BACKGROUND:Selecting high-quality spermatozoa with optimal nuclear integrity is a crucial step in assisted reproductive technologies (ART). While density gradient centrifugation (DGC) is widely used, the associated centrifugation steps may induce cell stress, potentially damaging sperm DNA. Conversely, micro swim-up (MSU) is an innovative, centrifugation-free procedure using autonomous gamete migration in a polyvinylpyrrolidone medium. OBJECTIVES:This study aimed to compare the sperm selection technique MSU with the conventional method DGC in terms of sperm chromatin integrity in 90 semen samples. MATERIALS AND METHODS:Rapid progressive motility, sperm DNA fragmentation (sDF; SCD test), nuclear immaturity (aniline blue [AB] staining), and chromatin decondensation (chromomycin A3 [CMA3] staining) were evaluated before and after selection. RESULTS:Both procedures significantly improved motility and nuclear parameters compared to unselected samples. However, MSU outperformed DGC across all nuclear integrity markers. Specifically, sDF decreased from 18.50% (14.63-26.25) to 9.25% (6.00-16.00) with DGC and 1.00% (0.00-2.00) with MSU (p < 0.0001 MSU vs. DGC). Regarding nuclear immaturity, values improved from 21.00% (15.00-27.50) to 15.00% (8.00-18.00) with DGC and 5.00% (4.00-10.00) with MSU (p = 0.012 MSU vs. DGC). Similarly, chromatin decondensation decreased from 18.25% (10.88-23.63) to 13.50% (7.00-15.63) with DGC and 5.50% (2.75-10.00) with MSU (p = 0.0001 MSU vs DGC). DISCUSSION AND CONCLUSION:MSU selects spermatozoa with significantly superior nuclear integrity compared to DGC, markedly reducing DNA fragmentation and improving chromatin maturation. Given the direct impact of these parameters on ART outcomes, MSU might represent a valuable alternative to traditional centrifugation-based methods in clinical settings.

The paper aims to investigate the biological role of microRNAs secreted by preimplantation embryo into the blastocoel fluid and to detect a distinctive molecular signature for identifying embryos with the highest implantation potential. We carried on a multicenter retrospective study involving five European IVF centers. We collected 112 blastocoel fluid samples from embryos on day 5 post-fertilization, cultured individually, along with data on blastocyst grade and embryo transfer outcomes. Using a custom TLDA Array, we compared the expression levels of 89 miRNAs between 33 fluids from high-quality implanted embryos and 30 fluids from high-quality not-implanted embryos. Expression differences were assessed using SAM and t-test. Additionally, correlation and function enrichment analysis and network construction were conducted to identify the biological roles of deregulated microRNAs. We identified six up-regulated microRNAs in the blastocoel fluid from implanted embryos, significantly and positively correlated across all samples (r ≥ 0.7; P ≤ 0.05). They could take part in pluripotency circuits, regulating and being regulated by transcription factors associated with stemness, cell growth, and embryo development. The ROC curve analysis confirmed the potential of these miRNAs as implantation classifiers. The six miRNAs up-regulated in blastocoel fluid from implanted embryos may represent a functional molecular signature for evaluating blastocyst quality and identifying the most competent embryos. Their evaluation associated with non-invasive preimplantation genetic testing, integrating epigenetic and genomic analyses, could enhance implantation grade and allow for identification of the euploid embryo not able to implant.

Background and aims: Vaginal microbiomes have been classified into five different general categories, termed Community State Type (CSTs), with CST-III and CST-IV often associated with vaginal dysbiosis which makes women more prone to recurrent infections and assisted reproductive technology (ART) failure. Since a healthy microbiome is one of the key steps for successful reproduction, we investigated the impact of modulating the vaginal microbiota through the oral administration of probiotic formula consisting of a consortium of vaginal-specific lactobacilli and prebiotics (Personal Flora 2®). Methods: We recruited 32 women who had previous failed IVF cycles and were scheduled to undergo ART. We examined the composition of the vaginal microbiota before and after oral probiotic supplementation using 16S ribosomal RNA (rRNA) sequencing technology. Results: Our data show a noticeable modulation of the vaginal microbiome upon probiotic supplementation. In particular, precision probiotic intervention lowers the species diversity, favoring the dominance of Lactobacillus (p = 0.015) and Bifidobacterium (p = 0.000) whilst decreasing the percentage of Atopobium (p = 0.003), Gardnerella (p = 0.022), and Prevotella (p = 0.000). Conclusions: Although CST-III and CST-IV are generally considered detrimental, gynecologists should not refrain from performing IVF in these women if they have been previously subjected to a consortium of precision probiotics treatments, as the administration of specific probiotics reduces the presence of pathogenic bacteria promoting the increase in lactobacilli associated with a healthy vaginal ecosystem, which could impact pregnancy success.

RESEARCH QUESTION:What effect does the use of a rapid warming method of vitrified embryos have on survival and pregnancy? DESIGN:A comparison of two different warming embryos protocols: long protocol (group 1, n = 486) and a new fast protocol (group 2, n = 413) for blastocyst and cleavage stage frozen embryo transfers at two IVF centres: Assisted Reproductive Technology Centre, Careggi University Hospital Firenze and IVF Unit, 'Cervesi' Hospital Cattolica. Total pregnancy rate was considered primary outcome, and embryo survival rate, clinical pregnancy rate, miscarriage rate, ongoing pregnancy rate and mean time required for warming procedure were considered secondary outcomes. RESULTS:The same embryo survival rate was observed in the two groups. Total and clinical pregnancy rates seemed to be higher, but not statistically different, in group 2 compared with group 1 (29.5% versus 26.7% and 27.6% versus 22.6%, respectively). Conversely, a statistically significant reduction in miscarriage rate was found in group 2 compared with group 1 (18.0% versus 32.3%, P = 0.009). Similarly, a significant increase in pregnancy rate at 26 gestational weeks or over was observed in group 2 compared with group 1 (66.9% versus 80.3%, P = 0.016). Finally, a difference in time required for warming procedures was found in favour of group 2. CONCLUSIONS:The use of the fast warming protocol is a valid alternative to the classic warming protocol, and allows a reduction in the time needed to carry out the procedure and the workload of embryologists in an IVF laboratory.

Today, women’s infertility is considered a social disease in females, occurring not only as an effect of POF (premature ovarian failure) but also as CTRI (cancer treatment-related infertility) in oncologic patients. Several procedures for FP (fertility preservation) are currently adopted to prevent this condition, mostly based on utilization of retrieved eggs from the patients with subsequent IVF (in vitro fertilization) or cryopreservation. However, great interest has recently been devoted to OSCs (ovarian stem cells), whose isolation from female ovaries, followed by their in vitro culture, led to their maturation to OLCs (oocyte-like cells), namely, neo-oocytes comparable to viable eggs suitable for IVF. Translation of these data to FP clinical application creates new hope in the treatment of infertility. Thus, in line with the significant progress in using stem cells in the regenerative medicine field, neo-oogenesis via OSCs, which is currently unapplicable in fertility preservation procedures, will provide novel possibilities for young and adult females in motherhood programs in the future.

The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

The vaginal microbiota plays a critical role in the health of the female genital tract, and its composition contributes to gynecological disorders and infertility. Lactobacilli are the dominant species in the female genital tract: their production of lactic acid, hydrogen peroxide, and bacteriocins prevents the invasion and growth of pathogenic microorganisms. Several factors such as hormonal changes, age of reproduction, sexual practices, menstrual cycle, pregnancy, and antimicrobial drugs use can cause imbalance and dysbiosis of the vaginal microbiota. This review aims to highlight the impact of the vaginal microbiota in Assisted Reproductive Technology techniques (ART) and it examines the factors that influence the vaginal microbiota, the consequences of dysbiosis, and potential interventions to restore a healthy female genital tract.

Background: In this consensus-based position paper, the scientific societies Fondazione Italiana Linfomi (FIL) and Società Italiana della Riproduzione Umana (SIRU) reviewed main aspects and identified the optimal paths aimed to preserve and monitor fertility in patients diagnosed with lymphoma. Methods: The multi-disciplinary working group for this consensus-based position paper was composed by 5 onco-hematologists, 4 gynecologist or andrologists, 1 embryologist and 1 biologist, with the supervision of an international expert leader. A series of rank-ordering key questions were proposed according to the clinical relevance focusing on patients diagnosed with non-Hodgkin's lymphomas and Hodgkin lymphoma. The agreement among all the Panelists was scored by a web-based questionnaire according to the Delphi methodology (two rounds). All statements were newly discussed in a round robin way and confirmed for the drafting of the final recommendations. Results: Chemotherapy-induced gonadotoxicity is influenced by the type of agent, the dose intensity (high risk of infertility ≥80%, intermediate risk 40%–60%, low risk <20%, very low risk and unknown risk, and in women, also by age at treatment. The correct timing for onco-fertility counseling is as soon as possible, ideally at time of diagnosis, in order to increase patient's awareness and to allow timing optimization to apply FP techniques. An urgent referral pathway needs to be established between the Hematological and The Reproductive Centers with the aim to offer the onco-fertility counseling within 24–48 h. Blood and specialist exams to be performed during the pre-therapy counseling have been summarized. Oocyte cryopreservation is a well-established FP technique that should be proposed to patients after a personalized onco-fertility counseling, and the possibility to delay treatment of 10–14 days. Ovarian tissue cryopreservation could be proposed as a unique technique in patients with: therapeutic urgency, when chemotherapy has to be started within 10–14 day, when there is a high/moderate gonadotoxic risk, and if the patient's clinical conditions are feasible for surgery. To improve the safety of ovarian tissue transplantation for patients in complete and prolonged survival after lymphoma, the ovarian samples have to be analyzed in order to exclude the presence of neoplastic cells by using molecular and histological analyses prior to graft, especially for aggressive NHL histotypes. Post-pubertal males should be offered sperm cryopreservation. GnRHa should not be considered an alternative option for FP with cryopreservation techniques unless for women for whom these latter are contraindicated due to treatment start delay or safety issues. Indications on fertility tests to be carried out in the period following chemotherapy (1–5 years and >5 years) has been also discussed. Conclusion: These recommendations would be useful for clinicians who take care of young lymphoma patients to guarantee an evidence-based onco-fertility assessment and treatment during the oncologic pathway. The research was funded by: Ministry of Health, Italian Government, R.C. funds 2022, to the IRCCS Istituto Tumori "Giovanni Paolo II," Bari-Italy. Keywords: Late Effects in Lymphoma Survivors No conflicts of interests pertinent to the abstract.

Clinical embryologists are highly trained laboratory professionals with multiple roles, including laboratory, clinical, biobanking and quality system management. In most European countries, clinical embryologists are trained to work in Medically Assisted Reproduction (MAR) centres without a specifically dedicated educational path. The criteria required for employment vary according to the educational structure and the public or private nature of the centre. We have herein described the educational profile required by Italian clinical embryologists to work in MAR centres of the National Health System (NHS). Public centres currently represent 36% of all the Italian MAR clinics. According to the Italian law, a future clinical embryologist must achieve a 3-4 year unpaid post-graduate specialization in a different field, choosing from Genetics, Microbiology, Clinical Pathology or Nutrition. Accesses to the above-mentioned post-graduate courses are themselves very limited. Clinical embryologists are basically trained by senior colleagues. This situation makes inevitably difficult to recruit laboratory staff in NHS centres. Moreover, it represents an emblematic example of the need for an equal training curriculum, possibly ensuring a comparable education quality, mobility of trainees and dissemination of skills for clinical embryologists all over Europe.

Introduction:Fertility preservation (FP) and monitoring has considerable relevance in the multidisciplinary approach to cancer patients. In these consensus-based practical recommendations, the scientific societies Fondazione Italiana Linfomi (FIL) and Società Italiana della Riproduzione Umana (SIRU) reviewed the main aspects and identified the optimal paths which aim to preserve and monitor fertility in patients diagnosed with lymphoma at the different phases of the disease and during long-term survivorship. Methods:For the Panel, eleven experts were selected for their expertise in research and clinical practice on onco-fertility and lymphoma. The Panel's activity was supervised by a chairman. A series of rank-ordering key questions were proposed according to their clinical relevance and discussed among the Panel, focusing on patients diagnosed with non-Hodgkin's lymphomas and Hodgkin lymphoma. Agreement among all the Panelists on the content and terminology of the statements was evaluated by a web-based questionnaire according to the Delphi methodology. Results:From the literature review a total of 78 questions or sentences, divided into the 6 areas of interest, were identified. By applying the Gwet's AC, k was: Section 1: 0,934 (Very good); Section 2: 0,958 (Very good); Section 3: 0,863 (Very good); Section 4: 0,649 (Good); Section 5: 0,936 (Very good); Section 6 raw agreement 100%. Two rounds of Delphi allowed to provide the maximum agreement. All statements were newly discussed in a round robin way and confirmed for the drafting of the final recommendations. Discussion:These recommendations would be useful for onco-hematologists, gynecologists, urologists, and general practice physicians who take care of young lymphoma patients to guarantee an evidence-based oncofertility assessment and treatment during the oncologic pathway.

Intracytoplasmic sperm injection (ICSI) was initially introduced to overcome problems due of severe male factor infertility not being solved with conventional in-vitro fertilization (cIVF). However, recent years have witnessed an increasing use of ICSI by most assisted reproductive technique laboratories for non-male factor indications. Examples of the latter include previous fertilization failure after cIVF, few or poor-quality oocytes, immature oocytes, advanced maternal age, preimplantation genetics test (PGT), cryopreserved oocytes, and unexplained infertility. The replacement of cIVF with ICSI in several non-male factor infertility cases is probably because some reproductive specialists consider that ICSI is associated with better reproductive outcomes. Unfortunately, data on reproductive outcomes in favor of ICSI over cIVF are limited or absent. Therefore, the factors that can help define the use of one technique over the other should be identified. These should include the likelihood of fertilization failure, potential risks of the procedure, and its costs. In this review, we aim to highlight the current guidelines, advantages, and limitations of the use of cIVF/ICSI for infertility treatment. Additionally, we provide a comprehensive review of the use of ICSI in indications other than severe male factor infertility.

Follitropin delta (Δ) is a recombinant human follicle-stimulating hormone (rFSH), like natural human FSH, that can stimulate the development and growth of multiple follicles. Treatment with Follitropin-Δ may cause mild to severe adverse reactions, such as the risk of developing ovarian hyperstimulation syndrome, resulting in nausea, vomiting and diarrhea, weight loss, respiratory difficulty, stomach swelling and discomfort of the pelvic area, headaches, and fatigue. To date, the effects of a Follitropin-Δ overdosage are unknown, and no data are reported in the scientific literature or in the drug data sheet. Therefore, this study aimed to describe the effects of Follitropin-Δ overdosages in poorly responding women who underwent IVF cycles. This is a descriptive case series of four nulligravid, poorly responding patients, two of whom made requests for fertility preservation. Four poorly responding patients who were prescribed 20.0 µg/day of Follitropin-Δ for three consecutive days wrongly injected the total cartridge of 72 µg Follitropin-Δ every day. After the incorrect injection of Follitropin-Δ, the patients continued their controlled ovarian stimulation and underwent vaginal ovarian pick up. The analyzed patients had no side effects or adverse reactions. The evaluations reported in this case series showed that the accidental use of 72 µg/day of Follitropin-Δ for three days did not cause side effects or adverse reactions in poor responders.

After the controlled ovarian stimulation (COS), the number of cumulus oocyte complexes collected is lower than predicted. The aim of this study is to understand if there is a possible reason for that deficient ovarian response. It was hypothesized that this is associated with the SNP (single-nucleotide polymorphism) of the FSH receptor (FSHr), specifically c.2039A > G, resulting in Asn680Ser. Two groups of patients were enrolled for this purpose: the normal (n = 36) and abnormal responses (n = 31). To predict the number of retrievable oocytes, according to the anti-Mũllerian hormone (AMH) and the antral follicle count (AFC), the following formula was applied in a log scale: the number of oocytes retrieved = 2.584 − 0.015 × (age) − 0.035 × (FSH) + 0.038 × (AMH) + 0.026 × (AFC). Then, when the number of oocytes collected was less than 50% of the calculated value, it was proposed that the patients result in an abnormal response. DNA sample blood was collected from the women, and then the genetic assessment for the Asn680Ser of the FSHr was evaluated in both groups. The differences between the two categories were statistically analyzed with an independent samples t test, a Mann–Whitney U test and a Chi-squared test. In a patient with an abnormal response, a significant prevalence of the amino acid serine at position 680 of the FSHr compared to the counterpart group (p < 0.05) was detected. In conclusion, according to the results, the genetic evaluation of the FSHr could represent an accurate and predictive feature for patients undergoing assisted reproductive technology treatment.

Aims:The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2',7'-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments. Main methods:The fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2',7'-dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status. Key findings:The 2',7'-dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407-12769) vs. 1356 (range 470-2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage. Significance:Based on our pilot experimental data, the microplate-based 2',7'-dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality.

Background and aims: Failure of the embryo to implant causes about three-fourths of lost pregnancies. Female genital tract microbiota has been associated to Assisted Reproductive Technologies (ART) outcomes. The objective of this study was to analyze the microbiota of human cervical swab and to correlate these findings with the ART outcomes. Materials and Methods: In this study, 88 cervical swabs were collected from women undergoing ART cycles, with various causes of infertility, at the beginning of the ART protocols. After microbial DNA extraction, V3–V4 variable regions of the 16S rRNA gene were amplified and sequenced on the Illumina MiSeq platform. PEnalized LOgistic Regression Analysis (PELORA) was performed to identify clusters of bacterial populations with differential abundances between patients with unfavorable and favorable pregnancy outcome groups, respectively. Results: We identified a core of microorganisms at lower taxonomic levels that were predictive of women’s pregnancy outcomes. Statistically significant differences were identified at species levels with Lactobacillus salivarius, Lactobacillus rhamnosus among others. Moreover the abundance of Lactobacillus crispatus and iners, respectively increased and decreased in favorable group as compared to unfavorable group, resulted within the core of microorganisms associated to positive ART outcome. Although the predominance of lactobacilli is generally considered to be advantageous for ART outcome, we found that also the presence of Bifidobacterium (together with the other lactobacilli) was more abundant in the favorable group. Discussion: Cervix is colonized by microorganisms which can play a role in ART outcomes as seen by an overall decrease in embryo attachment rates and pregnancy rates in both fertile and infertile women. If confirmed in a larger cohort, the abundance of these bacteria can be useful not only as a marker of unfavorable pregnancy outcome but also they may open the way to new interventional strategies based on genital tract microbiota manipulation in order to increase the pregnancy rates in woman undergoing assisted reproductive technologies.

Abstract Background Total fertilization failure represents a particularly frustrating condition for couples undergoing in vitro fertilization. With the aim of reducing the occurrence of total fertilization failure, intracytoplasmic sperm injection (ICSI) has become the first choice over conventional in vitro fertilization (IVF) procedures although evidence of improved results is still debated and its use in couples without male factor infertility is not recommended. Among the strategies potentially useful to promote the use of conventional IVF, we herein call attention to the late rescue ICSI, which consists in performing ICSI after 18–24 h from conventional insemination on oocytes that show no signs of fertilization. This treatment has however been reported to be associated with a low success rate until recent observations that embryos derived from late rescue ICSI may be transferred after cryopreservation in a frozen-thawed cycle with improved results. The aim of the present study was to assess whether frozen embryos deriving from rescue ICSI performed about 24 h after conventional IVF may represent a valuable option for couples experiencing fertilization failure. Methods A systematic review on the efficacy of late rescue ICSI was performed consulting PUBMED and EMBASE. Results Including twenty-two original studies, we showed that clinical pregnancy rate per embryo transfer and implantation rate obtainable with fresh embryo transfers after rescue ICSI are not satisfactory being equal to 10 and 5%, respectively. The transfer of cryopreserved rescue ICSI embryos seems to offer a substantial improvement of success rates, with pregnancy rate per embryo transfer and implantation rate equal to 36 and 18%, respectively. Coupling rescue ICSI with frozen embryo transfer may ameliorate the clinical pregnancy rate for embryo transfer with an Odds Ratio = 4.7 (95% CI:2.6–8.6). Conclusion Results of the present review support the idea that r-ICSI coupled with frozen embryo transfer may overcome most of the technical and biological issues associated with fresh transfer after late r-ICSI, thus possibly representing an efficient procedure for couples experiencing fertilization failure following conventional IVF cycles. Trial registration Prospero registration ID: CRD42021239026 .

Novel anti-cancer treatments have improved the survival rates of female young patients, reopening pregnancy issues for female cancer survivors affected by the tumor treatment-related infertility. This condition occurs in approximately one third of women of fertile age and is mainly dependent on gonadotoxic protocols, including radiation treatments. Besides routine procedures such as the hormonal induction of follicular growth and subsequent cryopreservation of oocytes or embryos, the ovarian protection by gonadotropin-releasing hormone (GnRH) agonists during chemotherapy as well as even gonadal shielding during radiotherapy, other innovative techniques are available today and need to be optimized to support their introduction into the clinical practice. These novel methods are hormone stimulation-free and include the ovarian cortex cryopreservation before anti-cancer treatments and its subsequent autologous reimplantation and a regenerative medicine approach using oocytes derived in vitro from ovarian stem cells (OSCs). For both procedures, the major benefit is related to the prompt recruitment and processing of the ovarian cortex fragments before gonadotoxic treatments. However, while the functional competence of oocytes within the cryopreserved cortex is not assessable, the in vitro maturation of OSCs to oocytes, allows to select the most competent eggs to be cryopreserved for fertility restoration.

The manuscript by Berkovitz et al . (1), re-opens the debate on a niche, but important, issue in in vitro fertilization concerning the cryopreservation of a very small number of spermatozoa form an ejaculate or with a surgical origin.