These results were presented at the 35 th annual meeting of the European Society of Human Reproduction and Embryology ABSTRACT 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.

We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.

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 40x10 6 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 58.9x10 6 ; 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 (65.9x10 6 vs. 62.3x10 6 ; 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).

Purpose Several culture media are available to be used in ART. However it is uncertain whether embryos would preferably benefit from one type of medium or the association of different media. Methods We performed this study to evaluate the impact of simultaneous transfer of embryos independently cultured in two distinct culture media, on pregnancy outcome. A total of 722 couples who underwent infertility treatment were sequentially allocated into three groups: those who had half of the embryos individually cultured in MEM and the other half cultured in sequential media (MEM + Seq Group) (n0 243); those who had all embryos cultured only in sequential medium (Seq Group) (n0239); and those who had all embryos cultured only in MEM (MEM Group) (n0240). Results The pregnancy rate was higher in the MEM + Seq group (51.8 %) than the Seq group (36.7 %) (p<0.001). However the pregnancy rate observed in the MEM group was similar to the others (44.2 %). When a logistic regression test was applied it demonstrated that the number of transferred embryos did not interfere in the pregnancy rates. Conclusions Our results suggests that offering different culture conditions for sibling embryos with subsequent transfer of embryos that were kept in distinct culture media, might increase pregnancy rates in assisted reproduction cycles.

After human embryonic stem cells were successfully obtained from frozen embryos, donation for research became a new alternative for surplus embryos. The aim of this study was to analyze the impact of Brazil's Biosecurity Law on the availability of frozen embryos donated for embryonic stem cell research, through the observation of the number of embryos donated for that purpose by patients of a private clinic and by obtaining data from the national Health Surveillance Agency's online registers. A total of 43 couples donated 245 embryos for research between 2005 and 2010. Couples kept their frozen embryos for 3.35 years before donating them to research and the mean age of the women was 36.54 when donation took place. A total of 37 couples (86%) that donated embryos for research achieved pregnancy during IVF fresh cycle. In 2008 in Brazil, 573 surplus frozen embryos, from 50 IVF clinics, were donated for human embryonic stem cell research and in 2009, 455 from 31 IVF clinics were donated. A reduction in embryo donation, therefore, was observed. We believe it is necessary to release information and promote public discussion to clarify the potential of embryonic stem cell research, as a strategy to increase the number of embryo donations for research.