AIM:The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men.METHODS:A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up.RESULTS:There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ (P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ (P = 0.02).CONCLUSIONS:The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs.

ART: assisted reproductive technology; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; PGD: preimplantation genetic diagnosis; PZD: partial zona dissection; ZP: zona pellucida; SUZI: subzonal insemination; PVS: perivitelline space; AH: assisted hatching; LAH: laserassisted hatching; ZT: zona thinning; UV: ultraviolet; IR: infrared; PCR: polymerase chain reaction; FISH: fluorescent in situ hybridization; NGS: next generation sequencing; QC: quality control; QA: quality assurance.

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Background/aim: Many technical factors can affect intracytoplasmic sperm injection (ICSI) outcomes. The role of the injection micropipette could be of vital importance in ICSI programs. The main goal was to compare ICSI pregnancy outcomes between commercial and home-made injection micropipettes in a large population with male factor infertility.Materials and methods: Five-hundred and eleven ICSI cycles with severe male factor were included in this retrospective study. ICSI cycles were divided into two groups: A (home-made micropipettes, n = 267) and B (commercial micropipettes, n = 244). Rates of fertilization, embryo formation, and chemical and clinical pregnancies were compared between the groups. The independent samples t-test, chi-square test, and Fisher's exact test were used, whenever appropriate, for statistical analysis.Results: A total of 3621 MII oocytes were retrieved, of which 2003 were fertilized. The rate of normal fertilization was significantly higher in group A (57.9%) compared to group B (52.5%). However, the rate of embryo formation showed an increase in group B compared to group A (90.4% and 85.9%, respectively, P = 0.002). In addition, the clinical pregnancy outcomes improved in group B. Conclusion:Our findings indicate that clinical pregnancy improves when commercial injection micropipettes are used in ICSI programs.

Fertility in cattle includes the ability of the uterus to provide the appropriate environment for pregnancy success, including the transport of spermatozoa before fertilization. Confocal laser endomicroscopy technology (Cellvizio) has been previously used in small ruminants to visualise labelled spermatozoa invivo in the uterine horns of ewes (Druart et al. 2009 Reprod. 138, 15-53). Nevertheless, to the best of our knowledge, no invivo study has reported a live visual analysis of the behaviour of spermatozoa in the bovine uterus upon AI. The aim of this study was to develop an experimental procedure to label bull spermatozoa and to visualise their progression in the uterine horns of dairy cows, using Cellvizio and uterine cervix catheterization. Fresh ejaculated bull spermatozoa were double-labelled with octadecyl rhodamine B chloride and MitoTracker Green FM dyes before dilution and freezing processing. At each step of the process, semen quality was evaluated with a semen quality analyzer (SQA-V b, Medical Electronic System) and compared to nonlabelled bull reference semen. The specificity of the labelling was validated invitro. We also developed a specific device to insert the Cellvizio fibre probe into the genital tract, in order to image vagina, cervix, and uterine body, as well as the proximal, middle, and distal parts of the uterine horn, including the utero-tubal junction (UTJ), before and after AI with fresh and frozen labelled spermatozoa. Then, 10 heifers were oestrus synchronized. Video sequences were recorded at oestrus (n=5), just before AI, at the time of AI (7-12h after the onset of oestrus), 30 and 120min after AI, and 14 days after oestrus (n=5; luteal phase), to investigate the effect of the steroid-primed uterine environment on the distribution and progression of spermatozoa in the genital tract. Using our approach, labelled spermatozoa were detected (1) at the day of oestrus and 14 days after oestrus, (2) in the uterine body at the time of AI, (3) in various parts of the genital tract 30min after AI, and (4) in the UTJ, the cervix, and the vagina 120min after AI. In addition, labelling the spermatozoa did not alter the fertilizing capacity: 4 of the 5 oestrus females were pregnant at Day 35. Additionally, an IVF test showed a blastocyst rate of 45.8% for labelled semen vs. 39.4% for the control group (Carvalho et al. 2017 Reprod. 154, 695-710). In conclusion, our study provides a method to phenotype in situ the transport of spermatozoa in the genital tract of heifers. Further investigations are in progress to optimize video quality, to develop an algorithm for an automatic analysis (spermatozoa count, speed, and path) of the recorded sequences.