The assisted reproduction success rate is affected by several factors including the age of the women, oocyte quality and maturation state, as well as sperm quality. Imaging of the meiotic spindle may be crucial for determining the oocyte maturation and the optimal time of oocyte fertilization by intracytoplasmic sperm injection (ICSI). A new accurate and non-invasive method for selecting quality maturated oocytes based on meiotic spindle imaging is for women over 35 years of age will be introduced. The evaluation of efficiency using meiotic spindle visibility in polarized light and its relative position to the polar body as indicator of oocyte maturity will be monitored and the optimal time for ICSI will be defined.

One of the main strategies of infertility treatment is in vitro fertilization (IVF) . The IVF success rate is affected by several key factors including the age of the women, oocyte quality and maturation state, as well as sperm quality. Several authors have suggested that the presence, position and retardance of the optically birefringent meiotic spindle (MS) are related to oocyte developmental competence, affecting the quality of fertilization and embryo development. Oocytes with normal spindle morphology are significantly more likely to produce euploid embryos compared with oocytes with meiotic spindles that are not visible or abnormal. Adjusting the timing of intracytoplasmic sperm injection (ICSI) based on MS morphology has also been proposed. It was shown that adjusting the time of ICSI based on the position of the MS with respect to the polar body (PB) increases the probability of successful fertilization for women over 35 years of age. For the oocytes of patients over 35 years of age, the longer incubation time enabled a greater number of oocytes to become fully mature (MII phase) and prepared for ICSI. In this situation, the MS status was an important indicator of oocyte immaturity.

Patient will be randomized into three groups using Research Electronic Data Capture (REDCap): group 1, standard ICSI without MS imaging; group 2, MS imaging followed by standard ICSI, group 3, MS imaging and ICSI fertilization according MS status. Oocytes from group 3 patients with MS evaluation will be fertilized according to MS status either 5-6 hours after ovum pick-up (OPU) or 7-8 hours after OPU. Oocytes without MS evaluation will all fertilized 5-6 hours after OPU. MS evaluation will take place in pre-prepared glass-bottomed dishes with about 5μl medium with the HEPES buffer covered with paraffin oil, put to heat one hour before use. The oocyte will be rotated using a needle so that Polar Body (PB) and MS are both well visible, and photographs will be taken using optical microscope. The MS status and the angle (α) between MS and PB will be obtained 3-4 hours after oocyte pick-up (OPU). At the same time, the polarized-light microscopy image will be acquired (polarized light microscopy at ×100 magnification. Specially, for oocytes from group 3 patients with PB/MS in close proximity (angle between PB and MS < 5◦) or MS not visible will be predicted as immature oocytes. For these oocytes, ICSI will be performed 4-5 h after the polarization microscopy evaluation, i.e., 7-8 h after OPU. For oocytes with MS clearly visible and PB/MS not in close proximity (angle between PB and MS > 5◦) ICSI will be performed typically 2-3 h after the polarization microscopy evaluation, i.e., 5-6 h after OPU, as for patients from 1 and 2 groups. ICSI will be performed according to standard protocol using ICSI/holding micropipettes (Microtech IVF, Czech Republic), polyvinylpyrrolidone (ICSI™, Vitrolife, Sweden), and Eppendorf (Hamburg, Germany) micromanipulation system equipped with thermoplate (Tokaohit, Japan). The oocytes will be cultivated individually, and their order preserved, so that the other outcomes (data from timelapse, clinical results) can be associated with individual oocytes. We will also monitor the time from the human chorionic gonadotropin (hCG) administration to the completion of the actual fertilization. The oocytes will be denuded (HYASE-10X™, Vitrolife, Sweden) after OPU, and the maturation stage will be examined. Germinal Vesicle (GV) stage oocyte will not be included. Oocytes and embryos in time-lapse incubator from the Japanese manufacturer Astec will be cultivated in a timelapse dish Origio, Denmark Sage 1-Step™, Origio, Denmark under paraffin oil (OVOIL™, VITROLIFE, Sweden) at 37.0 °C, 6% CO2 and 5%O2. Fertilization after ICSI will be defined as the presence of two pronuclei and 2 polar body 16-20 h post ICSI. Embryos will be cultivated for 122-144 h.

Pilot findings will be confirmed in a randomized control trial, i.e. to the efficiency of using MS visibility and relative position to the polar body as indicators of oocyte maturation in order to optimize ICSI timing will be evaluated.

Inclusion Criteria:

• Women who underwent ICSI;
• Spermiogram containing at least 0.5 mil/ml sperms;
• Morphlogy: normal morphology >1%;
• Stimulated cycles

Exclusion Criteria:

• Age under 35 years or over 40;
• Native cycles;
• Severe uterine abnormalities (submucosal fibroid, fibroid ≥5cm, uterine septum, endometrial polyps, uterus duplex)