Oocytes and sperm
The oocytes evaluated in this study were collected from patients between March and November 2020 in the Centre of assisted reproduction (CAR), Department of Obstetrics and Gynecology of the First Faculty of Medicine, Charles University and General Teaching Hospital, Prague 2, Czech Republic. All subjects participating in the study provided informed consent. The study was conducted in accordance with the Declaration of Helsinki. All the experimental protocols were approved by a Department of Obstetrics and Gynecology of the First Faculty of Medicine, General University Hospital, Prague and by Ethic Committee of the General University Hospital, Prague (the list is enclosed). During this research we recorded the following two datasets: 1) Oocyte utilization rates - the number of high - quality embryos per oocyte with a detected PB 32,33. 2) Patients clinical pregnancy rate – the detection of an embryonic sac during a transvaginal scan ≥5 weeks after embryo transfer after one cycle of IVF.
The sampled population were all patients of CAR. Patients were randomly divided into two groups before IVF/oocyte OPU: retrospective (R) and control (C). The selection criteria ensured that the results would not be affected by their number and quality. Key criteria for inclusion were, planned ICSI and a spermiogram containing at least 1 mil/ml of progressively motile spermatozoa measured during previous spermiogram evaluation. In both groups (R,C) oocytes with microscopically observable PB were taken into consideration. In the C group the status of the meiotic spindle was not determined before ICSI and ICSI was performed 6-8 hrs after OPU.
In the R group, after denudation at 3-4 hours after Ovum Pick-Up (OPU) the research team examined each oocyte using an optical microscope with a polarization filter in order to determine the angle between meiotic spindle and PB (α) and assessed the oocyte maturation using the standard method. MS evaluation lasted on average 3-5 minutes. R group patients with oocytes having α > 30º (thus a higher probability to be fully mature) were fertilized (ICSI) after a shorter time after the polarization microscopy control (typically a maximum of 2 hours). In R group 69 patients were identified with oocytes having α ≤30ºthat were considered as insufficiently mature in Telophase I, and ICSI for these patients was performed 4 hours after the polarization microscopy evaluation.
We evaluated the pregnancy rates and average oocytes utilization rates (number of high-quality embryo per oocyte with detected PB) in stratified patients. Each pregnancy rate was calculated as the ratio of numbers of pregnant patients to all patients belonging to a specific group. Average oocytes utilization rates were calculated as the number of high-quality embryos 32,33 divided by the number of collected oocytes with microscopically detected polar bodies averaged over the number of patients from specific groups.
The cumulus oophorus complexes were cultivated in 4-well plate (NUNC) using Sage Fertilization medium ™, Origio, Denmark Sage 1-Step™, Origio, Denmark under paraffin oil (OVOIL™, Vitrolife, Sweden) et 37.0º C and 6% CO2.
The oocytes were denuded (HYASE-10X™, Vitrolife, Sweden,) after pick-up and the and the maturation stage was examined. GV stage oocyte were not included. M1 were matured similarly to cumulus oophorus. ICSI was performed to standard protocol using ICSI/holding micropipettes (#002-5-30/#001-120-30, Microtech IVF, Czech Republic), polyvinylpyrrolidone (ICSI™, Vitrolife, Sweden), and Eppendorf (Hamburg, Germany) micromanipulation system equipped with thermoplate (Tokaohit, Japan).
We cultivated embryos for 2-5 days. Embryos were cultivated in 4-well plate (NUNC) using Sage 1-Step™, Origio, Denmark under paraffin oil (OVOIL™, Vitrolife, Sweden) et 37.00 C and 6% CO 2.
For both groups, high quality embryos were used for subsequent transfer or freezing defined as cleavage embryos - gr.1-3 with a blastocyst with good expansion of blastocoel cavity and the integrity of both the inner cell mass and trophectoderm cells. Blastocysts with embryoblast and trophoblast C were excluded in blastocysts 32,33.
The R group comprised of 124 patients between 22 and 42 years old (mean age 35 years). 77 patients from R group were >35 years old, 68 patients from R group were older than 35 years and younger than 40 years. The total number of oocytes with evaluated meiotic spindles collected from this group was 540. Evaluation of meiotic spindle was carried out 3-4 hrs after OPU. Based on the visualisation of MS in polarized light, patients in the study were divided into four sub-groups according to meiotic spindles and PB positions: 1) Patients with more than 40% oocytes with α ≤ 30º; 2) Patients with 50% or more of oocytes with α = 45º (±15º); 3) Patients with at least 10% oocytes α >60º; 4) Patients with some oocytes immature or with a poorly visible MS. For the majority cases the oocytes of a single patient belonged to several groups.
The quality of sperm was also monitored so that correlation between sperm quality and utilization and pregnancy rates could be evaluated: (N) normospermia (46%); (A) asthenozoospermia (10%); (O) oligospermia (10%); (OA) oligoastenozoospermia (4%), (OTA) Oligo‐astheno‐teratozoospermia (30%).
Optical microscopy
Using an optical microscope 36-38 with a Nikon CEE GmbH polarizing filter, the research team could effectively image the mutual position of PB and the dividing spindle in oocytes that were collected from patients under standard conditions within the IVF cycle.
Statistical evaluation of the data
To quantify the statistical significance of the collected data, we calculated the p values as; the rates of obtaining test results that were at least as extreme as the results actually observed during the test assuming that the null hypothesis was correct (i.e., the rate that we falsely rejected the null hypothesis). This means that even though the results may show effect in terms of numbers, we assume that this is caused by random variations and that this difference is therefore not a real, statistical one. The results were controlled by using the MaxStat Pro 3.6 program and Pearson Calculator 39. Patients average utilization rates were calculated as average over ratios of number of quality embryos and the number of oocytes collected from each patient.
The t-value (signal to noise ratio where the signal is the difference between the sample mean and the denominator, the noise, is the standard error of the mean) as significance of the difference between the compared groups of patients was calculated using Pearson Calculator. The t-values (normal distribution) and z-value (binary distribution) were calculated for all the compared group of patients according to their pregnancy rates, utilization rates and spindles visualisation. We also used Pearson's c square test for comparison of R group pregnancy rates and expected pregnancy rates - according to results obtained in 27-30 IVF pregnancy rates observed in one cycle of IVF. Pearson's chi-squared test was used to determine whether there is a statistically significant difference between the expected values and the observed values. The correlation between sperm quality, patient’s age and both utilization rate and probability of patient’s clinical pregnancy were also calculated.