Global Practice Patterns in the Evaluation of Non-Obstructive Azoospermia: Results of a World-Wide Survey and Expert Recommendations.

PURPOSE: Non-obstructive azoospermia (NOA) represents the persistent absence of sperm in ejaculate without obstruction, stemming from diverse disease processes. This survey explores global practices in NOA diagnosis, comparing them with guidelines and offering expert recommendations. MATERIALS AND METHODS: A 56-item questionnaire survey on NOA diagnosis and management was conducted globally from July to September 2022. This paper focuses on part 1, evaluating NOA diagnosis. Data from 367 participants across 49 countries were analyzed descriptively, with a Delphi process used for expert recommendations. RESULTS: Of 336 eligible responses, most participants were experienced attending physicians (70.93%). To diagnose azoospermia definitively, 81.7% requested two semen samples. Commonly ordered hormone tests included serum follicle-stimulating hormone (FSH) (97.0%), total testosterone (92.9%), and luteinizing hormone (86.9%). Genetic testing was requested by 66.6%, with karyotype analysis (86.2%) and Y chromosome microdeletions (88.3%) prevalent. Diagnostic testicular biopsy, distinguishing obstructive azoospermia (OA) from NOA, was not performed by 45.1%, while 34.6% did it selectively. Differentiation relied on physical examination (76.1%), serum hormone profiles (69.6%), and semen tests (68.1%). Expectations of finding sperm surgically were higher in men with normal FSH, larger testes, and a history of sperm in ejaculate. CONCLUSIONS: This expert survey, encompassing 367 participants from 49 countries, unveils congruence with recommended guidelines in NOA diagnosis. However, noteworthy disparities in practices suggest a need for evidence-based, international consensus guidelines to standardize NOA evaluation, addressing existing gaps in professional recommendations.

Algorithm-based individualization methodology of the starting gonadotropin dose in IVF/ICSI and the freeze-all strategy prevent OHSS equally in normal responders: a systematic review and network meta-analysis of the evidence.

PURPOSE: Ovarian hyperstimulation syndrome (OHSS) represents a rare but dangerous condition associated with controlled ovarian stimulation (COS) in IVF/ICSI. Over the last decades, many strategies have been introduced into clinical practice with the objective of preventing this potentially life-threatening condition. Among these, the freeze-all policy has gained great popularity, thanks to improvements in vitrification. Nevertheless, not all clinics have adequate skills in vitrification procedures and patients may be dissatisfied with a longer time to pregnancy. METHODS: This study is a systematic review and network meta-analysis of randomized controlled trials comparing different strategies of ovarian stimulation in IVF/ICSI cycles (freeze-all policy, algorithm-based individualization of the starting dose, experience-based individualization of the starting dose, standard dose) in terms of reduction of OHSS, in normal responders. RESULTS: The results indicate that only the algorithm-based individualization of the starting gonadotropin dose reduces OHSS similarly to the freeze-all strategy. CONCLUSION: Albeit in the era of the freeze-all policy, the personalization of the starting gonadotropin dose obtained by the use of algorithms should be pursued as a valid and safe option for IVF.

Cryoablation of Venous Malformations: A Systematic Review.

This study aimed to review systematically the efficacy, safety, and technical aspects of cryoablation in the treatment of venous malformations (VMs) and to provide the groundwork for future studies. A literature search for clinical studies utilizing percutaneous cryoablation of VMs was performed. All clinical studies related to primary or secondary treatment of VMs with percutaneous cryoablation were included in this review. These selected studies were evaluated for patient characteristics, cryoablation technique, technical success, lesion size and pain scores before and after cryoablation, and adverse outcomes. Random effects analysis of postprocedural changes in lesion volume and pain scores was performed. There were 54 patients with 55 cases of cryoablation of VMs. Of these cases, 27 recorded changes in lesion volume and 31 recorded changes in pain scores. The weighted mean postprocedural decrease in lesion size was 92.0% (raw average, 71.7%). The weighted mean reduction in pain score was 77% (raw average, 78.2%). Considering all treated cases (55), complete resolution of symptoms was seen in 35 cases (63.6%) and overall (complete or partial) improvement in 52 cases (94.5%). Common postprocedural symptoms included pain, bruising, swelling and numbness lasting less than 2 weeks. There were two major adverse events (3.7%), with both cases due to persistent dysesthesia. Patients with a history of prior sclerotherapy demonstrated lower preprocedural and postprocedural pain scores (4.7 and 1.3) than patients without prior treatments (5.8 and 2.8). Cryoablation of VMs appears to be potentially safe and effective on limited short-interval follow-up.

A randomized controlled trial investigating the use of a predictive nomogram for the selection of the FSH starting dose in IVF/ICSI cycles.

The number of oocytes retrieved is a relevant intermediate outcome in women undergoing IVF/intracytoplasmic sperm injection (ICSI). This trial compared the efficiency of the selection of the FSH starting dose according to a nomogram based on multiple biomarkers (age, day 3 FSH, anti-Müllerian hormone) versus an age-based strategy. The primary outcome measure was the proportion of women with an optimal number of retrieved oocytes defined as 8-14. At their first IVF/ICSI cycle, 191 patients underwent a long gonadotrophin-releasing hormone agonist protocol and were randomized to receive a starting dose of recombinant (human) FSH, based on their age (150 IU if ≤35 years, 225 IU if >35 years) or based on the nomogram. Optimal response was observed in 58/92 patients (63%) in the nomogram group and in 42/99 (42%) in the control group (+21%, 95% CI = 0.07 to 0.35, P = 0.0037). No significant differences were found in the clinical pregnancy rate or the number of embryos cryopreserved per patient. The study showed that the FSH starting dose selected according to ovarian reserve is associated with an increase in the proportion of patients with an optimal response: large trials are recommended to investigate any possible effect on the live-birth rate.

The carriers of the A/G-G/G allelic combination of the c.2039 A>G and c.-29 G>A FSH receptor polymorphisms retrieve the highest number of oocytes in IVF/ICSI cycles.

PURPOSE: The objective of this study was the elucidation of the possible role of the single-nucleotide polymorphisms (SNP) at position -29 and 2039 of the FSH receptor gene (FSHR) as independent predictive markers of ovarian response. Indeed, the tailoring of reproductive treatments is crucial for both maximizing the success of IVF patients and obtaining a reduction in hypo- or hyper-response rates. METHODS: This prospective, observational study analyzed the association of -29 and 2039 FSHR polymorphisms with the number of retrieved oocytes in 140 patients attending an IVF/ICSI cycle for severe male factors (≤5,000,000 spermatozoa/mL) or tubal factors at the ANDROS Day Surgery Clinic, Palermo, Italy. RESULTS: The results of this study demonstrate that the genetic combination of A/G for polymorphism c.2039 A>G with G/G for polymorphism c.-29 G>A is significantly associated with the highest number of collected oocytes (p = 0.03). This association was significant even after controlling for the effect of other clinical variables. CONCLUSIONS: The A/G-G/G allelic variant, identified as an independent variable, if confirmed in a larger number of patients, could be considered as a new genetic biomarker, which could increase the efficacy of prediction models for ovarian stimulation.

The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures.

PURPOSE: The aim of this study was to investigate the most suitable sperm preparation technique to apply in order to obtain a spermatozoon population with minimal DNA damage during in vitro fertilization procedures. We compared four preparation techniques: direct swim-up (DSU), pellet swim-up (PSU), density gradient (DG), and density gradient followed by swim-up (DG-SU), evaluating the effects of each technique on the DNA damage rate, evaluated by DNA fragmentation index of the spermatozoa obtained. METHODS: In this observational study, 98 semen samples from couples undergoing IVF/ICSI cycles were included. Data were collected between April and November 2014 at the ANDROS Day Surgery Clinic, Palermo, Italy. RESULT(S): The percentages of DNA fragmentation were 18.30 ± 10.8 in raw samples, 6.6 ± 5.7 after DSU, 4.2 ± 3.8 after PSU, 12.9 ± 9.9 after DG, and 3.7 ± 4.0 after DG-SU respectively. Compared to the raw evaluation, all the preparation techniques significantly decreased the total rate of the DNA fragmentation (DSU Z = -8.60, P < 0.008; PSU Z = -8.54, P < 0.008; DG Z = -6.42, P < 0.008, and DG-SU Z = -8.60, P < 0.008, respectively). Comparing them, spermatozoa with intact DNA after PSU and DG-SU were significantly higher than after DSU (Z = -7.12, P < 0.008; Z = -6.59, P < 0.008, respectively) and after DG (Z = -8.41, P < 0.008; Z = -8.60, P < 0.008, respectively). The difference between PSU and DG-SU was not significant (Z = -2.21, P = 0.03). CONCLUSION(S): There are, above all, two techniques of sperm preparation which allow for the recovery of spermatozoa with the lowest DNA fragmentation rate. Furthermore, given low costs and reduced time, we believe that PSU is the best option in the treatment of semen samples during IVF/ICSI.

Infertility-related stress, anxiety and ovarian stimulation: can couples be reassured about the effects of psychological factors on biological responses to assisted reproductive technology?

The aim of this prospective, longitudinal study was to examine the association between couples' pre-treatment psychological characteristics (state anxiety and infertility-related stress levels of both partners) and ovarian response during assisted reproductive technology treatment in a well-controlled sample. A total of 217 heterosexual couples (434 patients), suffering from primary infertility and undergoing their first assisted reproductive technology treatment at the Reproductive Medicine Unit of ANDROS Day Surgery Clinic in Palermo (Italy), were recruited. Psychological variables were assessed using the State Scale of State-Trait Anxiety Inventory (STAI-S) and the Fertility Problem Inventory (FPI). The number of follicles ≥ 16 mm in diameter, evaluated by transvaginal ultrasound scan on the eleventh day of the workup, was chosen as the outcome measure. No association between women's level of anxiety and infertility-related stress, and the number of follicles ≥ 16 mm in diameter was found. Moreover, the male partner's infertility stress and anxiety did not influence the relationship between the woman's infertility-related stress, anxiety level and ovarian response. Fertility staff should reassure couples that the woman's biological response to ovarian stimulation is not influenced by either partner's level of psychological distress.

A "four-cycle program" improves the estimate of the cumulative pregnancy rate and increases the number of actual pregnancies in IUI treatment: a cohort study.

OBJECTIVE: To demonstrate that reduction of the cumulative dropout rate (CDR) improves the accuracy of the estimate of the cumulative pregnancy rate (CPR) in a set of four intrauterine insemination (IUI) cycles ("four-cycle program") and increases the total number of pregnancies obtained. STUDY DESIGN: Single-centre retrospective observational cohort study of couples who underwent IUI cycles at the Andros Day Surgery Clinic, Palermo, from 1997 to 2011. The main outcome measure was the calculation of the CPR, with life table analysis, firstly by giving the same probability of pregnancy to the dropouts as the patients who continued the treatment (usual method) and secondly by considering this probability null (conservative method). The difference between these two methods was used to verify the accuracy of the estimate. RESULT(S): In the 15 years, 924 couples underwent 2956 cycles carried out consecutively in a set of four cycles. The CDR was 16%. The CPR was 31.4% with the usual method and 29.1% with the conservative method. The difference between the two estimates was not significant, indicating a high reliability of the results and a good accuracy of the calculation. Furthermore, maintenance of a low CDR permits improvement of the CPR, as was demonstrated by considering scenarios with worse dropout rates. CONCLUSION(S): The "four-cycle program" results in a reduction in the CDR, allowing a better estimation of the CPR, and increases the number of actual pregnancies in IUI. The CPR should become the focus for reporting outcome rates in IUI cycles. Reduction of the dropout rate allows us to give the patient a more reliable and accurate estimate of the pregnancy rate.

Gene expression of stem cells at different stages of ontological human development.

OBJECTIVES: To compare multipotent mesenchymal stem cells (MSCs) obtained from chorionic villi (CV), amniotic fluid (AF) and placenta, with regard to their phenotype and gene expression, in order to understand if MSCs derived from different extra-embryonic tissues, at different stages of human ontological development, present distinct stemness characteristics. STUDY DESIGN: MSCs obtained from 30 samples of CV, 30 of AF and 10 placentas (obtained from elective caesarean sections) were compared. MSCs at second confluence cultures were characterized by immunophenotypic analysis with flow cytometry using FACS CANTO II. The expression of the genes Oct-4 (Octamer-binding transcription factor 4, also known as POU5F1), Sox-2 (SRY box-containing factor 2), Nanog, Rex-1 (Zfp-42) and Pax-6 (Paired Box Protein-6), was analyzed. Real-time quantitative PCR was performed by ABI Prism 7700, after RNA isolation and retro-transcription in cDNA. Statistical analysis was performed using non-parametric test Kruskal-Wallis (XLSTAT 2011) and confirmed by REST software, to estimate fold changes between samples. Each gene was defined differentially expressed if p-value was <0.05. RESULTS: Cells from all samples were negative for haematopoietic antigens CD45, CD34, CD117 and CD33 and positive for the typical MSCs antigens CD13, CD73 and CD90. Nevertheless, MSCs from AF and placentas showed different fluorescence intensity, reflecting the heterogeneity of these tissues. The gene expression of OCT-4, SOX-2, NANOG was not significantly different among the three groups. In AF, REX-1 and PAX-6 showed a higher expression in comparison to CV. CONCLUSIONS: MSCs of different extra-embryonic tissues showed no differences in immunophenotype when collected from second confluence cultures. The expression of OCT-4, NANOG and SOX-2 was not significantly different, demonstrating that all fetal sources are suitable for obtaining MSCs. These results open new possibilities for the clinical use of MSCs derived from easily accessible sources, in order to develop new protocols for clinical and experimental research.

Endometrial expression of selected genes in patients achieving pregnancy spontaneously or after ICSI and patients failing at least two ICSI cycles.

The objective of this study was to identify the endometrial gene expression profile in receptive phase, which could represent a useful prognostic tool for selecting IVF patients. Endometrial expression of 47 selected genes biopsied during the window of implantation in natural cycles was compared between patients who achieved a successful pregnancy spontaneously or after subsequent intracytoplasmic sperm injection (ICSI) cycles and patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed significantly different levels of expression in 19 genes, five implicated in apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence for these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile, which could be used in the future as a prognostic tool for IVF patients. Gene expression analysis technology has opened new important perspectives on the study of the physiological processes of different tissues and organs. Specifically for the endometrium, it would be really interesting to find out an endometrial gene expression profile of receptive phase, which could be used in future as a useful prognostic tool for selecting IVF patients. To achieve this aim, the objective of the present paper was the comparison of endometrial expression in natural cycles of 47 selected genes between the biopsies of patients who achieved a successful pregnancy, either spontaneously or after subsequent ICSI cycles, and those of patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed a significant different expression in 19 genes: five implicated in programmed cell death, known as apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence of these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile which could be used in the future as a prognostic tool for IVF patients.

Conjoined twins in a trichorionic quadruplet pregnancy after intracytoplasmic sperm injection and quarter laser-assisted zona thinning.

OBJECTIVE: To describe the occurrence and management of conjoined twins (CTs) in a trichorionic quadruplet pregnancy after the transfer of three embryos obtained by intracytoplasmic sperm injection (ICSI) that had undergone assisted zona hatching (AZH) using a noncontact laser. DESIGN: Case report. SETTING: Private and public IVF centers. PATIENT(S): A 38-year-old nulliparous woman. INTERVENTION(S): IVF, diagnosis by two-dimensional, three-dimensional, and four-dimensional ultrasound, selective termination at 12 weeks gestation. MAIN OUTCOME MEASURE(S): Clinical follow-up and delivery. RESULT(S): After diagnosis, counselling, and selective termination, the subsequent prenatal course was uneventful, and two healthy boys were delivered by elective cesarean section at 38 weeks gestation. CONCLUSION(S): Monozygotic twinning (MZT) and CTs could complicate multifetal pregnancies obtained by assisted reproduction techniques. The relationship between CTs, IVF, and AZH should be better studied to fully understand the mechanisms that lead to MZT (with the possible role of AZH) and CTs in MZT, in particular. Three-dimensional and four-dimensional ultrasound improve prenatal assessment of the condition. Selective embryo reduction is a safe procedure for the management of these pregnancies.

Hormesis effect of trace metals on cultured normal and immortal human mammary cells.

An in vitro study was conducted to determine the effects of variable concentrations of trace metals on human cultured mammary cells. Monolayers of human mortal (MCF-12A) and immortal (MDA-MB231) mammary epithelial cells were incubated in the absence or presence of increasing concentrations of arsenic (As), mercury (Hg) and copper (Cu) for 24-h, 72-h, 4-d, and 7-d. The MTT assay was used to assess viability for all time periods and cell proliferation was monitored for 4-d and 7-d studies. Monolayers were also labeled with rhodamine-110 (R-6501), Sytox green, and Celltiter blue fluorescent dyes as indicators for intracellular esterase activity, nucleic acid staining, and cell reduction/viability, respectively. Total incubation time with chemical plus dyes was 24 h. For 24-h and 72-h studies, cells were seeded in 96-well plates, after which confluent monolayers were exposed to increasing concentrations of chemicals. For 4-d and 7-d studies, cells were seeded in 12-well plates at 1/3 confluent density (day 0) and exposed to increasing concentrations of metals on day 1. All cells were counted on days 4 and 7. In addition, test medium was removed from select groups of cultures on day 4, replaced with fresh medium in the absence of chemical (recovery studies), and assays were performed on day 7 as above. The data suggest that there is a consistent protective and/or stimulating effect of metals at the lowest concentrations in MCF-12A cells that is not observed in immortal MDA-MB231 cells. In fact, cell viability of MCF-12A cells is stimulated by otherwise equivalent inhibitory concentrations of As, Cu, and Hg on MDA-MB231 cells at 24-h. Whereas As and Hg suppress proliferation and viability in both cell lines after 4-d and 7-d of exposure, Cu enhances cell proliferation and viability of MCF-12A cells. MDA-MB231, however, recover better after 4-days of toxic insult. In addition, nutritional manipulation of media between the cell lines, or pretreatment with penicillamine, did not alter the hormesis effect displayed by MCF-12A. Growth of these cells however was not maintained in the alternative medium. The study demonstrates that a hormesis effect from trace metals is detectable in cultured mammary cells; fluorescent indicators, however, are not as sensitive as cell proliferation or MTT in recognizing the subtle responses. Also, sensitivity of mammary cells to lower concentrations of Cu, a biologically important trace metal, may play an important role in controlling cellular processes and proliferation. The ability to detect this in vitro phenomenon implies that similar processes, occurring in vivo, may be responsible for the development, induction, or enhancement of human cancers.