A novel solution for freezing small numbers of spermatozoa using a sperm vitrification device.

STUDY QUESTION: Does a novel sperm vitrification device (SpermVD) provide an efficient method for freezing a small number of human spermatozoa from men suffering from non-obstructive azoospermia? SUMMARY ANSWER: The novel SpermVD is an efficient and simple carrier method for freezing a small number of spermatozoa in low-volume droplets, reducing post-thaw search time from hours to minutes, allowing a 96% recovery rate and leading to successful use of sperm for fertilization. WHAT IS KNOWN ALREADY: Previous methods for cryopreservation of small numbers of human spermatozoa (e.g. mini-straws, ICSI pipette, alginate beads, cryoloop) have been proposed as a solution for cases of severe male infertility. Many drawbacks have prevented their widespread use, including cumbersome preparation and sperm retrieval procedures, and the fact that the thawed spermatozoa are not immediately available for micromanipulation and required additional treatment which posed excess risk of harm. STUDY DESIGN, SIZE, DURATION: We conducted a feasibility experiment of the novel SpermVD and a prospective cohort study of ICSI cycles in men suffering from non-obstructive azoospermia in two outpatient fertility IVF clinics, from 2015 through 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent extended ejaculate search prior to the day of oocyte retrieval, and any single motile spermatozoa found was transferred to 0.8 µl droplets of 1:1 washing medium/cryoprotectant on the SpermVD, then plunged into liquid nitrogen for cryopreservation. In patients with non-obstructive azoospermia who underwent surgical TESE, both the motile and immotile spermatozoa found underwent cryopreservation using the SpermVD. On the day of oocyte retrieval, the SpermVD was thawed, directly transfered to the ICSI plate and retrieved spermatozoa were used for the ICSI procedure. MAIN RESULTS AND THE ROLE OF CHANCE: The prospective cohort included 44 cases. We used the SpermVD to vitrify 631 spermatozoa, of which 540 (86%) were motile. The average number of frozen spermatozoa per patient was 14.3 ± 9.3. After thawing, we retrieved 607 spermatozoa, producing a recovery rate of 96%. The average number of thawed spermatozoa was 13.8 ± 9.2. The recovery of 180 thawed motile sperm accounted for 33% of all frozen motile spermatozoa. The fertilization rate was 59%. Of 44 oocyte retrieval procedures, 24 (55%) clinical pregnancies were achieved. The delivery rate (not including three ongoing pregnancies) was 32% and the miscarriage rate was 29%. LIMITATIONS, REASONS FOR CAUTION: Although we presented the SpermVD on 44 cases, a larger cohort would provide more information. Moreover, we cryopreserved only motile sperm from the ejaculates and not immotile sperm, thus limiting the knowledge regarding the efficacy of the VD for immotile sperm from this source. WIDER IMPLICATIONS OF THE FINDINGS: The novel SpermVD is a simple efficient carrier, optimizing the protocol for freezing a small number of spermatozoa. It may allow for the routine use of frozen spermatozoa after TESE for men suffering from non-obstructive azoospermia and thus avoid repeated TESE surgeries. Furthermore, in cases of non-obstructive azoospermia, routine cryopreservation of the retrieved spermatozoa prior to the IVF cycle may avoid the risk of cycle cancelation and thus decrease the number of unnecessary oocyte retrieval procedures. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding. There are no competing interests. TRIAL REGISTRATION NUMBER: IRB no 00119-16-ASMC.

Shear stress-induced transcriptional regulation via hybrid promoters as a potential tool for promoting angiogenesis.

Among the key effects of fluid shear stress on vascular endothelial cells is modulation of gene expression. Promoter sequences termed shear stress response elements (SSREs) mediate the responsiveness of endothelial genes to shear stress. While previous studies showed that shear stress responsiveness is mediated by a single SSRE, these endogenous promoters often encode for multiple SSREs. Moreover, hybrid promoters encoding a single SSRE rarely respond to shear stress at the same magnitude as the endogenous promoter. Thus, to better understand the interplay between the various SSREs, and between SSREs and endothelial-specific sequences (ESS), we generated a series of constructs regulated by SSREs cassettes alone, or in combination with ESS, and tested their response to shear stress and endothelial specific expression. Among these constructs, the most responsive promoter (NR1/2) encoded a combination of two GAGACC/SSREs, the Sp1/Egr1 sequence, as well as a TPA response element (TRE). This construct was four- to five-fold more responsive to shear stress than a promoter encoding a single SSRE. The expression of constructs containing other SSRE combinations was unaffected or suppressed by shear stress. Addition of ESS derived from the Tie2 promoter, either 5' or 3' to NR1/2 resulted in shear stress transcriptional suppression, yet retained endothelial specific expression. Thus, the combination and localization order of the various SSREs in a single promoter is crucial in determining the pattern and degree of shear stress responsiveness. These shear stress responsive cassettes may prove beneficial in our attempt to time the expression of an endothelial transgene in the vasculature.