Heads and Tails: Requirements for Informative and Robust Computational Measures of Sperm Motility

Abstract

The ability of sperm to self-propel through the female reproductive tract is essential for natural fertilisation. Despite this fact, manual assessments of motility are limited in scope and computer-aided sperm analysis (CASA) systems are not routinely used in clinical practice. One significant factor hindering the clinical use of CASA systems is the lack of evidence linking motility measures and fertility outcomes. To progress these technologies, we need to address the variations in the way samples are prepared and imaged, and whether current kinematic parameters provide the physiological insight necessary for establishing these links. In this manuscript, we discuss how preparation (sample viscosity, temperature, concentration), acquisition (chamber depth, frame rate, duration) and analysis can dramatically affect CASA results. With the aim of advancing the clinical application of CASA, we outline the requirements for obtaining measurements that can be compared between samples and systems. We further highlight how the introduction of flagellar tracking can form the basis of increasingly insightful diagnostics; by combining kinematic data with mathematical modelling experimentally intractable details can be uncovered, such as metabolic requirements of motility from a single cell to the population level.

Appendix

The characteristic sperm tracks presented in this paper are taken from semen samples provided by unscreened normozoospermic donors recruited at Birmingham Women’s and Children’s NHS Foundation Trust after giving informed consent. Full details of the experimental procedures for each cell shown in Figs. 1–7 can be found in this appendix.

Figure 1—Cells were suspended in supplemented Earle’s balanced salt solution (sEBSS) without phenol red, and supplemented with 2.5 mM Na pyruvate and 19 mM Na lactate (06-2010-03-1B; Biological Industries, Kibbutz Beit HaEmek, Israel), and 0.3% weight/volume charcoal delipidated bovine serum albumin. Cells were imaged in a 10 μm depth chamber (10-01-04-B-CE; Leja Products B.V., Nieuw-Vennep, The Netherlands) using an Olympus (BX-50) microscope and negative-phase contrast microscopy (objective 20× 0.40 Ph1 BM ∞/0.17 WD 1.2), and recorded using a Hamamatsu Photonics C9300 CCD camera (pixel size 7.4 μm at 285.2 Hz).

Figure 2—(a) Cells were selected using a swim-up, where a 500 μL aliquot of supplemented Earle’s balanced salt solution (sEBSS) without phenol red, and supplemented with 2.5 mM Na pyruvate and 19 mM Na lactate (06-2010-03-1B; Biological Industries, Kibbutz Beit HaEmek, Israel), and 0.3% weight/volume charcoal delipidated bovine serum albumin was placed in a 5 mL round-bottom tube (Corning, Falcon 352058). A 300 μL aliquot of semen was pipetted to the bottom of the tube, inclined and left in the incubator for 30 min at 37 °C. Cells were imaged in a 10 μm depth chamber using a Nikon (Eclipse 80i) microscope and negative-phase contrast microscopy at 10× magnification (10× 0.2 Ph1 BM ∞/-WD7.0) and an Andor Zyla 5.5 (Andor, Oxford UK) microscopy camera at 200 Hz with pixel size 6.5 μm × 6.5 μm.

Figure 2—(b) Cells were suspended in supplemented Earle’s balanced salt solution (sEBSS) without phenol red and supplemented with 2.5 mM Na pyruvate and 19 mM Na lactate (06-2010-03-1B; Biological Industries, Kibbutz Beit HaEmek, Israel), and 0.3% weight/volume charcoal delipidated bovine serum albumin, with the addition of 1% methylcellulose (M0512, Sigma-Aldrich, Poole, UK, specified so that an aqueous 2% solution gives a nominal viscosity of 4000 centipoise or 4 Pa s at 20 °C). The cells were loaded by capillary action into flat-sided borosilicate capillary tubes (VITROTUBES, 2540, Composite Metal Services, Ilkley, UK) with length 50 mm and inner dimensions 4 × 0.4 mm. One end of the tube was sealed with CRISTASEAL (Hawksley, Sussex, UK #01503-00). Cells were selected for imaging by immersing the open end of the capillary tube into a 1.5 mL Beem capsule (Agar Scientific, UK) containing a 200 μL aliquot of raw semen, within 30 min of sample production. Incubation was performed for 30 min at 37 °C in 6% CO₂. Cells were imaged at 2 cm migration distance into the capillary tube and in the surface accumulated layer 10–20 μm from the inner surface of the capillary tube at 10× magnification using a Nikon (Eclipse 80i) microscope and negative-phase contrast microscopy and an Andor Zyla microscopy camera (pixel size 6.5 × 6.5 μm) at 200 Hz.

Figure 3—The sample was counted according to WHO guidelines (WHO 2010) and diluted to a concentration of 10 M/mL in of supplemented Earle’s balanced salt solution (sEBSS) without phenol red, and supplemented with 2.5 mM Na pyruvate and 19 mM Na lactate (06-2010-03-1B; Biological Industries, Kibbutz Beit HaEmek, Israel), and 0.3% weight/volume charcoal delipidated bovine serum albumin. Cells were imaged in a 10 μm depth chamber using a Nikon (Eclipse 80i) microscope and negative-phase contrast microscopy (objectives 10× 0.2 Ph1 BM ∞/-WD7.0), using a Basler Microscopy ace camera (acA 1300-200uc) at 169 Hz with pixel size 4.8 × 4.8 μm, using Pylon Viewer (v.5.0.11.10913, Basler AG, Ahrensburg, Germany).

Figure 4—See Fig. 1.

Figure 5—See Fig. 3.

Figure 6—See Fig. 5.

Figure 7—(a) See Fig. 2a.

Figure 7—(b) See Fig. 2b.