Viable acrosome-intact human spermatozoa in the ejaculate as a marker of semen quality and fertility status

Abstract

This work was supported by grants from the Innovation Fund Denmark. M.G. and S.K. work at ChemoMetec, which produces the image cytometer used in the study, M.G. hold shares in the company. The other authors have no conflict of interest.

Expression of the O-Glycosylation Enzyme GalNAc-T3 in the Equatorial Segment Correlates with the Quality of Spermatozoa

“…The most significant association was observed with motility and morphology, while the association with sperm concentration was less significant. To further integrate the semen parameters into one combined clinical relevant parameter, we used the guidelines from the World Health Organisation (WHO) on normal levels of semen quality to differentiate men into groups according to the number of sperm defects and categories of high, intermediate, and low semen quality as it has recently been done in other publications [ 24 , 27 ]. This showed that men with a poor semen quality indeed had a significantly lower fraction of GalNAc-T3-positive spermatozoa in their ejaculate.…”

“…Semen samples were collected from 206 men representing a broad spectrum of semen quality as described before [ 27 ]. Samples originated from an unselected and random sub-sample of men from infertile couples ( N = 125), men from an ongoing study of reproductive health in the general Danish population ( N = 68), and fertile men ( N = 13) who previously fathered a child.…”

“…Semen samples from laboratory QC-donors were allowed to liquefy and mixed thoroughly before the motile spermatozoa were purified from the ejaculate by the swim-up technique using human tubular fluid (HTF + ) medium containing 97.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO 4 , 0.37 mM KH 2 PO 4 , 2.04 mM CaCl 2 , 0.33 mM Na-pyruvate, 21.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, and 4 mM NaHCO 3 , adjusted to pH 7.3–7.4 with NaOH, as described elsewhere [ 27 ]. After incubation for 1 h at 37 °C, 10% CO 2 the upper swim-up fraction was carefully collected, and concentration was determined using image cytometry [ 35 , 36 ].…”

“…For capacitation, the cell concentration was adjusted to 10 × 10 6 cells/mL in HTF + medium containing 25 mM NaHCO 3 and 3 mg/mL (3% [ v / v ]) human serum albumin (HSA, Irvine Scientific, Santa Ana, CA, USA) and incubated for at least 3 h at 37 °C in a 10% CO 2 atmosphere. After capacitation, the sperm cells were induced to undergo acrosome reaction by addition of 2 μM of the calcium ionophore ionomycin (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 30 min at 37 °C at 300 rpm shaking on a thermal mixer [ 27 ].…”

The Acrosomal Status of Density Purified Spermatozoa Differentiates Men from Couples in IVF and ICSI Treatment and Is Associated with Fecundity

“…Traditionally, assays to investigate the functionality of the acrosome involve quite laborious manual procedures, and they have not been used in the clinic or in larger clinical studies. Recently, we have developed an assay based on image cytometry, which allows automatization and easy determination of the percentage of viable acrosome-intact spermatozoa in an ejaculate, an indicator of the acrosomal status [15,16]. This assay exploits the advantage of PSA coupled to fluorescein isothiocyanate (FITC) to flow via membrane pores into the acrosomal compartment of reacting acrosomes and to stabilize the acrosomal matrix allowing easy detection of spermatozoa undergoing the acrosome reaction [17].…”