Towards a more sustainable balance between optimal live birth rate and supernumerary embryos in ART treatments.

PURPOSE: To assess the relation between number of inseminated oocytes and cumulative live birth rate (CLBR) in order to provide guidance for limiting the number of surplus blastocysts. METHODS: The study was a retrospective, single-center cohort analysis of 1223 ART complete cycles. Cycles were stratified according to female age (≤ 34, 35-38, and 39-42 years) and number of inseminated oocytes (1-5, 6-10, and > 10). Inclusion criteria were indication for IVF/ICSI with own spermatozoa and blastocyst culture up to day 6 of all embryos. RESULTS: In patients younger than 35 years, insemination of more than ten oocytes produced an increase in overall blastocyst number, CLBR (40.3%, 54.3%, and 75.8%, respectively, for each oocyte group) and surplus embryo rate (12.9%, 27.8%, and 49.7% of cases for each group). Instead, in the middle age group, the use of more than ten oocytes was solely associated with an increase in the rate of surplus embryos (1.25%, 21.33%, and 28.68% of cases after stratification for oocyte number). In older patients, neither CLBR (9.1%, 23.9%, and 24.7%, respectively) nor rate of surplus embryos (2.0%, 7.1%, and 13.4% of cases for each group) were higher in cycles with more than ten inseminated oocytes. CONCLUSION: In women up to 38 years, sustainable CLBR are achieved while limiting the number of inseminated oocytes and the resulting blastocysts remaining unused. Based on this notion, novel treatment strategies could pursue high outcome rates, while alleviating the problems derived from surplus stored embryos.

Human 1PN and 3PN zygotes recapitulate all morphokinetic events of normal fertilization but reveal novel developmental errors.

STUDY QUESTION: Does mono- (1PN) and tri-pronuclear (3PN) fertilization recapitulate the morphokinetic changes of normal bi-pronuclear (2PN) fertilization? SUMMARY ANSWER: Abnormal fertilization retraces the overall choreography of normal fertilization but reveals novel morphokinetic phenomena and raises scientifically and clinically relevant questions. WHAT IS KNOWN ALREADY: ART has allowed the extracorporeal observation of early human development. Time-lapse technology (TLT) has revealed the complexity of the morphokinetic changes underpinning fertilization and the importance of this process for the genetic and cellular integrity of the embryo. Abnormal fertilization has remained neglected, despite its relevance to the physiology and pathology of early human development. STUDY DESIGN, SIZE, DURATION: This retrospective study involved TLT observation of normally (2PN, N = 2517) and abnormally (1PN, N = 41; 3PN, N = 27) fertilized oocytes generated in ICSI cycles performed between October 2019 and December 2020. Oocyte retrieval was carried out after clomiphene citrate-based minimal ovarian stimulation. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 1231 couples treated for diverse infertility causes. The fraction of male factor cases was substantial (36.1%). Microinjected oocytes were assessed by a combined TLT-culture system. Oocytes not suitable for TLT assessment, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared between groups. MAIN RESULTS AND THE ROLE OF CHANCE: Extrusion of the second polar body (PBII) was observed in almost all 2PN/1PN (99.9% and 100.0%, respectively) and in a vast majority of 3PN zygotes (92.1%). Rates of PBII fusion with the ooplasm were much higher in 1PN and 3PN zygotes (P < 0.0001 versus 2PN). The cytoplasmic wave was observed not only in 2PN and 3PN but also in 1PN zygotes (positivity rates of 99.8% and 100% and 82.9%, respectively; P < 0.0001). More rarely, 2PN and 1PN zygotes emitted a third polar body (PBIII). The average times of this event were comparable. The presence and position of the cytoplasmic halo were comparable among the three classes of zygotes. In the 1PN group, the single PN was maternally or paternally derived in 17 and 24 zygotes, respectively, while in the vast majority of 3PN zygotes (121/127) the supernumerary PN was of maternal origin. Average times of maternal PN appearance were comparable, while average times of paternal PN appearance were delayed in 3PN zygotes (P = 0.0127). Compared with the control group, the area of the maternal PN was larger in 1PN zygotes, but smaller in 3PN zygotes (P < 0.0001). The paternal PNs displayed the same trend (P < 0.0001), although such values were consistently smaller than maternal PNs. The area of the third PN in the 3PN group was on average more than 50% smaller than those of maternal and paternal PNs. In maternal PNs of 3PN zygotes, nucleolus precursor bodies (NPBs) aligned along the area of PN juxtaposition at a lower rate compared with the 2PN group. The rate of NPB alignment was ∼50% smaller in 1PN zygotes (P = 0.0001). In paternal PNs, the rates of NPB alignment were not statistically different among the three groups. Asynchronous PN breakdown was increased in 3PN compared with 2PN zygotes (P = 0.0026). In 1PN zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 h at the time of the first cleavage (P < 0.0001). Higher rates of abnormal cleavage patterns and blastomere fragmentation (P < 0.0001) were observed in 1PN compared to 2N and 3PN zygotes. Cleavage progression was increasingly affected after abnormal fertilization, especially 1PN, finally resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN, 1PN and 3PN embryos, respectively (P < 0.0001). Both maternal and paternal ages were higher in cases involving 3PN fertilization. LIMITATIONS, REASONS FOR CAUTION: The study data were obtained from ICSI, but not standard IVF, treatments carried out in a single centre. The study findings therefore require independent verification. WIDER IMPLICATIONS OF THE FINDINGS: This study reports the first detailed morphokinetic map of human abnormal fertilization. Collectively, this evidence prompts new scientific hypotheses and raises clinical questions relevant to the aetiology and the treatment of abnormal fertilization. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Fine-tuning IVF laboratory key performance indicators of the Vienna consensus according to female age.

PURPOSE: To test the validity of the Vienna consensus laboratory key performance indicators (KPIs) to monitor the outcome of treatments involving women of different age ranges. METHODS: The retrospective cohort study included 862 complete IVF/ICSI cycles carried out between January 2014 and May 2021. All embryos of each cycle cohort were subject to extended culture. The overall population was divided into two groups according to female age: the Vienna consensus (≤ 39 years) and older female age (≥ 40 years). We compared outcomes of a selection of the Vienna performance indicators (PIs) and KPIs, with a focus on measures relevant to embryo cleavage and blastocyst formation. A possible association between total good blastocyst development rate (TGBDR) and cumulative clinical pregnancy rate (CPR) was also assessed. RESULTS: No differences were observed in fertilization and embryo cleavage KPIs between the Vienna consensus and the older female age group (standard IVF fertilization, 67.2 vs. 67.3; ICSI fertilization, 72.3 vs. 75.3; day 2 development, 57.6% vs 58.7%; day 3 development, 52.4% vs. 50.7%, respectively). TGBDR was lower in the older female age group (45.5% vs. 33.4% p < 0.001). Multivariate logistic regression analysis indicated female age as a factor independently associated with TGBDR. Clinical outcomes significantly decreased with increasing female age. CONCLUSION: The study suggests that, while most laboratory outcome measures are reliably applicable irrespective of female age, KPIs describing extended embryo culture should be fine-tuned in consideration of older female age.

The Association of Kinetic Variables with Blastocyst Development and Ploidy Status.

BACKGROUND: Despite a plethora of studies conducted so far, a debate is still unresolved as to whether TLM can identify predictive kinetic biomarkers or algorithms universally applicable. Therefore, this study aimed to elucidate if there is a relationship between kinetic variables and ploidy status of human embryos or blastocyst developmental potential. METHODS: For conducting this retrospective cohort study, the normal distribution of data was verified using Kolmogorov-Smirnov test with the Lilliefors' amendment and the Shapiro-Wilk test. Kinetic variables were expressed as median and quartiles (Q1, Q2, Q3, Q4). Mann-Whitney U-test was used to compare the median values of parameters. Univariate and multiple logistic regression models were used to assess relationship between blastocyst developmental potential or ploidy status and kinetics. Several confounding factors were also assessed. RESULTS: Blastocyst developmental potential was positively correlated with the t4-t3 interval (s2) (OR=1.417, 95% CI of 1.288-1.560). s2 median value was significantly different between high- and low-quality blastocysts (0.50 and 1.33 hours post-insemination, hpi, respectively; p=0.003). In addition, timing of pronuclear appearance (tPNa) (OR=1.287; 95% CI of 1.131-1.463) had a significant relationship with ploidy changes. The median value of tPNa was statistically different (p=0.03) between euploid and aneuploid blastocysts (Euploid blastocysts=8.9 hpi; aneuploid blastocysts=10.3 hpi). CONCLUSION: The present findings are in line with the study hypothesis that kinetic analysis may reveal associations between cleavage patterns and embryo development to the blastocyst stage and ploidy status.

The paternal toolbox for embryo development and health.

The sperm is essential for reconstitution of embryonic diploidy and highly specialized developmental functions. Immediately after gamete fusion, the sperm-borne PLC-zeta triggers activation, generating intracellular free Ca2+ oscillations. Mutations in the PLC-zeta encoding gene are associated with the absence of this factor in mature sperm and inability to achieve fertilization. Sperm play also a role in the greater game of the choreography of fertilization. In the human, the sperm centrioles are introduced into the oocyte environment with gamete fusion. They interact with the oocyte cytoskeletal apparatus to form a functional pair of centrosomes and ultimately regulate pronuclear juxtaposition in preparation for the first cleavage. As a consequence, the fidelity of chromosome segregation during the first cell divisions depends on the function of sperm centrioles. Sperm DNA integrity is essential for embryo development and health. Damaged DNA does not impact on the sperm fertilization ability following ICSI. However, detrimental effects emerge at pre- and post-implantation stages. Sperm-specific epigenetic factors also play an active role in the regulation of embryonic development, as shown by correlations between reduced embryo morphological quality and incorrect chromatin packaging during spermiogenesis or abnormal methylation of sperm CpG islands. This functional landscape demonstrates that the contribution of the sperm to development goes far beyond its well-established role in fertilization. Clinical studies confirm this view and indicate sperm function as a crucial aspect of research to increase the efficacy of assisted reproduction treatments.

Embryo morphokinetic score is associated with biomarkers of developmental competence and implantation.

PURPOSE: To study embryo morphokinetics in relation to release in spent media of molecules with possible roles in development and implantation (miR-20a, miR-30c, and sHLA-G). METHODS: Data were obtained from embryos generated in standard IVF and ICSI cycles. The Eeva system was used for embryo assessment, based on early morphokinetic parameters and producing a score (1-5, best-worst) corresponding to higher/medium/lower chances of development to blastocyst. miRNAs - mm miR-20a-5p and miR-30c-5p - and sHLA-G were quantified in 25 µl of spent blastocyst media (SBM) collected before vitrification or transfer. Statistical analyses were performed applying Kolmogorov-Smirnov, Shapiro-Wilk, and Spearman's correlation coefficient tests, where appropriate. RESULTS: SBM were collected from a total of 172 viable blastocysts. Their analysis showed that concentration of miR-20a was progressively lower as Eeva score increased and probability of development to blastocyst decreased (P = 0.016). The opposite trend was observed in the case of miR-30c, i.e., concentration was higher as score increased and chances of development to blastocyst decreased (P = 0.004). Analysis of sHLA-G revealed a negative correlation with Eeva score, i.e., levels were progressively lower as Eeva score increased and probability of development to blastocyst decreased (R = - 0.388, N = 141, P = 0.001). CONCLUSION: Our data suggest that morphokinetic algorithms that predict development to blastocyst stage, in fact, also identify embryos with molecular and cellular profiles more consistent with developmental functions.

Sperm count affects cumulative birth rate of assisted reproduction cycles in relation to ovarian response.

PURPOSE: To explore the possible influence of sperm quality, as assessed by prewash total sperm count (TSC), on cumulative success rates in assisted reproduction cycles. METHODS: Retrospective study carried out in private IVF centre. Seven hundred sixty-five couples undergoing complete ICSI cycles, i.e. whose all embryos were transferred or disposed of. Couples were characterised by male infertility and female age younger than 36 years. Couples with a combination of female and male infertility factors were excluded. The primary outcome measure was cumulative live birth rate. Secondary outcomes were cumulative pregnancy and miscarriage rates. No specific interventions were made. RESULTS: Higher TSC values have a positive impact on cumulative success rates in cycles characterised by few retrieved oocytes (1 to 5), while does not influence the outcome of cycles with a normal (6 to 10) or high (> 10) number of retrieved oocytes. CONCLUSIONS: The study highlights the importance of sperm quality for the efficacy of assisted reproduction treatments. This influence may remain relatively cryptic in association with normal or high ovarian response, but emerge decisively in cases of reduced ovarian response, suggesting a relationship between ovarian response and oocyte ability to compensate for paternal-derived deficiencies.

DNA Fragmentation in Viable and Non-Viable Spermatozoa Discriminates Fertile and Subfertile Subjects with Similar Accuracy.

Sperm DNA fragmentation (sDF) negatively affects reproduction and is traditionally detected in total sperm population including viable and non-viable spermatozoa. Here, we aimed at exploring the ability of DNA fragmentation to discriminate fertile and subfertile men when detected in viable (viable sDF), non-viable (non-viable sDF), and total spermatozoa (total sDF). We revealed sDF in 91 male partners of infertile couples and 71 fertile men (max 1 year from natural conception) with LiveTUNEL coupled to flow cytometry, able to reveal simultaneously DNA fragmentation and cell viability. We found that the three sDF parameters discriminated fertile and subfertile men with similar accuracy and independently from age and basal semen parameters: AUCs (area under the curves) (95% CI) were: 0.696 (0.615-0.776), p < 0.001 for total sDF; 0.718 (0.640-0.797), p < 0.001 for viable sDF; 0.760 (0.685-0.835), p < 0.001 for non-viable sDF. We also found that total and non-viable but not viable sDF significantly correlated to age and semen quality. In conclusion, the three sDF parameters similarly discriminated fertile and subfertile men. Viable spermatozoa with DNA fragmentation are likely cells able to fertilize the oocyte but failing to properly support subsequent embryo development. Non-viable sDF could be a sign of a subtler damage extended beyond the non-viable cells.

Alternative patterns of partial embryo compaction: prevalence, morphokinetic history and possible implications.

RESEARCH QUESTION: The morula stage is a poorly understood developmental stage. In the morula, cell compaction can involve all or only some blastomeres, with largely unknown implications. Here, the prevalence, underlying morphokinetic mechanisms and possible consequences of partial compaction, were investigated. DESIGN: Preimplantation genetic testing for aneuploidies (PGT-A) cycles of women whose embryos were observed by time-lapse technology were studied. PGT-A data, generated by array comparative genomic hybridization analysis and assessed in three age groups (≤34, 35-39 and ≥40 years), were obtained from trophectoderm biopsies after development to blastocyst stage. RESULTS: Compaction occurred according to three modalities: (i) full compaction, with all blastomeres included (FCM); partial compaction (partially compacted morula [PCM]), with blastomeres (ii) excluded from the outset (excluded-PCM) or (iii) extruded after compaction (extruded-PCM). Partial compaction occurred more frequently than full compaction. Excluded-PCM displayed the slowest morphokinetics at most stages and were most often associated with abnormal cleavage. After compaction, embryo degeneration was more frequently associated with cell extrusion. In excluded-PCM, loss of ≥2 cells impacted blastocyst rate. In embryos of both younger and middle age groups, no statistical differences were observed in the rate of aneuploidy in relation to the three compaction groups, unlike what observed in ≥40 years women. Implantation rates after transfer of euploid blastocysts were not statistically different between the three groups. CONCLUSIONS: Alternative modalities of incomplete compaction were detected. Such patterns are characterized by different morphokinetic behaviours overarching the entire preimplantation development, and by different developmental abilities.

Male factor infertility impacts the rate of mosaic blastocysts in cycles of preimplantation genetic testing for aneuploidy.

PURPOSE: In this study, we tested the hypothesis that, in PGT-A cycles, decreased semen quality is associated with increased rates of mosaic blastocysts. METHODS: In a retrospective analysis, three hundred and forty PGT-A cycles are divided into study groups according to semen quality. Cycles were initially divided into two groups, discerning couples with absence of male factor of infertility (non-male factor: NMF; N = 146 cycles) from couples with a male factor of infertility (MF; N = 173 cycles). Couples with severe male factor (SMF) infertility (n = 22) were assessed separately. Embryos were cultured to the blastocyst stage and chromosomally assessed by array comparative genomic hybridization (aCGH). The study did not involve specific interventions. RESULTS: The reproductive outcome of MF and NMF groups did not indicate statistically significant differences. However, while no differences were found between MF and NMF groups in terms of euploid or aneuploid blastocysts rates, a significantly higher rate of mosaic blastocysts was observed in the MF group (3.6% vs. 0.5%, respectively; P = 0.03). A similar pattern of results was observed in the SMF group when compared with those of the other PGT-A cycles taken together (no SMF). In particular, a significantly higher rate of mosaic blastocysts was observed in the SMF group (7.7% and 1.8%, respectively; P = 0.008). CONCLUSIONS: The study outcome strongly suggests that compromised semen quality is associated with increased rates of mosaic blastocysts analysed in PGT-A cycles. Sperm assessment appears therefore as an important factor in the determination of embryo development and for a more precise prognostic assessment of PGT-A cases.

Effect on Sperm DNA Quality Following Sperm Selection for ART: New Insights.

In the female reproductive tract, male gametes undergo a natural sperm selection process in order to discriminate spermatozoa on the basis of their quality to maximize the chances of successful reproduction. With the introduction of assisted reproductive technology (ART), scientists and clinicians developed diverse sperm selection strategies focusing on the isolation of competent spermatozoa. With increasing understanding of sperm functions and fertilization mechanism and evolution of available technologies, the initial simple sperm preparation protocols were complemented, and sometimes replaced, by new sperm-sorting techniques. In particular, while in the early years the focus was on obtaining motile spermatozoa, in later years, especially after the introduction of intracytoplasmic sperm injection (ICSI), the focus shifted to the isolation of functional and "healthy" spermatozoa, considering some other important factors, such as sperm DNA integrity. Sperm DNA damage, as well as chromatin structure alterations, in fact, is related to decreased reproductive ability of men, in natural as well as in assisted reproduction.

Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.

OBJECTIVE: To determine the pregnancy outcome potential of mosaic embryos, detected by means of preimplantation genetic screening (PGS) with the use of next-generation sequencing (NGS). DESIGN: Retrospective study. SETTING: Genetics laboratories. PATIENT(S): PGS cycles during which either mosaic or euploid embryos were replaced. INTERVENTION(S): Blastocysts were biopsied and processed with the use of NGS, followed by frozen embryo transfer. Trophectoderm (TE) biopsies were classified as mosaic if they had 20%-80% abnormal cells. MAIN OUTCOME MEASURE(S): Implantation, miscarriage rates, and ongoing implantation rates (OIRs) were compared between euploid and types of mosaic blastocysts. RESULT(S): Complex mosaic embryos had a significantly lower OIR (10%) than aneuploidy mosaic (50%), double aneuploidy mosaic (45%), and segmental mosaic (41%). There was a tendency for mosaics with 40%-80% abnormal cells to have a lower OIR than those with <40% (22% vs. 56%). However, few embryos (n = 34) with a mosaic error in 40%-80% of the TE sample were replaced. There was no difference between monosomic and trisomic mosaics or between entire chromosome mosaicism or segmental mosaicism. Implantation rates were significantly higher (70% vs. 53%), miscarriage rates lower (10% vs. 25%), and OIRs higher (63% vs. 40%) after euploid embryo transfer than after mosaic embryo transfer. CONCLUSION(S): Forty-one percent of mosaic embryos produced an ongoing implantation. Complex mosaic blastocysts had a lower OIR than other mosaics. Mosaic monosomies performed as well as mosaic trisomies and mosaic segmental aneuploidies. The results suggest that embryos with >40% abnormal cells and those with multiple mosaic abnormalities (chaotic mosaics) are likely to have lower OIRs and should be given low transfer priority.

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling.

Variation of DNA Fragmentation Levels During Density Gradient Sperm Selection for Assisted Reproduction Techniques: A Possible New Male Predictive Parameter of Pregnancy?

Predicting the outcome of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) is one main goal of the present research on assisted reproduction. To understand whether density gradient centrifugation (DGC), used to select sperm, can affect sperm DNA integrity and impact pregnancy rate (PR), we prospectively evaluated sperm DNA fragmentation (sDF) by TUNEL/PI, before and after DGC. sDF was studied in a cohort of 90 infertile couples the same day of IVF/ICSI treatment. After DGC, sDF increased in 41 samples (Group A, median sDF value: 29.25% [interquartile range, IQR: 16.01-41.63] in pre- and 60.40% [IQR: 32.92-93.53] in post-DGC) and decreased in 49 (Group B, median sDF value: 18.84% [IQR: 13.70-35.47] in pre- and 8.98% [IQR: 6.24-15.58] in post-DGC). PR was 17.1% and 34.4% in Group A and B, respectively (odds ratio [OR]: 2.58, 95% confidence interval [CI]: 0.95-7.04, P = 0.056). After adjustment for female factor, female and male age and female BMI, the estimated OR increased to 3.12 (95% CI: 1.05-9.27, P = 0.041). According to the subgroup analysis for presence/absence of female factor, heterogeneity in the association between the Group A and B and PR emerged (OR: 4.22, 95% CI: 1.16-15.30 and OR: 1.53, 95% CI: 0.23-10.40, respectively, for couples without, n = 59, and with, n = 31, female factor).This study provides the first evidence that the DGC procedure produces an increase in sDF in about half of the subjects undergoing IVF/ICSI, who then show a much lower probability of pregnancy, raising concerns about the safety of this selection procedure. Evaluation of sDF before and after DGC configures as a possible new prognostic parameter of pregnancy outcome in IVF/ICSI. Alternative sperm selection strategies are recommended for those subjects who undergo the damage after DGC.

Annexin V magnetic-activated cell sorting versus swim-up for the selection of human sperm in ART: is the new approach better then the traditional one?

PURPOSE: To investigate whether the sperm fertilizing potential can be improved by selecting a non-apoptotic fraction using magnetic activated cell sorting (MACS), and to compare the results with the conventional swim-up method. METHODS: Twenty five male patients attending the andrology laboratory for sperm DNA fragmentation analysis. The sperm were prepared by density gradient centrifugation (DGC) and subsequently divided into three aliquots. The first was further separated into Annexin V-negative (non-apoptotic) fraction using MACS, the second was further processed by swim-up, while the third was left unseparated as a control. The impact of the combination of DGC with the two sperm preparation techniques on sperm quality was evaluated by comparing 'rapid progressive' motility, normal morphology according to Tygerberg's strict criteria and DNA integrity (by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling [TUNEL]) for each aliquot. RESULTS: Sperm preparation that combines DGC with conventional swim-up method can provide sperm of higher quality in terms of motility, morphology and extent of DNA fragmentation compared to the Annexin V-negative (non-apoptotic) fraction derived from the combination of DGC with MACS. CONCLUSIONS: Integrating MACS as a part of sperm preparation technique will not improve sperm fertilizing potential to the same extent as the traditional swim-up separation procedure.

Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART.

Cryopreservation of human spermatozoa-introduced in the 1960's-has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

Sperm-hyaluronan-binding assay: clinical value in conventional IVF under Italian law.

Hyaluronan has recently been employed in the development of a commercial diagnostic kit for assessing sperm maturity, the so-called sperm-hyaluronan-binding assay (HBA). The aim of this study was to evaluate the usefulness of this test, in addition to routine semen analysis, to identify patients with poor reproductive prognosis in conventional IVF. Furthermore, the ability of hyaluronan to select spermatozoa with low DNA fragmentation was investigated. A total of 60 IVF patients were analysed with regard to reproductive outcome, sperm parameters, HBA score and sperm DNA fragmentation. The DNA fragmentation analysis was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay on the total sperm population and on the hyaluronan-bound spermatozoa obtained from the same samples. No relationship between hyaluronan binding and fertilization, cleavage, good-quality embryos, implantation, clinical pregnancy, miscarriages and biochemical pregnancy rates was found. Otherwise, correlations between spermatozoa hyaluronan binding and morphology (P < 0.01) and a significant difference between DNA fragmentation of the total sperm population and DNA fragmentation of the hyaluronan-bound spermatozoa (P = 0.029) were found. The results underline the ability of hyaluronan to select spermatozoa with higher DNA integrity and morphology. Nevertheless, the clinical value of the HBA in the management of male infertility seems to be limited.

Impact of intracytoplasmic morphologically selected sperm injection on assisted reproduction outcome: a review.

To date, several publications have focused their attention on a new method for observing spermatozoa called 'motile sperm organelle morphology examination' (MSOME), which enables the evaluation of the fine nuclear morphology of motile spermatozoa in real time at high magnification (>x6000). As a consequence, a new microinjection procedure called intracytoplasmic morphologically selected sperm injection (IMSI) has been developed. The aim of the present work is therefore to evaluate the efficacy of the IMSI technique in the light of the current literature, focusing attention on the potential clinical application of the selection of strictly morphologically normal spermatozoa in patients undergoing conventional intracytoplasmic sperm injection treatments. In addition, a brief analysis of preliminary data regarding the relationship between IMSI and assisted reproduction treatment outcome is presented.