were analysed for enriched pathways using Cytoscape/Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Reactome tools to prioritise candidates for endometrial receptivity. These were used for validation on women with (n = 9) and without (n = 13) adenomyosis. Results: Functional enrichment analysis of 173, 42 and 151 genes associated with endometriosis, adenomyosis and healthy women, respectively, revealed signalling by interleukins and interleukin-4 and interleukin-13 signalling pathways, from which annotated LIF, JUNB, IL6, FOS, IL10 and SOCS3 were prioritised. Selected genes showed downregulated expression levels in adenomyosis compared to the control group, but without statistical significance. Conclusion: This is the first integrative study providing putative candidate genes and pathways characterising endometrial receptivity in women with adenomyosis in comparison to healthy women and women with endometriosis.

Purpose To synthesise data from genome-wide studies reporting molecular signature of eutopic endometrium through the phases of the menstrual cycle in endometriosis. Methods Extraction of data from publications reporting genetic signatures characterising endometrium associated with endometriosis. The nomenclature of extracted differentially expressed transcripts and proteins was adopted according to the HUGO Gene Nomenclature Committee (HGNC). Loci were further sorted according to the different phases of the menstrual cycle, i.e. menstrual (M), proliferative (P), secretory (S), early-secretory (ES), mid-secretory (MS), late-secretory (LS), and not specified (N/S) if the endometrial dating was not available. Enrichment analysis was performed using the DAVID bioinformatics tool. Results Altered molecular changes were reported by 21 studies, including 13 performed at the transcriptomic, 6 at proteomic, and 2 at epigenomic level. Extracted data resulted in a catalogue of total 670 genetic causes with available 591 official gene symbols, i.e. M = 3, P = 188, S = 81, ES = 82, MS = 173, LS = 36, and N/S = 28. Enriched pathways included oestrogen signalling pathway, extracellular matrix organization, and endothelial cell chemotaxis. Our study revealed that knowledge of endometrium biology in endometriosis is fragmented due to heterogeneity of published data. However, 15 genes reported as dysregulated by at least two studies within the same phase and 33 significantly enriched GO-BP terms/KEGG pathways associated with different phases of the menstrual cycle were identified. Conclusions A multi-omics insight into molecular patterns underlying endometriosis could contribute towards identification of endometrial pathological mechanisms that impact fertility capacities of women with endometriosis.

STUDY QUESTION What has the ESHRE programme ‘ESHRE Certification for Clinical Embryologists’ achieved after 10 years? SUMMARY ANSWER The post-exam analysis showed a pass rate of 60% for Clinical and 50% for Senior Clinical Embryologists and a high level of internal consistency of all exams, leading to a total of 773 certified Clinical and 493 Senior Clinical Embryologists over the decade. WHAT IS KNOWN ALREADY In an ESHRE survey on the educational and professional status of Clinical Embryology in Europe, it was found that education of laboratory personnel working in the field of assisted reproduction is highly variable between countries. In 2008, ESHRE introduced a programme, curriculum and certification in the field of Clinical Embryology. Knowledge gained by postgraduate study of recommended literature, following a clear curriculum, is verified by a written two-level exam for obtaining a certificate for Clinical (basic) or Senior Clinical (advanced) Embryologists. With a total of 1266 certificates awarded over a period of 10 years and recognition by the Union Européenne des Médecins Spécialistes and their Council for European Specialists Medical Assessment, the ESHRE Clinical Embryology exams have become an internationally recognized educational standard in the field of Clinical Embryology. STUDY DESIGN, SIZE, DURATION A retrospective analysis of all applications for ESHRE Clinical (2009–2018) and Senior Clinical Embryologist Certification (2008–2018) and exam results of the first decade was carried out by the Steering Committee for Clinical Embryologist Certification. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 2894 applications for ESHRE Certification for Clinical Embryologists and the results of 10 exams for the Clinical (1478 candidates) and 11 exams for Senior Clinical (987 candidates) levels were analysed. A detailed post-exam retrospective analysis was performed regarding difficulty, discrimination and reliability levels of 1600 multiple-choice questions (MCQs) with a single best answer among four options, from eight different curriculum topics (Basic cell biology, Genetics, Developmental biology, Female reproduction, Male reproduction, IVF laboratory, Cryopreservation and Laboratory management), representing the core theoretical knowledge of Clinical Embryology. Difficulty levels of the MCQs were subsequently compared regarding each topic and each yearly exam. The participation and success rates in the ESHRE Clinical Embryology exams were also assessed in terms of the educational and geographic backgrounds of candidates. MAIN RESULTS AND THE ROLE OF CHANCE Over the 10 years studied, the mean pass rate for the Clinical Embryologist exam was 60% (range 41–86%), and for the Senior Clinical Embryologist exam was 50% (range 34–81%). On average, 63% European candidates and 35% non-European candidates passed the Clinical Embryologist exam, while 52% European candidates and 31% non-European candidates passed the Senior Clinical Embryologist exam. The candidates’ educational level impacted on the success of the Clinical Embryologist exam but not of the Senior Clinical Embryologist exam. The mean difficulty indices by study topic showed that in the period of 10 years, there were no statistically significant differences between topics, for either the Clinical or Senior Clinical Embryologist exams. However, the overall exam difficulty varied between years. Reassuringly, the exam MCQ discrimination and reliability indices always showed a high level of internal consistency in all exams. LIMITATIONS, REASONS FOR CAUTION Some data from the initial ESHRE certification programme were not obtained electronically, in particular data for education, implying tables and figures reflect the specified valid data periods. Several countries exhibit different study profiles for those working in ART laboratories, such that laboratory technicians/technologists predominate in some countries, while in others only biologists and medical doctors are allowed to work with human embryos. Such differences could consequently affect the exam performance of candidates from specific countries. WIDER IMPLICATIONS OF THE FINDINGS The ESHRE exams on Clinical Embryology are the most widely, internationally accepted tests of knowledge in the rapidly growing area of human reproduction. Clinical Embryology is increasingly recognized as a specific discipline for scientific staff who are collaborating closely with clinicians in managing human infertility through medically assisted reproduction. The analysis of the first 10 years of application of a two-level exam for Clinical Embryology shows a consistent high quality and reliability of the exam and MCQs used. These results represent an important follow-up of the quality of the ESHRE Certification programme for Clinical Embryologists, and convincingly position Clinical Embryology in the wider group of health disciplines that are harmonized through professional bodies such as ESHRE and European Board & College of Obstetrics and Gynaecology. The exams provide a clear step towards the increasing professional recognition and establishment of Clinical Embryology within health systems at both European and international level. STUDY FUNDING/COMPETING INTEREST(S) No competing interest. All costs of the Steering Committee meetings were covered by ESHRE.

Vitrified human blastocysts show varied re-expansion capacity after warming. This prospective observational study compared behaviour of artificially collapsed blastocysts (study group patients, n = 69) to that of blastocysts that were vitrified without artificial collapse (control group patients, n = 72). Warmed blastocysts were monitored by time-lapse microscopy and blastocoel re-expansion speed and growth patterns compared between study and control groups. These parameters were also retrospectively compared between blastocysts that resulted in live birth and those that failed. Artificially collapsed blastocysts re-expanded on average 15.01 µm2/min faster than control blastocysts (P = 0.0013). Warmed blastocysts expressed four different patterns of blastocoel growth. The pattern showing contractions at the end of culture was observed to have a lower prevalence in control blastocysts, which coincided with the lower incidence of hatching in this group. Re-expansion speed and prevalence of growth patterns were comparable between blastocysts that did and did not result in a live birth. This was seen in the study and control groups. Despite faster re-expansion and different growth patterns of artificially collapsed blastocysts, live birth rate did not differ between groups. However, this result should be interpreted with caution due to the small sample size and high risk of bias.

Purpose The purpose of this study was to find out the most important prognostic factors for achieving a pregnancy after in vitro fertilization (IVF) in women with history of repeated unsuccessful IVF attempts. Methods We analyzed factors affecting pregnancy rate in a retrospective study including 429 IVF/ICSI cycles performed in women younger than 40 years with at least three previous consecutive failed IVF/ICSI attempts. Results Clinical pregnancy was observed in 140/429 (32.6%) cycles. Clinical pregnancy rate (CPR) was significantly higher in cycles with LEI compared to cycles without LEI before embryo transfer (44.4 vs 26.54%, p = 0.007). The CPR was also higher in cycles with day 5 blastocyst- compared with day 3 cleavage-stage embryo transfers (45.51 vs 26.54%, p

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst's volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts' volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Purpose The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in selected genes, responsible for hormonal regulation of folliculogenesis, are associated with response to controlled ovarian hyperstimulation (COH) and clinical characteristics of women enrolled in in vitro fertilization (IVF) programs. Methods In a cross-sectional study, 60 (IVF) patients underwent COH by using gonadotropin-releasing hormone (GnRH) antagonist and recombinant follicle-stimulating hormone (rFSH) protocol. Patients were classified into three groups: poor-responders (according to Bologna criteria), normo-responders (≤ 15 oocytes), and hyper-responders (> 15 oocytes). Genotyping of SNPs AMH rs10407022, AMHR rs3741664, FSHR rs1394205 and rs6166, and ESR1 rs2234693 was performed using high-resolution melting analysis (HRMA). Basal FSH (bFSH), estradiol (E2), and anti-Müllerian hormone (AMH) were measured by enzyme-linked immunosorbent assay (ELISA). Results Patients with GG genotype of FSHR rs1394205 had significantly lower AMH level (P = 0.016) and required higher rFSH dose per oocyte compared to women with AA or AG genotype (P = 0.036). We also found higher frequency of GG genotype of FSHR rs1394205 in poor- (76.5%) than in hyper-responders (37.5%, P = 0.002). Patients with AA genotype of FSHR rs6166 had higher level of measured bFSH compared to those with AG or GG genotypes (P = 0.043). Women with GG genotype of AMHR rs3741664 required higher rFSH dose in comparison with patients carrying genotypes AA or AG (P = 0.028). Conclusions The GG genotype at position rs1394205 is associated with poor ovarian response to COH. Patients with this genotype may require higher doses of rFSH for ovulation induction.

Study question What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? Summary answer It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. What is known already Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. Study design, size, duration A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. Participants/materials, setting, methods The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. Main results and the role of chance In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. Limitations, reasons for caution Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. Wider implications of the findings This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. Study funding/competing interests None. Trial registration number No.

A reduction in the number of embryos transferred is the most important step in decreasing multiple gestation rates after medically assisted reproduction. Slovenia has implemented insurance company regulations that regulate single-embryo transfer in selected good-prognosis couples. The aim of the present study was to evaluate its effects on the Slovenian population compared with cross-border patients, who are not affected by the insurance company policy. Ultimately, 2403 couples undergoing IVF or intracytoplasmic sperm injection were included in the retrospective analysis. Patients were classified according to their origin. The decision about the number of embryos transferred and the treatment success were evaluated. The implementation of the policy favouring single-embryo transfer resulted in a significant decrease in the twin birth rate in Slovenian patients (24.4% before policy versus 6.7% after policy implementation, P 0.001). Although in cross-border patients twin birth rates have declined through the study period, they remained significantly higher compared with Slovenian patients (23.1% versus 6.7%, P 0.001). The data demonstrate that insurance company policies favouring single-embryo transfer are an effective tool in decreasing multiple gestation rates. Similar mechanisms should be implemented in the cross-border patient population. A reduction in the number of embryos for transfer is the most important step in decreasing multiple gestation rates after medically assisted reproduction. Slovenia has implemented insurance company regulations that regulate single-embryo transfer in selected good-prognosis couples. This means that in patients with female partner younger than 36 years, undergoing their first or second treatment attempt, a single top-quality should be transferred in order for national insurance company to reimburse six IVF/intracytoplasmic sperm injection (ICSI) cycles. The aim of the present study was to evaluate the effect of this regulation on the Slovenian patient population compared with cross-border patients, who are not affected by insurance company policy. Ultimately, 2403 couples undergoing IVF or ICSI procedure were included in the analysis. Patients were classified according to their origin. The decision about the number of embryos for transfer and treatment success was evaluated. The implementation of the policy favouring single-embryo transfer resulted in a significant decrease in the twin birth rate in Slovenian patients (24.4% before versus 6.7% after policy implementation). Although in cross-border patients, twin birth rates have declined through the study period, they remained significantly higher compared with Slovenian patients (23.1% versus 6.7%). Our data demonstrate that insurance company policies favouring single-embryo transfer are an effective tool in decreasing multiple gestation rates. Similar mechanisms should be implemented in the cross-border patient population.

Spherical crystallization of drugs Spherical crystallization of drugs is the process of obtaining larger particles by agglomeration during crystallization. The most common techniques used to obtain such particles are spherical agglomeration and quasi-emulsion solvent diffusion. Ammonia diffusion systems and crystallo-co-agglomeration are extensions of these techniques. By controlling process parameters during crystallization, such as temperature, stirring rate, type and amount of solvents, or excipient selection, it is possible to control the formation of agglomerates and obtain spherical particles of the desired size, porosity, or hardness. Researchers have reported that the particles produced have improved micromeritic, physical, and mechanical properties, which make them suitable for direct compression. In some cases, when additional excipients are incorporated during spherical crystallization, biopharmaceutical parameters including the bioavailability of drugs can also be tailored.

Objective To assess whether the embryo cultivation at different oxygen tensions had any effect on intracytoplasmic sperm injection (ICSI) outcome. Design Prospective randomized trial. Setting University clinical center. Patient(s) Women undergoing ICSI (n = 647). Intervention(s) Culturing of embryos, either at 6% CO 2 , 5% O 2 , 89% N 2 , or at 6% CO 2 in air. Main Outcome Measure(s) The primary outcome was ongoing pregnancy rates (PR). Secondary outcomes: cumulative PRs, implantation, and embryo quality, for both treatment groups and clinical outcomes for subgroups (optimal cycles, poor responders, older women). Result(s) Although low oxygen resulted in a higher proportion of good day 2 embryos (0.547 ± 0.021 vs. 0.39 ± 0.019) and optimal blastocysts (0.162 ± 0.199 vs. 0.083 ± 0.128), the ongoing PRs (31.6% vs. 27.1%) and implantation rates (28.8% vs. 25.2%) were similar in both oxygen groups. Low oxygen caused a higher cumulative PR (38% vs. 28.3%) in the main group and a higher PR in the poor responder subgroup (23% vs. 9.8%) with embryo transfers performed mostly on day 3. Conclusion(s) The use of reduced oxygen in IVF is reasonable, irrespective of the duration of embryo culture. It improves embryo development and cumulative PR and is also recommended in poor responding cycles.

Objective To evaluate the rate and type of aneuploidies of chromosomes 13, 16, 18, 21 and 22, with respect to the length of in vitro maturation (IVM) period, and to compare the results to previously published studies on aneuploidy rates of unfertilized, uninseminated mature oocytes and first polar bodies. Study design Two hundred and twelve immature germinal vesicle stage oocytes were assigned to two groups. After successful IVM, depending on their maturational period of 24 h (Group A) or 36 h (Group B), chromosomal analysis was performed by five color fluorescence in situ hybridization (FISH). In Groups A and B the rates of aneuploid oocytes were calculated and compared by chi-square test. Also the rates of hyperhaploidy, hypohaploidy, disomy and nullisomy were determined and compared by chi-square test. The difference was considered statistically significant at p -value of Results The prolonged IVM did not significantly affect the aneuploidy rate compared to the shorter maturation period (48.1% and 45.0%, respectively). Regarding the unbalanced premature chromatid separation, no statistically significant difference was found between hyperhaploidy and hypohaploidy (14.8% versus 8.3%). For chromosome nondisjunction, higher frequency of disomy than nullisomy was observed (30.6% versus 14.8%; p Conclusions The aneuploidy rate of IVM GV-oocytes is comparable to the aneuploidy rate of in vivo matured oocytes and first polar bodies, regardless of the length of maturation period. This suggests that the immature oocytes can be used in infertility treatment after they complete maturation.

Background Anti-Müllerian hormone (AMH) is a marker of the ovarian reserve with promising prognostic potential in reproductive medicine. We aimed to evaluate the prognostic ability of AMH for predicting excessive or poor responses to ovarian stimulation using gonadotrophin-releasing hormone (GnRH) agonist and GnRH antagonist protocols in patients undergoing medically assisted reproduction (MAR) procedures. Methods This retrospective analysis included 623 women who underwent ovarian stimulation for medically assisted reproduction. AMH level measurements were acquired from all couples within six months of the initiation of ovarian stimulation. Results AMH was significantly correlated with the number of retrieved oocytes, and age was not relevant in a multivariate regression analysis (unstandardized regression coefficient of 1.130, 95 % confidence interval 0.977-1.283). AMH was a better predictor of both excessive (>19 oocytes) and poor (

The testicular sperm from biopsy and frozen/thawed tissue are frequently immotile. The purpose of our retrospective study was to assess the effect of short exposure of testicular samples with only immotile sperm to pentoxifylline (PF)-sperm motility stimulator. In 77 of 294 (26.2%) testicular sperm ablation/testicular sperm extraction-intracytoplasmic sperm injection (TESA/TESE-ICSI) cycles in patients with azoospermia, only immotile sperm were found in biopsies even after 2 hours of incubation of tissue in the medium. These 77 cycles were divided into 2 groups. In group 1 (cycles between 1999 and 2001; n = 30), ICSI was performed with untreated immotile sperm. In group 2 (cycles between 2002 and 2004; n = 47), immotile testicular sperm were treated for 20 minutes with pentoxifylline (PF) (1.76 mM) before ICSI. Both groups had the same proportion of ICSI cycles with fresh, frozen/thawed, and aspirated testicular sperm. The overall pregnancy rate of TESA/TESE-ICSI did not vary during the study period. In 45 of 47 (95.7%) testicular samples with total immotility, the sperm started to move 20 minutes after PF treatment. The mean time required for ICSI was shortened in the PF group (30 minutes [minimum 10, maximum 90] vs 120 minutes [minimum 60, maximum 240]) due to easier identification of motile sperm. In comparison with the nontreated group, the PF group had a higher fertilization rate (66% vs 50.9%; P < .005) and mean number of embryos per cycle (4.7 ± 3.3 vs 2.7 ± 2.1; P < .01). The clinical pregnancy rate per cycle in PF and non-PF groups was 38.3% and 26.7%, respectively. By using PF in cases of only immotile testicular sperm we can cause movement of testicular sperm, allow easier identification of vital sperm, shorten the procedure, improve fertilization rates, and increase the number of embryos.

This prospective randomized study compared the effectiveness of a flexible single-dose gonadotrophin-releasing hormone (GnRH) antagonist (cetrorelix) and a single-dose long GnRH agonist (goserelin) protocol for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. All patients from the waiting list were successively included in the study, pre-programmed with an oral contraceptive, and randomized into goserelin and cetrorelix groups. Depending on the date on which their menstrual period started, patients took oral contraceptives for one or two cycles. Ultimately, 236 patients in the first group received a single dose of depot preparation of goserelin and 224 patients received a single 3 mg dose of cetrorelix in the late follicular phase, when the mean follicle diameter exceeded 12 mm. The mean number of ampoules of FSH and the duration of stimulation was statistically significantly lower in the cetrorelix group than in the goserelin group (25.9 versus 34.5, and 9.6 versus 12.2 days, P0.01). The mean number of oocytes retrieved was similar (6.7 +/- 4.5 versus 7.2 +/- 4.6, NS). Similar results were observed in fertilization rates, blastulation rates and blastocyst transfer rates in both groups. Clinical pregnancy and delivery rates per cycle were higher in the goserelin group (34.3 and 30.1%) than in the cetrorelix group (31.9 and 28.3%), but the differences were not statistically significant. The flexible single-dose GnRH antagonist protocol is an advantageous alternative to the long GnRH agonist protocol, with similar efficacy, shorter duration, a significant reduction in the number of FSH ampoules used and without the menopause-like effects of the GnRH antagonist.

Objective: To determine whether extended culture of embryos to blastocysts has any benefit in cycles with only one or two created embryos. Design: Retrospective analysis of cycles comparing outcomes of day 2 and day 5 transfers. Our day 2 group was from the year 1999 and our day 5 group, from the year 2000. Setting: Assisted reproductive technology program of a teaching hospital. Patient(s): All patients, irrespective of age, who had developed one or two embryos. Intervention(s): Stimulated IVF, intracytoplasmic sperm injection, or testicular sperm extraction and intracytoplasmic sperm injection cycles with 2-day culture in universal IVF medium (n = 133) or 5-day culture in BlastAssist media (MediCult, Jyllinge, Denmark; n=132). Main Outcome Measure(s): Pregnancy, implantation, and take-home baby rates. Result(s): In the groups of 2-day and 5-day culture, embryo transfer was performed in 98% and in 57% of cycles, respectively. However, the total implantation rate per created embryo (18% vs. 18%), the pregnancy rate per cycle (23% vs. 21%), and the take-home baby rate (69.4% vs. 71.4%) did not differ between the day 2 and day 5 groups. Conclusion(s): Extended culture of embryos does not improve or decrease their capacity for implantation but only allows for better selection and is therefore not necessary in cycles with fewer than three embryos.

Purpose: The purpose was to test the adequacy of unstimulated cycles for intracytoplasmic sperm injection (ICSI) and to evaluate implantation and pregnancy rates in three different age groups.

BACKGROUND: The aim of the study was to test the influence of 2- and 5-day cultivation of a single oocyte on the pregnancy rate in a non-stimulated cycle. METHODS: A retrospective chart review of 391 consecutive patients undergoing IVF and intracytoplasmic sperm injection in unstimulated cycles was performed. The embryos were kept in MediCult universal IVF medium for day 2 transfers and in BlastAssist System for day 5 transfers. RESULTS: The oocyte recovery rate in the group for 2-day cultivation and in the group for 5-day cultivation was similar, being 79.4 (162/204) and 83.6% (154/187) respectively. The same is true of the fertilization rate (73.8 versus 77.7%). The blastulation rate was 52.8%. The embryo transfer rate per cycle was higher when day 2 embryos were transferred: 64.8% (105/162) compared with 35.7% (55/154) if blastocyst-stage embryos were transferred. The pregnancy rate per transferred embryo was higher when a blastocyst was transferred (40.0%) instead of a day 2 embryo (23.8%). CONCLUSION: The expected pregnancy rate calculated per embryo available on day 2 of cultivation was similar in both groups (23.8 versus 22.2%) and it was not affected by oocyte culture to the blastocyst stage.

Objectives The aim of the study was to investigate whether endometrial thickness, endometrial pattern, endometrial movement and serum estradiol and progesterone affect the outcome of in vitro fertilization. Methods Prospectively collected data of endometrial thickness, endometrial pattern and subendometrial activity were analyzed in 122 consecutive in vitro fertilization cycles. All measurements were made on the day of hCG administration (day −2), day of embryo transfer (day +2), day +6 and day +12. Results It would appear that pregnant women tend to have a thicker endometrium on day +12 than non-pregnant women. There appears to be no appreciable difference between pregnant and non-pregnant women in endometrial movement and thickness, nor in serum estradiol and progesterone on day +2 and day +6. In combining three echographic variables (thickness, pattern and movement) and hormone level (estradiol and progesterone), only progesterone on day +12 could be used to predict the outcome. Conclusions Subendometrial contractility plays no important role in implantation on the day of embryo transfer or 4 days later. Copyright © 2001 International Society of Ultrasound in Obstetrics and Gynecology

Objective To determine the diagnostic value of the following sperm function tests in predicting the fertilizing ability of spermatozoa in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI): hyaluronan-binding assay (HBA), DNA fragmentation (Halosperm), and hyperactivity. Design Prospective study. Setting University medical center. Patient(s) 133 couples undergoing infertility treatment with IVF/ICSI. Intervention(s) Analysis of sperm DNA fragmentation, hyaluronan-binding ability, and hyperactivation on washed semen samples used for the insemination of oocytes. Main Outcome Measure(s) Correlation between the results of sperm function tests and the fertilization rate (FR) or embryo quality (EQ) after IVF and ICSI. Comparison of the sperm DNA fragmentation, hyperactivation, and hyaluronan binding ability between cycles with less than 50% (group 1) and more than 50% (group 2) of oocytes fertilized after IVF. Result(s) Both FR and EQ in IVF cycles negatively correlated with sperm DNA fragmentation. Furthermore, a positive correlation was observed between FR and hyaluronan-binding ability or induced hyperactivity. The semen samples from the IVF cycles with low FR (group 1) were characterized by statistically significantly higher sperm DNA fragmentation and lower hyaluronan-binding ability in comparison with semen samples from the group with high levels of fertilization (group 2). In ICSI cycles, no relationship was found between sperm function tests and FR or EQ. Conclusion(s) The Halosperm test, the HBA test, and induced hyperactivity are useful in predicting the ability of spermatozoa to fertilize oocytes in IVF and are helpful in distinguishing semen samples suitable for IVF or ICSI.

In vitro matured (IVM) human oocytes are very sensitive to culturing conditions. There are many external factors influencing their nuclear and cytoplasmic maturation, which can lead to morphologic or genetic abnormalities. Even oocytes themselves can possess some intrinsic abnormalities in biochemical cell cycle regulation which prevent them from reaching maturity in vivo. The following chapter will describe different morphological characteristics and their possible connection to aneuploidy, fertilization and embryo development. Also it will cover some aspects of oocyte IVM and describe mechanisms leading to aneuploidy. The second part of the chapter will be devoted to fluorescent in situ hybridization (FISH) analysis of IVM oocytes and first polar bodies (PB1) with the emphasis on aneuploidy occurrence in oocytes with prolonged cultivation.

Investigation of one complex pathological condition is definitively a challenging task, but trying to find the connection(s) between two is even more difficult. This is very true in the case of infertility and endometriosis. Both conditions have numerous symptoms, very diverse clinical pictures and multifactorial etiologies. The first step toward understanding the connection between the two is to prove the correlation between them. The next task is to try to understand the mechanisms by which they affect each other, which involves examination and comparison of numerous variables specific for each condition. The results are still the subject of many controversies.

Prolonged cultivation of embryos to the blastocyst stage has become a routine practice in the human in vitro fertilization program (IVF) since 1999, when the first commercial sequential media were developed. The culture systems have been improved many times and today most of the blastocyst culture media enable the embryos to reach the blastocyst stage in more than 50% of cases (Gardner et al., 1998; Kovacic et al., 2004). The advantages of blastocyst culture are in the possibilities for selection of embryos that have an activated genome (Braude et al., 1988), higher predictive values for implantation on the basis of their morphological appearance as compared with earlier embryos (Gardner and Schoolcraft, 1999; Kovacic et al., 2004) and in a reduction in the number of transferred embryos without compromising pregnancy rate (Gardner et al., 2000a). Blastocyst is also a stage that is better synchronized with endometrial receptivity for its implantation (Croxatto et al., 1978; Gardner et al., 2000c). By replacement of embryos in the blastocyst stage, their exposure to hyperstimulated milieu and consequent endometrial contractions, which could be fatal for them, is significantly shortened (Lesny et al., 1998). Blastocysts also contain a larger number of cells than early stage embryos and should therefore have a better possibility of survival of cryopreservation (Veeck, 2003).

The tension of oxygen measured in the oviducts of several mammals was 5-8.7 %, but this drops in the uterine milieu to

OBJECTIVE: Programmed oocyte retrieval was performed in order to make the in vitro fertilization (IVF) program cheaper and work of the IVF team easier. METHOD: In a group of 77 patients included in the IVF program, the menstrual cycle was modified with estrogen-progesterone contraceptive pills. For this reason, it was possible to start the stimulation protocol in all patients on the same day. The stimulation protocol was a combination of clomiphene (100 mg) for 5 days and HMG (150 IU) every other day. Cycles were monitored by ultrasound only. RESULT: The implantation rate per embryo transfer was 22.4%. The number of embryos per embryo transfer was low (2.6 ± 1.4) and eliminated the need for cryopreservation. Fertilization rate (82%) and embryo transfer rate (87%) were high. The take home baby rate was 14.3%. CONCLUSION: Seventy percent of all punctures were performed in 3 days in the middle of the week. In our conditions, programed oocyte retrieval is associated with significant economic benefits.

Numerous studies show the beneficial effect of reduced oxygen on the culture of animal embryos in vitro. However, few similar studies have been carried out in humans, and the conclusions from these were contradictory. Using sibling human oocytes, a prospective study was carried out to analyse the effect of 5 and 20% oxygen on prolonged development of embryos. The outcomes measured were fertilization rate and proportion of morphologically optimal embryos, blastocysts and optimal blastocysts developing on day 5. The results were analysed separately for the group of IVF (n = 988 oocytes) and ICSI (n = 928 oocytes) cycles. It was found that low oxygen did not influence fertilization, but in comparison with 20% oxygen, it resulted in a significantly higher proportion of embryos being optimal on day 3 after IVF (59 versus 43.2%; P < 0.001) as well as after ICSI cycles (51.2 versus 28.5%; P < 0.001). In both methods, the lower oxygen concentration improved the blastulation rate (73.2 versus 63.1%; P < 0.05 and 67.4 versus 54.7%; P < 0.001) and increased the proportion of embryos reaching the stage of expanded blastocyst with normal inner cell mass on day 5 (31.1 versus 14.6%; P < 0.001 and 18.9 versus 11.4%; P < 0.01). The ratio of successful embryo development to optimal blastocyst stage on day 5 of culture, calculated for two oxygen concentrations, was 2.1 for IVF and 1.7 for ICSI, in favour of lower oxygen tension.