Technology for Biobanking of Epididymal Spermatozoa from Patient with Obstructive Azoospermia: Case Report about Baby Born after Conventional Freezing Only with a Nonpermeable Cryoprotectant 360 kDa Polyvinylpyrrolidone.

This publication reports, for the first time, the birth of a healthy child after intracytoplasmic sperm injection (ICSI) of motile spermatozoa after conventional ("slow") freezing of epididymal spermatozoa using 5% polyvinylpyrrolidone (PVP) of high molecular weight (360 kDa). Cryopreservation solution with 10% PVP was added to 30 µL of spermatozoa suspension in a 1:1 ratio, with a final PVP concentration of 5%. Then, polycarbonate capillaries for oocyte denudation with a diameter of 170 µm were filled with 60 µL of the resulting sperm suspension. After that, the capillaries were placed for 10 minutes at a height of 15 cm above liquid nitrogen and immersed into liquid nitrogen. To warm the spermatozoa, the capillaries were immersed in a water bath at a temperature of 40°C for 30 seconds. Oocyte fertilization was performed by ICSI. Zygotes were cultured in vitro for 5 days to the blastocyst stage. More than 100 spermatozoa were obtained after percutaneous epidydimal sperm aspiration, of which 80% were motile. After cryopreservation, storage for 3 months in liquid nitrogen, and thawing, 72% of the total sperm cells remained motile. Ten oocyte-cumulus complexes were found after follicle puncture, and eight metaphase II stage oocytes were fertilized using ICSI. After 18 hours, two pronuclei were found in seven (88%) of the oocytes. An analysis of the morphological characteristics of 5-day-old embryos showed that four (57%) of them reached the blastocyst stage. One embryo was transferred, and the remaining embryos were cryopreserved (vitrified). The onset of pregnancy was detected on the 14th day after embryo transfer, and one healthy girl (3300 g) was born at term.

Embryological Characteristics and Preimplantation Genetic Testing for Aneuploidy of Embryos Derived from Cryopreserved Oocytes of Women of Different Reproductive Ages.

Oocyte vitrification is widely used for female fertility preservation. However, the efficacy of this procedure may depend on the women's age. The aim of the study was to compare the morphology, viability of cryopreserved oocytes, and their fertilization outcomes (fertilization, blastulation rate, level of embryo chromosomal aneuploidy-preimplantation genetic testing for aneuploidy [PGT-A]) in women of different reproductive ages. The studied oocytes were divided into groups depending on the age of patients: up to 30 years (group 1), 30-35 years (group 2), 36-40 years (group 3), and older than 40 years (group 4). It has been shown that in women of older reproductive age, the number of oocytes with polymorphism of endo- and extracytoplasmic structures was higher compared with younger patients. This could reflect on their cryosurvival rate, which was the highest in group 1 (98.1%) and the lowest was in group 4 (47.4%). With increasing age, the fertilization rate of cryopreserved oocytes and subsequent blastulation was decreased. However, the number of embryos with an aneuploid chromosome set number was increased. The chromosome set number euploidy rate of the embryos obtained from cryopreserved oocytes of advanced age women (group 4) did not differ from the fresh group with the same age (31.2% vs. 24.4%, p > 0.05), but the number of euploid embryos per patient was less than one (0.8 ± 0.1). Therefore, the decision to cryopreserve the oocytes of a patient of older reproductive age should be made individually for each situation, taking into account the prospects of obtaining full-fledged embryos and the chances of pregnancy.

New method for cryoprotectant-free freezing of human oligoasthenoteratozoospremic spermatozoa with high-molecular polymer.

Data about cryoprotectant-free cryopreservation of human ICSI spermatozoa are limited. The aim of this investigation was to compare two technologies for cryopreservation of spermatozoa from men with oligoasthenoteratozoospermia: standard conventional freezing with 5% glycerol (freezing in glycerol) and cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone (PVP) (PVP-freezing). Capillaries with spermatozoa were cooled in vapor and then plunginged into liquid nitrogen. Head-, midpiece- and tail-abnormality of spermatozoa, mitochondrial membrane potential (MMP) and DNA fragmentation rates after cryopreservation were evaluated. After warming of spermatozoa, fertilization of oocytes (ICSI) was performed. It was detected the lower rate of morphological abnormalities of PVP-frozen spermatozoa in comparison with cells frozen with glycerol (34.6 ± 4.1% vs. 20.7 ± 4.7%, respectively) (P < 0.05). Quality of cells with high MMP after warming in spermatozoa frozen with glycerol was lower than in PVP-frozen spermatozoa (34.7 ± 4.2 vs. 54.5 ± 4.2%, respectively) (P < 0.05). It was established that the DNA fragmentation rate in PVP-frozen spermatozoa was significantly lower in comparison with spermatozoa frozen with glycerol (23.1 ± 2.5% vs. 38.8 ± 3.0%, respectively) (P < 0.05). After fertilization (ICSI) of oocytes, it was established that cleavage and blastulation rates were higher in oocytes after fertilization with PVP-frozen spermatozoa than with spermatozoa frozen with glycerol. Fertilization-, development to 8-blastomeres-, and blastocyst-rates were for PVP-frozen and spermatozoa frozen with glycerol, respectively: 94.4 ± 7.8 vs. 82.2 ± 6.2% (P > 0.1 with tendency to increasing), 90.0 ± 4.6 vs. 69.5 ± 5.1% (P < 0.05), and 45.4 ± 4.1% vs. 30.9 ± 3.3% (P < 0.05). It was concluded that permeable cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone can be applied successfully for cryopreservation of human oligoasthenoteratozoospremic spermatozoa.

The impact of cryopreservation on the morphology of spermatozoa in men with oligoasthenoteratozoospermia.

The cryopreservation of ejaculate can reduce the viability, motility, and morphological characteristics of the spermatozoa of infertile men. Oligoasthenoteratozoospermia (OAT) is the most common cause of male subfertility. The aim of this study was to evaluate the morphological characteristics and viability of progressive motile sperm fraction before and after cryopreservation, and to determine whether cryopreservation of progressive motile sperm fraction is effective in eliminating morphologically abnormal sperm in men with OAT. An increased proportion of spermatozoa with normal morphology in fresh progressive motile sperm fraction compared with fresh ejaculate has been observed. After cryopreservation, the motility was 65.5 ± 8.8%; the proportion of spermatozoa with normal morphology increased non-significantly compared with freshly prepared motile sperm fraction (35.6 ± 5.5%). Concurrently, the proportion of cryopreserved spermatozoa with head defects increased significantly by 1.7 times (to 38.4 ± 4.7%) and the proportion of almost all morphologically abnormal sperm cells, particularly spermatozoa with multiple abnormalities, was reduced significantly. These data appear to be a novel finding in the context of patients with OAT. Using such spermatozoa for in vitro fertilization leads to a significant decrease in both a number of embryos at the cleavage stage and the blastocysts formation rate. High-magnification sperm morphology examination and selection, IMSI, post-cryopreservation significantly increased the likelihood of successful oocyte fertilization and subsequent embryo development.

Cryopreservation of incomplete compacted morulae and preliminary biopsy of excluded fragments.

The complexity of predicting embryo development potential at the cleavage stages and the emergence of epigenetic risks during prolonged in vitro culture of pre-implantation embryos made it more advantageous to transfer embryos at the morula stage to the uterine cavity. The criteria for estimating embryos at this stage that allow prediction of cryopreservation outcomes have been poorly described. All day 4 embryos (n = 224) were graded 1, 2, 3, 4 or 5 according to blastomere compaction degree (BCD = 100, 75, 50, 25 or 0%, respectively) and the survival and blastocyst formation rate of these morulae were studied after cryopreservation. An inverse dependence was found between survival rate and BCD. Excluded fragments were characterized by low osmotic reaction during exposure to cryoprotective medium and, after freeze-thawing, they were destroyed. As damaged necrotic areas of the embryo can affect their further development rate we proposed blastomeres and biopsy fragments of incomplete compacted morula be removed before embryo cryopreservation. This step led to significant increase in the post-thawing survival rate up to 93.1 ± 4.1%, 75 ± 8.8% and blastocyst formation rate up to 85.2 ± 10.4%, 59.4 ± 5.2% in grade 2 and grade 3 embryos, respectively. There was no significant difference in grade 4 embryos. Therefore the removal of blastomeres and biopsy fragments in incomplete compacted morulae can improve cryopreservation outcomes of grade 2 and grade 3 embryos with BCD.