Cleavage-stage embryo micromanipulation in the clinical setting.

Embryo micromanipulation was developed after introduction of microinjection to overcome infertility. Embryo micromanipulation may be performed at any embryo stage from pronuclear to blastocyst. The technique started out as basic and turned out to be increasingly more complex. Embryo micromanipulation at the cleavage-stage includes a wide range of techniques, from opening the zona pellucida in order to improve the chance of implantation, to removing detrimental components from the embryo to enhance embryo development or blastomeres for preimplantation genetic diagnosis and embryo splitting. Evaluating the impact(s) of different micromanipulation techniques on epigenetics of the embryo and considering quality control during these techniques are important issues in this regard. This review aims to discuss the micromanipulation of cleavage-stage embryos in clinical assisted reproductive technology (ART). ABBREVIATIONS: 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.

Vitrification Increased Vacuolization of Human Spematozoa: Application of MSOME Technology.

BACKGROUND: Sperm vitrification is a technique of ice and cryoprotectant free cryopreservation by direct plunging of sperm suspension into liquid nitrogen (LN2). The aim of this study was to investigate the influence of cryoprotectant free-vitrification on human sperm fine structure by MSOME technology and the fertility potential by zona binding assay (ZBA). METHODS: 20 normo-ejaculates were prepared by swim up technique, and supernatants were divided into two parts of fresh and vitrified groups. For vitrification, sperm was dropped into LN2. Sperm motility, morphology, viability and MSOME were evaluated for each sample. In MSOM morphologically normal sperm (class 1), ≤2 small vacuoles (class 2), and one large vacuole or >2 small vacuoles (class 3) were evaluated. Also, fertility potential was evaluated by zona binding assay. Data was analyzed using paired t-test or Willcoxon's test and p-value <0.05 was considered significant. RESULTS: Vitrification significantly reduced both progressive motility, viability and morphology. Also, normal morphology of spermatozoa decreased significantly after vitrification. In MSOME evaluation, normal motile spermatozoa (Class 1) decreased from 23.00±12.44 to 16.00.56±10.79 after vitrification (p=0.008). Although spermatozoa classes 2 and 3 were increased, the difference was not significant. Moreover, fertility potential of motile spermatozoa was reduced after vitrification (9.0±13.87 vs. 13.40±22.73; p=0.07). CONCLUSION: Vitrification increased the rate of vacuolization in motile sperm head. Therefore, MSOME technology is recommended for assessment of sperm fine morphology in ICSI program used cryopreserved spermatozoa.

Performing ICSI with commercial microinjection pipettes enhanced pregnancy rates.

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.

The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa.

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.

Large nuclear vacuoles in spermatozoa negatively affect pregnancy rate in IVF cycles.

BACKGROUND: Recently, motile sperm organelle morphology examination (MSOME) criteria as a new real time tool for evaluation of spermatozoa in intracytoplasmic sperm injection (ICSI) cycles has been considered. OBJECTIVE: The aim was to investigate the predictive value of MSOME in in vitro fertilization (IVF) in comparison to ICSI cycles and evaluation of the association between MSOME parameters and traditional sperm parameters in both groups. MATERIALS AND METHODS: This is a cross sectional prospective analysis of MSOME parameters in IVF (n=31) and ICSI cycles (n=35). MSOME parameters were also evaluated as the presence of vacuole (none, small, medium, large or mix); head size (normal, small or large); cytoplasmic droplet; head shape and acrosome normality. In sub-analysis, MSOME parameters were compared between two groups with successful or failed clinical pregnancy in each group. RESULTS: In IVF group, the rate of large nuclear vacuole showed significant increase in failed as compared to successful pregnancies (13.81±9.7vs7.38±4.4, respectively, p=0.045) while MSOME parameters were the same between successful and failed pregnancies in ICSI group. Moreover, a negative correlation was noticed between LNV and sperm shape normalcy. In ICSI group, a negative correlation was established between cytoplasmic droplet and sperm shape normalcy. In addition, there was a positive correlation between sperm shape normalcy and non-vacuolated spermatozoa. CONCLUSION: The high rate of large nuclear vacuoles in sperm used in IVF cycles with failed pregnancies confirms that MSOME, is a helpful tool for fine sperm morphology assessment, and its application may enhance the assisted reproduction technology success rates.