How to optimize culture media osmolality during Assisted Reproductive Technologies treatments.

OBJECTIVE: The objective of our study was to compare the osmolality in sequential and single step culture media, used for in vitro human embryo culture, covered with mineral oil and paraffin, in dry and humid incubators. METHODS: We performed a prospective observational study. A total of 120 Petri dishes, with 960 droplets of culture media, were evaluated. Each dish was prepared with 4 droplets of single step medium and sequential medium. Sixty dishes were covered with mineral oil and 60 with paraffin oil. Half were incubated in a dry incubator and half in a humid. Osmolality was measured on days 1, 3, 5, 7. ANOVA test was performed for statistical analysis. RESULTS: Osmolality results for single step and sequential medium, that were covered with both mineral and paraffin oil and placed in the dry incubator, significantly increased throughout the study time (D7>D5>D3). In the humid incubator, the results were similar for all periods. Osmolality was significantly lower in humid incubator, in all periods, when droplets were covered with both oils. When both culture media were placed in the humid incubator, no variation was detected, using both oils. However, when single step medium was placed in the dry incubator, covered with mineral oil, we observed a higher osmolality than the covered with paraffin oil. CONCLUSIONS: TWe can conclude that humid incubator is better for maintaining osmolality and paraffin oil protect single step media from evaporation in dry incubator.

Ultrastructural analysis of Lyophilized Human Spermatozoa.

OBJECTIVE: Lyophilization is potentially more practical and cost-effective alternative for sperm preservation. However, there are no studies that evaluate the ultrastructure of human spermatozoa after lyophilization. Therefore, the aim of our study was to evaluate the ultrasctructure of lyophilized spermatozoa using Transmission Electron Microscopy. METHODS: From a total of 21 donated seminal samples, 30 aliquots were originated and divided into two aliquots so that one could have been submitted to cryopreservation/thaw and the other for lyophilization/rehydration. The liquefied aliquots were homogenized at room temperature. Samples assigned for cryopreservation were placed in straws and samples assigned for lyophilization were placed in the appropriate vials. Cryopreservation samples were placed at -30oC for 30 minutes subsequently for 30 minutes at vapour phase and then plunged into liquid nitrogen. Lately, were warmed in water bath at 37oC for 10 minutes followed by 10 minutes centrifugation. The pellet was resuspended and analysed in a Makler chamber. The semen vials assigned for lyophilization were loaded into a pre-fixed freeze-drying chamber. Following lyophilization, vials were removed from the freeze-drying chamber and kept at 4oC until rehydration. TEM was performed after rehydration and thawing. Sperm samples were fixed, rinsed in buffer, post fixed and dehydration was carried out in escalating concentrations of alcohol solution, acetone and then, embedding in Epon resin. Ultrathin sections were stained and examined in a Transmission Electron Microscope. RESULTS: Analysis of sperm after freezing/thawing using Transmission Electron Microscopy showed lesions to the midpiece, with some mitochondria degeneration and random rupture of plasma membrane. In the head, we identified intact plasma membrane, nucleus and acrosome, as in the flagellum all main structures remained intact including the plasma membrane, the longitudinal columns of dense fibers and the semicircular fibers. Analysis by Transmission Electron Microscopy showed that spermatozoa heads had ruptured plasma membranes, absence of acrosomes, nuclei with heterogeneous and decompressed chromatin. Mitochondria were deteriorated in the midpiece. Longitudinal columns of dense fibers were absent in the flagellum. Axonemes, in cross-sections, were disrupted with disorganized structures. CONCLUSIONS: To our knowledge, our study demonstrated, for the first time, the structure of the human spermatozoa after lyophilization using Transmission Electron Microscopy. The use of a fixed lyophilization protocol with media containing cryoprotectants might explain the damage to the structures. More studies are necessary to improve the results of sperm lyophilization. In the future, the use of lyophilization of spermatozoa might reduce the costs of fertility preservation, since there will be no need for storage space and transportation is simpler.

Prevalence of human papillomavirus (HPV) in the semen of patients submitted to assisted reproductive technology treatment in a private clinic in Brazil.

OBJECTIVE: The aim of our study was to identify the prevalence of HPV in the semen of men submitted to ART treatment and look into the possible impacts of the virus on sperm parameters. METHODS: Thirty-five patients treated for infertility from March to August 2016 were invited to join the study. Samples with a minimum concentration of 40x106 spermatozoa per milliliter were included in the study. After the evaluation of semen parameters, DNA extraction and PCR were performed to verify the presence of HPV by electrophoresis in 8% polyacrylamide gel. RESULTS: Patient age ranged from 27 to 68 years (mean 39.2 years). Semen analysis showed a mean volume of 2.5mL; mean concentration of 58.9x106; and mean motility of 51.8%. HPV DNA was identified in seven semen samples from 25 patients (28%). Ten samples with DNA concentrations below 10ng/µL were excluded from the study due to poor amplification quality. There was no statistical difference in sperm concentration when HPV-negative and HPV-positive samples were compared (65.9x106 vs. 62.3x106; p=0.70). However, sperm motility was significantly higher in HPV-positive semen (65% vs. 46.6%; p=0.02). CONCLUSIONS: HPV prevalence was 28% in the semen of patients submitted to ART treatment. HPV-positive samples had statistically increased motility compared to negative samples (65% vs. 46.6%; p=0.02).