Cryopreservation of human semen by inherently-controlled icing probability: Or how the surface profile of superhydrophobic carbon soot coatings and the sperm volume affect the outcome of slow freezing?

The restoration of initial functionality of human spermatozoa subjected to cryopreservation is challenging, because the deleterious intracellular icing and the occurrence of osmotic shocks due to prolonged exposure to increased concentrations of intracellular solutes are oppositely dependent on the cooling rate. This longstanding problem could be overcome if using superhydrophobic soot coatings delaying the heat transfer rate, reducing the ice formation probability and triggering balanced and timely dehydration of the cells, but the effect of their surface profile and sperm volume on the success rate of slow freezing is unclear. Here, we show for the first time that the two-factor freezing injury is entirely avoidable by tailoring the solid-to-gas voids (pores) fraction in the soot, leading to increased nucleation free energy barrier, presumable incipiency of ice crystals with controllable shape and size and hence, fully (100 %) recovered post-thaw sperm motility. It is demonstrated that the reason for such a unique scientific result is the selection of soot coatings with appropriate morphochemical features, hypothetically (not directly proven yet) inducing equilibrium among the solution composition and ice crystals formation, retarding the undesirable compression of liquid-filled "slush ice" channels surrounding the cytoplasm and impeding the ice recrystallization. The novel insights introduced in this article open endless horizon for customizing and revolutionizing the technical protocols in cryobiology.

Na,K-ATPase Atp1a4 isoform is important for maintaining sperm flagellar shape.

PURPOSE: The aim of this study is to investigate the mechanisms by which the testis specific Na,K-ATPase ion transport system (Atp1a4) controls sperm morphology and shape. METHODS: Sperm from wild-type (WT) and Atp1a4 knockout (Atp1a4 KO) mice were analyzed morphologically, using light, transmission, and scanning electron microscopy; and functionally, applying sperm osmotic challenge and viability tests. In addition, a sperm proteomic study was performed. RESULTS: Light microscopy confirmed that sperm lacking Atp1a4 present a bend at the junction of the mid- and principal piece of the flagellum. This bend had different degrees of angulation, reaching occasionally a complete flagellar retroflexion. The defect appeared in sperm collected from the cauda epididymis, but not the epididymal caput or the testis. Transmission and scanning electron microscopy revealed a dilation of the cytoplasm at the site of the bend, with fusion of the plasma membrane in overlapping segments of the flagellum. This was accompanied by defects in the axoneme and peri-axonemal structures. Sperm from Atp1a4 KO mice showed an abnormal response to hypoosmotic challenge with decreased viability, suggesting reduced capacity for volume regulation. Exposure to Triton X-100 only partially recovered the flagellar bend of Atp1a4 KO sperm, showing that factors other than osmotic regulation contribute to the flagellar defect. Interestingly, several key sperm structural proteins were expressed in lower amounts in Atp1a4 KO sperm, with no changes in their localization. CONCLUSIONS: Altogether, our results show that Atp1a4 plays an important role in maintaining the proper shape of the sperm flagellum through both osmotic control and structurally related mechanisms.

The Negative Impact of Higher Body Mass Index on Sperm Quality and Erectile Function: A Cross-Sectional Study Among Chinese Males of Infertile Couples.

The objective of the current study was to explore the role of body mass index (BMI) in sperm quality and erectile function in Chinese males of infertile couples. A total of 28 fertility centers in different regions of China were enrolled. Patient data were collected from June 1, 2017, through October 31, 2017. Semen analyses and demographic data were collected and the five-item International Index of Erectile Function (IIEF-5) questionnaire was used to evaluate the erectile function in participants with different BMIs. In total, 3,174 Chinese men of infertile couples with an average age of 33.11 ± 6.08 years were enrolled. The occurrence of obesity, overweight, normal weight, and underweight were 5.4%, 36.6%, 56.8%, and 1.2%, respectively. In addition to hypertension and diabetes, primary infertility, a longer course of infertility, and chronic prostatitis were risk factors for obesity. Compared with men of normal weight, adjusted odds ratios (ORs) for men with obesity, overweight, and underweight for semen volume <2 ml were 2.53 (95% CI [1.61, 3.97]), 1.33 (95% CI [1.09, 1.62]), and 0.84 (95% CI [0.29, 2.43]); for sperm progressive motility (A + B) (%) <32, the ORs were 1.60 (95% CI [1.16, 2.22]), 1.30 (95% CI [1.12, 1.51]), and 1.03 (95% CI [0.54, 1.98]); and for IIEF-5 ≤ 21, the ORs were 1.52 (95% CI [1.10, 2.10]), 1.11 (95% CI [0.96, 1.30]), and 0.62 (95% CI [0.31, 1.26]), respectively. Obesity was associated with lower semen volume, lower sperm motility, and erectile dysfunction in Chinese males of infertile couples.

Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species.

Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 µL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler® counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks' balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 µL, whereas membrane integrity required a minimum of 2 µL (at 1 × 106 cells/mL). Thus, <5 µL of sperm suspension (after dilution to 30-150 µL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.

Associations of hypoosmotic swelling test, relative sperm volume shift, aquaporin7 mRNA abundance and bull fertility estimates.

Mammalian sperm are exposed to a natural hypoosmotic environment during male-to-female reproductive tract transition; although this activates sperm motility in vivo, excessive swelling can harm sperm structure and function. Aquaporins (AQPs) is a family of membrane-channel proteins implicated in sperm osmoregulation. The objective was to determine associations among relative sperm volume shift, hypoosmotic swelling test (HOST), sperm aquaporin (AQP) 7 mRNA abundances, and sire conception rate (SCR; fertility estimate) in Holstein bulls at a commercial artificial insemination center. Three or four sires for each full point SCR score from -4 to +4 were included. Each SCR estimate for study bulls (N = 30) was based on > 500 services (mean ± SEM) of 725 ± 13 services/sire). Sperm from a single collection day (two ejaculates) from these commercial Holstein bulls were used. Relative mRNA expression of AQP7 in sperm was determined by polymerase chain reaction. Mean relative sperm volume shift and percentage of sperm reacted in a HOST (% HOST) were determined (400 sperm per bull) after incubating in isoosmotic (300 mOsm/kg) and hypoosmotic (100 mOsm/kg) solutions for 30 min. There was no correlation between %HOST and SCR (r = 0.28 P > 0.1). However, there was a positive correlation between relative sperm volume shift and SCR (r = 0.65, P < 0.05). Furthermore, AQP7 mRNA abundance was positively correlated to both relative volume shift (r = 0.73; P < 0.05) and to SCR (r = 0.67; P < 0.05). The mRNA expressions of AQP7 and relative sperm volume shift differed (P < 0.05) among low- (<2 SCR), average- (-2 to +2) and high- (>2) fertility sire groups. In conclusion, bulls with higher SCR had significantly greater AQP7 mRNA abundance in frozen-thawed sperm. This plausibly contributed to greater regulation of sperm volume shift, which apparently conferred protection from detrimental swelling and impaired functions.