Time-lapse embryo monitoring: does it add to standard in-vitro fertilization/intra cytoplasmic sperm injection?

Since the first pioneering studies on time-lapse systems (TLSs) for embryo incubation, many things have changed. Two main factors influence the development of modern time-lapse incubators for human in-vitro fertilization (IVF): 1) the switch from traditional cell culture incubators to benchtops incubators, more suitable for human IVF; and 2) the improvement of imaging technology. Another major factor for the increase in the utilization of TLSs in IVF labs over the last decade was the advances in computer/wireless and smartphone/tablet technology, which allowed patients to see the footage of their growing embryos. Hence, more user-friendly features have allowed their introduction and routine use in IVF labs while image-capturing software has enabled storage and providing additional information to the patients concerning the development of their embryos. This review aims to describe the history and the different TLSs available in the market, to summarize the research and clinical results obtained by using this technology, and to reflect on how this technology is changing the modern IVF laboratory. The current limitations of TLSs will be also reviewed.

Oocytes evaluation and in-vitro fertilization/intra cytoplasmic sperm injection outcomes.

The identification of markers capable of evaluating oocyte quality, its maturation, function, and embryo progression and implantation potential has frequently initiated research interest. However, to date, univocal criteria of oocyte competence do not exist. A major cause of low oocyte quality is evidently advanced maternal age. However, other factors may influence oocyte competence. Among these are obesity, lifestyle factors, genetic and systematic pathologies, ovarian stimulation protocols, laboratory procedures, culture, and environmental conditions. The morphological and maturational evaluation of oocytes is probably the most widely used. Several morphological features, both cytoplasmic (cytoplasmic pattern and hue, presence of vacuoles, refractile bodies, granulation, and smooth endoplasmic reticulum clusters) and extra-cytoplasmic (perivitelline space, zona pellucida thickness, oocyte shape, and polar bodies), have been proposed to distinguish oocytes with the best reproductive potential among a cohort. No single abnormality seems to be sufficiently predictive of the developmental capacity of the oocyte. Some abnormalities such as cumulus cells dysmorphisms, central granulation, vacuoles, and smooth endoplasmic reticulum clusters, however, seem to be associated with poor developmental potential of the embryo, although oocyte dysmorphisms are very common and the data in the literature is limited and provide conflicting views. Other criteria involving gene expression of cumulus cells as well as the metabolomic analysis of spent culture media have been explored. Also, sophisticated technologies such as polar bodies biopsy, meiotic spindle visualization, mitochondrial activity, oxygen consumption, and measurement of glucose-6-phosphate dehydrogenase activity have been proposed. Many of these approaches, however, remain largely research-based and have not found widespread application in clinical service. Due to the lack of consistent data for the assessment of oocyte quality and competence, probably oocyte morphology and oocyte maturity remain important indicators to determine oocyte quality. The aim of this review was to provide spherical attributes and evidence on recent and present research on the topic by analyzing the current methods for evaluation of the oocyte quality, and the impact of oocyte quality on reproductive outcomes. Additionally, current limitations of oocyte quality evaluation are highlighted and insights on future research are provided to optimize the selection techniques of oocytes to improve ART outcomes.

The number and rate of euploid blastocysts in women undergoing IVF/ICSI cycles are strongly dependent on ovarian reserve and female age.

STUDY QUESTION: Can the possibility of having at least one euploid blastocyst for embryo transfer and the total number of euploid blastocysts be predicted for couples before they enter the IVF programme? SUMMARY ANSWER: Ovarian reserve and female age are the most important predictors of having at least one euploid blastocyst and the total number of euploid blastocysts. WHAT IS KNOWN ALREADY: The blastocyst euploidy rate among women undergoing ART has already been shown to significantly decrease with increasing female age, and the total number of euploid embryos is dependent on the blastocyst cohort size. However, the vast majority of published studies are based on retrospective analysis of data. STUDY DESIGN, SIZE, DURATION: This prospective analysis included 847 consecutively enrolled couples approaching their first preimplantation genetic testing for aneuploidies (PGT-A) cycle between 2017 and 2020. Only couples for whom ejaculated sperm was available and women with a BMI of <35 kg/m2 were included in the study. Only the first cycle was included for each patient. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at an IVF centre where, for all patients, the planned treatment was to obtain embryos at the blastocyst stage for the PGT-A programme. The impact of the following covariates was investigated: a woman's serum AMH level, age, height, weight and BMI and a man's age, height, weight, BMI, sperm volume and sperm motility and morphology. The analysis was performed with a machine learning (ML) approach. Models were fit on the training set (677 patients) and their predictive performance was then evaluated on the test set (170 patients). MAIN RESULTS AND ROLE OF CHANCE: After ovarian stimulation and oocyte insemination, 40.1% of couples had at least one blastocyst available for the PGT-A. Of 1068 blastocysts analysed, 33.6% were euploid. Two distinct ML models were fit: one for the probability of having at least one euploid blastocyst and one for the number of euploid blastocysts obtained. In the training set of patients, the variable importance plots of both models indicated that AMH and the woman's age are by far the most important predictors. Specifically, a positive association between the outcome and AMH and a negative association between the outcome and female age appeared. Gradient-boosted modelling offers a greater predictive performance than generalized additive models (GAMs). LIMITATIONS, REASONS FOR CAUTION: The study was performed based on data from a single centre. While this provides a robust set of data with a constant ART process and laboratory practice, the model might be suitable only for the evaluated population, which may limit the generalization of the model to other populations. WIDER IMPLICATIONS OF THE FINDINGS: ML models indicate that for couples entering the IVF/PGT-A programme, ovarian reserve, which is known to vary with age, is the most important predictor of having at least one euploid embryo. According to the GAM, the probability of a 30-year-old woman having at least one euploid embryo is 28% or 47% if her AMH level is 1 or 3 ng/ml, respectively; if the woman is 40 years old, this probability is 18% with an AMH of 1 ng/ml and 30% with an AMH of 3 ng/ml. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by an unrestricted grant from Gedeon Richter. The authors declared no conflict of interests. TRIAL REGISTRATION NUMBER: N/A.

Can sperm quality influence embryo development and its ploidy? Analysis of 811 blastocysts obtained from different sperm sources.

The aim of our study was to evaluate the correlation between sperm quality and ploidy status of the derived blastocysts. We performed a retrospective analysis on a restricted pool of patients enrolling only those who had no female factors. Male patients with genetic factors affecting spermatogenesis were also excluded. We chose a maternal age ≤38 years to decrease the female factor, therefore the male factor was the main component of sterility. We divided the patients in four groups based on semen quality and comparing fertilization, pregnancy and euploidy rates above all. In total, 201 intracytoplasmic sperm injection (ICSI) cycles were enrolled in the study. Cycles were divided into four groups, according to semen source: normal semen, oligoasthenoteratozoospermia (OAT), cryptospermia or non-obstructive azoospermia (NOA). An extremely statistically lower fertilization rate was found in NOA patients. Unexpectedly, no differences were detected in blastocyst formation, euploidy, aneuploidy and mosaicism rates among the four groups. Interestingly, we also found a higher abortion rate comparing NOA to normal semen with an odds ratio of 4.67. In our study no statistically significant differences among the analyzed groups were found, showing little or no effect at all using spermatozoa from different semen sources or quality. This may be linked to the oocyte competence of fixing sperm DNA damage and it could be hypothesized that only sperm with a good rate of DNA integrity are able to fertilize the oocyte, explaining why poor quality semen is reflected in a low fertilization rate without effect on ploidy.

Which factors influence the success rate of egg donation programmes with imported vitrified oocytes?

RESEARCH QUESTION: Which factors influence the success rate of egg donation programmes with imported vitrified oocytes? DESIGN: Observational longitudinal cohort study of 431 oocyte donation cycles conducted between January 2015 and February 2019. A total of 398 couples underwent an IVF cycle with imported donated vitrified eggs. All consecutive oocyte donation cycles conducted at the Centre for Reproductive Medicine of the European Hospital in Rome, Italy, were included. RESULTS: A univariable analysis was conducted. Among the demographic characteristics studied, body mass index (BMI) was significantly able to influence outcome. In 49% of unsuccessful cycles, men were overweight, compared with 39.4% in cycles with a positive beta-HCG test (P = 0.03). The relationship between female or donor BMI and treatment outcome, however, was not statistically significant. The day on which the embryo is transferred affects the outcome of the cycle: transferring on day 2 versus day 5 reduces the probability of clinical pregnancy. The relationship between male BMI classes and semen parameters was studied: obese men have a significantly lower sperm concentration than normal-weight men (P = 0.006 after Bonferroni correction). No statistically significant differences were found in semen volume (P = 0.722), sperm morphology (P = 0.100) and motility (P = 0.179) in obese men compared with normal weight men. CONCLUSION: In addition to the number of oocytes available after warming, male BMI, semen characteristics and blastocyst transfer are critical parameters able to influence the reproductive outcomes of egg donation programmes with imported vitrified oocytes.

The paradox of the Italian clinical embryologist in the national public health system: hints towards harmonization of a postgraduate educational curriculum.

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.

Cross-Validation of Next-Generation Sequencing Technologies for Diagnosis of Chromosomal Mosaicism and Segmental Aneuploidies in Preimplantation Embryos Model.

Detection of mosaic embryos is crucial to offer more possibilities of success to women undergoing in vitro fertilization (IVF) treatment. Next Generation Sequencing (NGS)-based preimplantation genetic testing are increasingly used for this purpose since their higher capability to detect chromosomal mosaicism in human embryos. In the recent years, new NGS systems were released, however their performance for chromosomal mosaicism are variable. We performed a cross-validation analysis of two different NGS platforms in order to assess the feasibility of these techniques and provide standard parameters for the detection of such aneuploidies. The study evaluated the performance of MiseqTM Veriseq (Illumina, San Diego, CA, USA) and Ion Torrent Personal Genome Machine PGMTM ReproSeq (Thermo Fisher, Waltham, MA, USA) for the detection of whole and segmental mosaic aneuploidies. Reconstructed samples with known percentage of mosaicism were analyzed with both platforms and sensitivity and specificity were determined. Both platforms had high level of specificity and sensitivity with a Limit Of Detection (LOD) at ≥30% of mosaicism and a showed a ≥5.0 Mb resolution for segmental abnormalities. Our findings demonstrated that NGS methodologies are capable of accurately detecting chromosomal mosaicism and segmental aneuploidies. The knowledge of LOD for each NGS platform has the potential to reduce false-negative and false-positive diagnoses when applied to detect chromosomal mosaicism in a clinical setting.

Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use.

OBJECTIVE: To study how the attributes of mosaicism identified during preimplantation genetic testing for aneuploidy relate to clinical outcomes, in order to formulate a ranking system of mosaic embryos for intrauterine transfer. DESIGN: Compiled analysis. SETTING: Multi-center. PATIENT(S): A total of 5,561 euploid blastocysts and 1,000 mosaic blastocysts used in clinical transfers in patients undergoing fertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation (gestational sac), ongoing pregnancy, birth, and spontaneous abortion (miscarriage before 20 weeks of gestation). RESULT(S): The euploid group had significantly more favorable rates of implantation and ongoing pregnancy/birth (OP/B) compared with the combined mosaic group or the mosaic group affecting only whole chromosomes (implantation: 57.2% vs. 46.5% vs. 41.8%; OP/B: 52.3% vs. 37.0% vs. 31.3%), as well as lower likelihood of spontaneous abortion (8.6% vs. 20.4% vs. 25%). Whole-chromosome mosaic embryos with level (percent aneuploid cells) <50% had significantly more favorable outcomes than the ≥50% group (implantation: 44.5% vs. 30.4%; OP/B: 36.1% vs. 19.3%). Mosaic type (nature of the aneuploidy implicated in mosaicism) affected outcomes, with a significant correlation between number of affected chromosomes and unfavorable outcomes. This ranged from mosaicism involving segmental abnormalities to complex aneuploidies affecting three or more chromosomes (implantation: 51.6% vs. 30.4%; OP/B: 43.1% vs. 20.8%). Combining mosaic level, type, and embryo morphology revealed the order of subcategories regarding likelihood of positive outcome. CONCLUSION(S): This compiled analysis revealed traits of mosaicism identified with preimplantation genetic testing for aneuploidy that affected outcomes in a statistically significant manner, enabling the formulation of an evidence-based prioritization scheme for mosaic embryos in the clinic.

Cryopreserved Gamete and Embryo Transport: Proposed Protocol and Form Templates-SIERR (Italian Society of Embryology, Reproduction, and Research).

Introduction: In Italy, the transport of cryopreserved biological material is controlled by several Decrees (Legislative Decree No. 191/2007 and No. 16/2010 and Health Ministry's Decree of October 10, 2012). Given the nature of their applications, the transport of reproductive cells has peculiar quality and safety requirements that must be applied universally, minimizing the chance of error. To standardize the cross-border shipping procedure to meet the quality, traceability, and safety criteria for cells and tissues, it is appropriate to establish a unified process using the same tools, forms, and communication channels. Methods: A working group has been created by SIERR. This "FOCUS Group" was constituted by representatives from Italian-assisted reproductive technology centers and sperm banks who worked together to define joint procedural steps and create specific forms to support the movement of cryopreserved samples. Results: The FOCUS Group identified the critical steps in the communication procedures between Italian centers and created the related forms: patient authorization, request from the recipient center, critical checks carried out by both sending and recipient centers, start of samples transfer, collection, transport and taking responsibility of the biological material, acknowledgment of samples arrival, and acknowledgement of any adverse event that occurred. Discussion: Indications on shipping between tissue institutions and legal responsibilities are important points and a working protocol with shared transport forms has been defined. Standard Operating Procedures are necessary in light of the increasingly widespread movement of biological samples between the various countries, and represent a valid means of support for the patients who could have a higher awareness of safety and traceability during each stage of gamete transport.

When embryology meets genetics: the definition of developmentally incompetent preimplantation embryos (DIPE)-the consensus of two Italian scientific societies.

A clear definition of developmentally incompetent preimplantation embryo (DIPE) in literature is still missing, while several scientific societies are discussing this challenging topic. From both a clinical and scientific perspective, the identification of embryos unfit for reproductive purpose is crucial. This aim should be pursued in light of all diagnostic technologies for embryo evaluation, encompassing also genetic analyses, of recent implementation in IVF. The Italian context is characterized by an unusual scenario: embryos can be discarded only if not viable and cannot be used for research purposes either. Therefore, thousands of embryos, diagnosed as affected and/or aneuploid as resulting from preimplantation genetic testing (PGT) and clinically not utilizable, are cryopreserved and stored indefinitely, with important psychological, legal, and financial implications. With the aim of updating the definition of DIPE, also on the basis of the embryo genetic status, the Italian Society of Embryology, Reproduction and Research (SIERR) and the Italian Society of Human Genetic (SIGU) reviewed the literature on this topic, found a consensus, and produced a list of relevant criteria.

The clinical use of time-lapse in human-assisted reproduction.

A major challenge in the assisted reproduction laboratory is to set up reproducible and efficient criteria to identify the embryo with the highest developmental potential. Over the years, several methods have been used worldwide with this purpose. Initially, standard morphology assessment was the only available strategy. It is now universally recognized that besides being a very subjective embryo selection strategy, morphology evaluation alone has a very poor prognostic value. More recently, the availability of time-lapse incubators allowed a continuous monitoring of human embryo development. This technology has spread quickly and many fertility clinics over the world produced a remarkable amount of data. To date, however, a general consensus on which variables, or combination of variables, should play a central role in embryo selection is still lacking. Many confounding factors, concerning both patient features and clinical and biological procedures, have been observed to influence embryo development. In addition, several studies have reported unexpected positive outcomes, even in the presence of abnormal developmental criteria. While it does not seem that time-lapse technology is ready to entirely replace the more invasive preimplantation genetic testing in identifying the embryo with the highest implantation potential, it is certainly true that its application is rapidly growing, becoming progressively more accurate. Studies involving artificial intelligence and deep-learning models as well as combining morphokinetic with other non-invasive markers of embryo development, are currently ongoing, raising hopes for its successful applicability for clinical purpose in the near future. The present review mainly focuses on data published starting from the first decade of 2000, when time-lapse technology was introduced as a routine clinical practice in the infertility centers.

Phthalates and Bisphenol A: Presence in Blood Serum and Follicular Fluid of Italian Women Undergoing Assisted Reproduction Techniques.

BACKGROUND: folliculogenesis is a strictly regulated process that may be affected by endocrine disrupting chemicals (EDCs) through sometimes not so clear molecular mechanisms. METHODS: we conducted a multicentric observational study involving six fertility centers across Italy, prospectively recruiting 122 women attending a fertility treatment. Recruited women had age ≤42 years, and normal ovarian reserve. Blood and follicular fluid samples were taken for EDCs measurement using liquid chromatography tandem mass spectrometry and each woman completed an epidemiological questionnaire. RESULTS: The main EDCs found were monobutyl phthalate (MBP) (median blood: 8.96 ng/mL, follicular fluid 6.43 ng/mL), monoethylhexyl phthalate (MEHP) (median blood: 9.16 ng/mL, follicular fluid 7.68 ng/mL) and bisphenol A (BPA) (median blood: 1.89 ng/mL, follicular fluid 1.86 ng/mL). We found that serum MBP concentration was significantly associated with the considered area (p < 0.001, adj. mean: 7.61 ng/mL, 14.40 ng/mL, 13.56 ng/mL; Area 1: Milan-Turin, Area 2: Rome-Naples; Area 3: Catania-Bari, respectively) but negatively with home plastic food packaging (p = 0.004). Follicular MBP was associated with irregular cycles (p = 0.019). No association was detected between EDCs and eating habits and other clinical and epidemiological features. CONCLUSIONS: This study represents the first Italian biomonitoring of plastic EDCs in follicular fluid, laying the basis for future prospective evaluation on oocyte quality before assisted reproduction techniques (ART).

Preimplantation Genetic Testing: Where We Are Today.

BACKGROUND: Preimplantation genetic testing (PGT) is widely used today in in-vitro fertilization (IVF) centers over the world for selecting euploid embryos for transfer and to improve clinical outcomes in terms of embryo implantation, clinical pregnancy, and live birth rates. METHODS: We report the current knowledge concerning these procedures and the results from different clinical indications in which PGT is commonly applied. RESULTS: This paper illustrates different molecular techniques used for this purpose and the clinical significance of the different oocyte and embryo stage (polar bodies, cleavage embryo, and blastocyst) at which it is possible to perform sampling biopsies for PGT. Finally, genetic origin and clinical significance of embryo mosaicism are illustrated. CONCLUSIONS: The preimplantation genetic testing is a valid technique to evaluated embryo euploidy and mosaicism before transfer.

Comparison of euploidy rates of blastocysts in women treated with progestins or GnRH antagonist to prevent the luteinizing hormone surge during ovarian stimulation.

STUDY QUESTION: Does the prevalence of euploid blastocysts differ between patients treated with progestin primed ovarian stimulation (PPOS) and those treated with conventional ovarian stimulation? SUMMARY ANSWER: The numbers of blastocysts and euploid blastocysts per patient and the number of euploid embryos per injected oocyte are similar for patients undergoing progestin-primed ovarian stimulation and for those undergoing conventional ovarian stimulation with GnRH antagonist. WHAT IS KNOWN ALREADY: New approaches to ovarian stimulation have been developed based on the use of drugs administrable by mouth instead of via injections. Attention has been dedicated to progestins to block the LH surge. Previous data regarding the number of oocytes retrieved and the number of good-quality embryos generated in PPOS have demonstrated similar outcomes when compared to conventional ovarian stimulation, even if some concerns regarding the quality of embryos have been advanced. STUDY DESIGN, SIZE, DURATION: This is a prospective non-inferiority age-matched case-control study. In a period of 6 months, a total of 785 blastocysts from 1867 injected oocytes obtained from 192 patients were available for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile women undergoing IVF and preimplanation genetic testing (PGT) cycles were included. Forty-eight patients were treated with PPOS, and for each of them three age-matched historical controls (n = 144) treated with a GnRH antagonist protocol were selected. PGT was performed according to next-generation sequencing technology. MAIN RESULTS AND THE ROLE OF CHANCE: Basal characteristics were similar in the two groups; a substantial similarity of the main outcome measures in the two treatment groups has also been found. The rate of formation of euploid blastocysts per oocyte was 21% in both the two treatment groups. The percentage of patients with euploid embryos and the total number of euploid blastocysts per patient (median and interquartile range, IQR) in the PPOS group were 38.7 (25.5-52.9) and 2 (1.3-3.1), respectively. These figures were not significantly different in women treated with the GnRH antagonist protocol i.e. 42 (28-53.8) and 2.1 (1.3-2.9), respectively. LIMITATIONS, REASONS FOR CAUTION: This was a case-control study which may limit the reliability of the main findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results encourage the use of PPOS, especially for oocyte donation, for fertility preservation and for patients in which total freezing of embryos is foreseen, for those expected to be high responders or candidates for preimplantation genetic testing. However, studies aiming to investigate the effect of PPOS on the live birth rate are warranted. STUDY FUNDING/COMPETING INTEREST(S): None.

Time of morulation and trophectoderm quality are predictors of a live birth after euploid blastocyst transfer: a multicenter study.

OBJECTIVE: To investigate whether the morphodynamic characterization of a euploid blastocyst's development allows a higher prediction of a live birth after single-embryo-transfer (SET). DESIGN: Observational cohort study conducted in two phases: training and validation. SETTING: Private in vitro fertilization centers. PATIENT(S): Euploid blastocysts: 511 and 319 first vitrified-warmed SETs from 868 and 546 patients undergoing preimplantation genetic testing for aneuploidies (PGT-A) in the training and validation phase, respectively. INTERVENTION(S): Data collected from time of polar body extrusion to time of starting blastulation, and trophectoderm and inner-cell-mass static morphology in all embryos cultured in a specific time-lapse incubator with a continuous medium. Logistic regressions conducted to outline the variables showing a statistically significant association with live birth. In the validation phase, these variables were tested in an independent data set. MAIN OUTCOME MEASURE(S): Live births per SET. RESULT(S): The average live birth rate (LBR) in the training set was 40% (N = 207/511). Only time of morulation (tM) and trophectoderm quality were outlined as putative predictors of live birth at two IVF centers. In the validation set, the euploid blastocysts characterized by tM <80 hours and high-quality trophectoderm resulted in a LBR of 55.2% (n = 37/67), while those with tM ≥ 80 hours and a low-quality trophectoderm resulted in a LBR of 25.5% (N = 13/51). CONCLUSION(S): Time of morulation and trophectoderm quality are better predictors of a euploid blastocyst's reproductive competence. Our evidence was reproducible across different centers under specific culture conditions. These data support the crucial role of morulation for embryo development, a stage that involves massive morphologic, cellular, and molecular changes and deserves more investigation.

How 1 h of abstinence improves sperm quality and increases embryo euploidy rate after PGT-A: a study on 106 sibling biopsied blastocysts.

PURPOSE: The aim of our study was to evaluate the influence of different ejaculatory abstinence time frames (several days versus 1 h) on semen parameters, blastocysts ploidy rate, and clinical results in assisted reproduction cycles on sibling oocytes. METHODS: This is a prospective study including 22 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between November 2015 and December 2018. Male partners with oligoastenoteratozoospermia produced two semen samples on the day of oocyte retrieval: the first one after several days of abstinence and the second, 1 h after the first one. Oocytes from each patient were divided into two groups: those in group 1 were injected with spermatozoa from the first ejaculate (N = 121) and oocytes in group 2 with spermatozoa from the second one (N = 144). Outcomes of aniline blue test, fertilization, blastocyst formation, ploidy rates, and clinical results were compared between the two groups. RESULTS: Semen volume resulted lower in the second sperm retrieval. Sperm concentration, motility, and morphology were similar in the two groups. A total of 106 blasotcysts were biospied. Higher blastocyst euploidy rates resulted in group 2 (43.6%) than in group 1 (27.5%). A higher percentage of mature chromatine was observed in group 2. CONCLUSION: Using spermatozoa from samples with a shorter abstinence could be a simple method to select higher quality spermatozoa, reducing aneuploidy rate in blastocysts. Prospective randomized controlled trials should be performed to confirm the potential advantage of using semen samples with short abstinence period to improve the outcome of assisted reproduction cycles.

The aging male: Relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time.

OBJECTIVE: the aim of our study was to put forward insights to treat any possible correlation among sperm quality, sperm DNA damage and male age as they may have fertility implications for men who choose to delay fatherhood. MATERIALS AND METHODS: Our study is a non-interventional retrospective analysis of 3124 semen samples from patients that were investigated for the conventional semen parameters. Tunel test assay was set up for the evaluation of the sperm DNA fragmentation index (DFI). We applied the Kappa index to compare both the 1999 and the 2010 World Health Organization (WHO) reference criteria to evaluate the competence of such semen parameters categorization during the standard routine of our laboratory. RESULTS: With regards to our findings, it is possible to underline a significant relationship between aging and semen volume (p = 0.001), motility (p = 0.009), semen viscosity (p < 0.003) and sperm DNA damage (p < 0.009). We found a trend when focusing on the semen concentration (p = 0.05). The analysis of sperm morphology did not show any influence with advancing age (p = 0.606). When comparing both the 1999 and the 2010 WHO scales we found no accordance in the appraisal of sperm morphology but a very good one in the evaluation of the other parameters. CONCLUSIONS: Conventional semen analysis represents the opportunity to draw up a proxy insight on the male fertility status even if semen quality can only indirectly assess the probability of pregnancy. Several studies have verified a decay in the male reproductive system, sperm quality and fertility with advancing age although the reported results are not yet conclusive. Our results substantially agree with those findings outlined in the literature. Moreover we find that the discrepancy between the two WHO reference scales would eventually lead to an improper diagnosis of infertility.

Testicular histopathology, semen analysis and FSH, predictive value of sperm retrieval: supportive counseling in case of reoperation after testicular sperm extraction (TESE).

BACKGROUND: To provide indicators for the likelihood of sperm retrieval in patients undergoing testicular sperm extraction is a major issue in the management of male infertility by TESE. The aim of our study was to determine the impact of different parameters, including testicular histopathology, on sperm retrieval in case of reoperation in patients undergoing testicular sperm extraction. METHODS: We retrospectively analyzed 486 patients who underwent sperm extraction for intracytoplasmic sperm injection and testicular biopsy. Histology was classified into: normal spermatogenesis; hypospermatogenesis (reduction in the number of normal spermatogenetic cells); maturation arrest (absence of the later stages of spermatogenesis); and Sertoli cell only (absence of germ cells). Semen analysis and serum FSH, LH and testosterone were measured. RESULTS: Four hundred thirty patients had non obstructive azoospermia, 53 severe oligozoospermia and 3 necrozoospermia. There were 307 (63%) successful sperm retrieval. Higher testicular volume, lower levels of FSH, and better histological features were predictive for sperm retrieval. The same parameters and younger age were predictive factors for shorter time for sperm recovery. After multivariable analysis, younger age, better semen parameters, better histological features and lower values of FSH remained predictive for shorter time for sperm retrieval while better semen and histology remained predictive factors for successful sperm retrieval. The predictive capacity of a score obtained by summing the points assigned for selected predictors (1 point for Sertoli cell only, 0.33 points for azoospermia, 0.004 points for each FSH mIU/ml) gave an area under the ROC curve of 0.843. CONCLUSIONS: This model can help the practitioner with counseling infertile men by reliably predicting the chance of obtaining spermatozoa with testicular sperm extraction when a repeat attempt is planned.