Human sperm motility in a microgravity environment

Abstract

Background and Aims: We carried out clinostat and parabolic flight experiments to examine the effects of a microgravity (µG) environment on human sperm motility.

Methods: Semen samples were obtained manually from 18 healthy men (aged 27.4 ± 5.4 years) who had given their informed consent. In clinostat experiments, samples that were left stationary were used as a stationary control. Samples rotated vertically and horizontally were used as a rotation control and a clinostat rotation, respectively. In parabolic flight experiments using a jet plane, sperm motility was compared for each parameter at µG, 1G and 2G. The state of 1G during the flight was used as a control. Sperm motility was determined using an automatic motility analyzer HT‐M2030 in a microgravity environment.

Results: All parameters of sperm motility tended to be lower in clinostat rotation compared with rotation control at both low‐speed and high‐speed, but the differences were not statistically significant. In parabolic flight, sperm motility and parameters of linear movement were decreased (P < 0.05). There was no significant difference between µG and 2G, but sperm motility was significantly decreased at µG than at 1G.

Conclusions: Our findings suggest that sperm motility is reduced under µG. (Reprod Med Biol 2005; 4: 161–168)

INTRODUCTION

AS THE SPACE age progresses, we need to determine whether humans can pursue a normal life in space, including reproductive function. We have been studying the effects of the environmental factors in space that are different from those on earth, such as gravity, solar rays, ultraviolet rays, electromagnetic waves, temperature and radioisotopes. We have been concerned with the effect of a long‐term stay in space on the human body, and have examined the effect of a µG environment on human sperm motility.

The parachute method (drop from a tower or balloon) and parabolic flight have been used to simulate a µG environment on earth. By these methods a gravity of 1 × 10⁻³ G can be produced. The clinostat, water submersion and suspension methods have been used to simulate a µG environment where the G level on the individual is unchanged. ¹

Egg laying and fertilization of amphibians, fish and birds in a µG environment has been found to be similar to that on earth. ² , ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ , ⁹ Interesting findings in the field of reproductive biology have been reported sporadically, for example, eggs developing for a certain period after fertilization in space. ¹⁰ Spermatogenesis in rats was unchanged after a 22‐day space flight. ¹¹ There is, however, little data concerning the effects on human reproduction.

A male rat was mated with a female rat 5 days after returning from a 2.5 to 3 month flight in the biosatellite Kosmos‐1129. The growth of the neonates was impaired, and the mortality was reportedly high. ¹² In 1996, a female rat was placed in the space shuttle from days 9–20 of gestation and was reported to have offspring with normal growth. ¹³ However, concerning sperm motility, there has been only a single report that the motility and linear movement of bovine sperm were increased significantly in a µG environment. ¹⁴ In the present study, we carried out clinostat and parabolic flight experiments to examine changes in human sperm motility in a simulated µG environment.

MATERIALS AND METHODS

Preparation of sperm

SPERM WERE PREPARED using a ‘layering method’ as follows. ¹⁵ Sperm were obtained manually from 18 healthy men, average age 27.4 ± 5.4 years (22–40 years) who had given their informed consent. The total numbers of specimen was 30. Sperm samples were left to stand for 30–40 min at room temperature to liquefy. To each tube containing 2.0 mL of human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA), 0.5 mL of liquefied sperm was added. Care was taken not to mix the two fluids. The test tubes were sealed with parafilm, tilted at a 30° angle, and incubated in 5% CO₂ air incubator at 37°C and 100% relative humidity (RH) for 1 h. The sperm that swam up to the medium were then recovered, and centrifuged at 230 g for 5 min to obtain sperm with high motility. The sperm were washed, and made to a final concentration of 5–10 × 10⁶/mL with HTF medium. They were used immediately for the clinostat experiments, but 2 h later for the parabolic flight experiments, because of the need to prepare an airplane for the experiment.

Analysis of sperm motility

Sperm motility was analyzed using an automatic motility analyzer HT‐M2030 (Hamilton‐Thorn Research, Beverly, MA, USA). A total of nine parameters were examined. These were: motility (%) and mean progressive motility (%) as parameters of motility rate; mean path velocity (µm s⁻¹), mean track speed (µm s⁻¹) and mean progressive velocity (µm s⁻¹) as parameters of progressive rate; mean linearity (%) and mean linear index (%) as parameters of linear movement; and mean lateral head displacement (µm) and mean head oscillation frequency (Hz) as parameters of head oscillation.

The automatic motility analyzer recorded the position coordinate data for each sperm on a Makler chamber ¹⁶ consecutively every 30 frames per second. Motility is the percentage of motile sperm among the total sperm. Progressive velocity is the distance per second from the starting point to ending point. Progressive motility is the percentage of sperm with a progressive velocity of 50 µm s⁻¹ or more. Path velocity is the curve obtained by connecting the mean coordinates of five consecutive frames divided by the measurement time. Track speed is defined as the total distance of the straight line connecting the consecutive coordinates on the frame divided by the measurement period (Fig. 1). Linearity and linear index are parameters for the linear movement of sperm. Linearity = progressive velocity ÷ track speed × 100; linear index = progressive velocity ÷ path velocity × 100. Lateral head displacement is defined as the doubled value of the mean of the three coordinates for each sperm head in each frame and the frames before and after that frame. Head oscillation frequency is the number of oscillations of the sperm head per second. The measurement values of mean progressive velocity, mean path velocity, mean track speed, mean linearity and mean linear index are the mean values for the motile sperm actually displayed by each instrument. Mean lateral head displacement and mean head oscillation frequency are measured in the motile sperm with a linear index of more than 50% as an amplitude of sperm vibration. ¹⁷

Figure 1.

This schema depicts the motion of human spermatozoa. Progressive velocity is the distance from the starting point to ending point. Progressive motility is the percentage of sperm with a progressive velocity of 50 µm s⁻¹ or more. Path velocity is the curve obtained by connecting the mean coordinates of five consecutive frames divided by the measurement time. Track speed () is defined as the total distance of the straight line connecting the consecutive coordinates on the frame divided by the measurement time.

Clinostat experiments

The clinostat is a rotating system, which converts gravity from a vector quantity to a scalar quantity. The specimen is rotated horizontally in a clinostat and rotated at a constant angular rate, which simulates a µG environment.

In the basic experiment, the clinostat was placed in an incubator with 5% CO₂ in air at 37°C and 100% RH. A 500 µL microtube (Eppendorf, Hamburg, Germany) with the specimen positioned in the direction of the rotating axis was fixed in the clinostat with a rotating axis in the horizontal direction. Specimens rotated in this direction were referred to as a clinostat rotation, and those rotated in a perpendicular direction were referred to as a rotation control. The rotations were 5 r.p.m. (hereinafter low‐speed) or 50 r.p.m. (hereinafter high‐speed). A stationary control was a specimen without rotation in the incubator under the same condition. Sperm motility was measured at 1G immediately after each rotation was stopped (Fig. 2).

Figure 2.

Schematic diagram of the clinostat used to produce a vector‐free gravitational environment. The clinostat was set in an incubator in 5% CO₂ at 37°C and 100% relative humidity. The clinostat rotation is the rotation in the perpendicular direction around a horizontal rotation axis. The rotation control is rotated in a horizontal direction. The microtube containing the specimen was fixed at the center along the rotation axis. HTF, human tubal fluid; (░), sperm in HTF.

Parabolic flight experiments

A parabolic flight is a flight that produces an almost gravity‐free (µG) state for 20–25 s on board a jet airplane in parabolic flight at a height of 29 000–21 000 feet. A µG environment can be obtained approximately 10 times per flight. The G level on board an airplane is 3 × 10⁻² G in the perpendicular direction and approximately 1 × 10⁻² G in the horizontal directions (forward‐backward, left‐right). When the instrument is suspended, theoretically a value of 10⁻³ G or less is obtained. The temperature within the airplane is adjusted to approximately 20–30°C, the RH to 10–40%, and the atmospheric pressure to 0.9 atmospheres. The measurement temperature is maintained at 30°C in the motility analyzer. Along with clinostat experiments, parabolic flight experiments are important in terrestrial studies of µG (Fig. 3).

Figure 3.

Flight curve obtained during a parabolic flight. The jet plane ascended rapidly from 21 000 to 29 000 feet and then descended rapidly in a parabolic flight. Hypergravity (2G) was produced during the ascent and immediately before reaching the maximum point microgravity (µG) was obtained, and persisted for approximately 20 s. This was repeated 10 times during each flight. One parabolic flight was for approximately 100 s.

Sperm samples were prepared in the same way as for the clinostat experiments. A motility analyzer HT‐M 2030 was fixed securely onto a 35 cm × 60 cm × 90 cm metal rack on board an airplane for the experiment, and could be adjusted according to changes in vibration and atmospheric pressure. The state of 1G (just before parabolic flight) during the flight was regarded as the control, and sperm motility was compared for each episode of µG and the immediately preceding 2G state. The parameters measured were the same as in the clinostat experiments. The motility of the motile sperm was also evaluated at 1G and µG according to the percentage of rapid sperm, medium sperm, slow sperm and static sperm. The sperm moving at the rates of over 25, 10–25, <10 and 0 µm s⁻¹ were regarded as rapid sperm, medium sperm, slow sperm and static sperm, respectively.

Statistical analysis

We used the paired t‐test for statistical analysis of sperm motility parameters for both the clinostat and parabolic flight studies, and the χ² test for the parameters showing the rate of motility (%) (rapid sperm, medium sperm, slow sperm, static sperm). Values with P < 0.05 were considered to be significantly different.

RESULTS

Clinostat experiments

1. Comparison of rotation control and clinostat rotation

AT LOW‐SPEED ROTATION, there was a tendency to decrease in clinostat rotation in all parameters excluding mean lateral head displacement and mean head oscillation frequency. At high‐speed rotation, all parameters other than mean lateral head displacement also showed a tendency to decrease. However, there were no significant differences between the rotation control and the clinostat rotation in any parameters (Table 1).

Table 1.

Change of human sperm motility in clinostat

	Low‐speed rotation (5 r.p.m)			High‐speed rotation (50 r.p.m)
	Stationary control	Rotation control	Clinostat rotation	Rotation control	Clinostat rotation
Motility (%)	30.8 ± 17.2	43.9 ± 27.9	35.8 ± 24.2	40.3 ± 21.8	39.6 ± 25.7
Mean progressive motility (%)	2.3 ± 3.1	4.3 ± 7.9	2.5 ± 4.6	4.3 ± 6.8	3.9 ± 6.7
Mean path velocity (µm s−1)	32.6 ± 12.2	31.2 ± 12.7	29.5 ± 10.0	34.8 ± 12.5	30.1 ± 9.4
Mean track speed (µm s−1)	44.5 ± 14.9	39.5 ± 13.4	39.2 ± 10.8	46.1 ± 13.2	38.7 ± 11.0
Mean progressive velocity (µm s−1)	25.7 ± 13.7	28.9 ± 13.1	24.0 ± 12.0	31.1 ± 11.4	25.7 ± 10.8
Mean linearity (%)	55.7 ± 14.3	63.5 ± 6.8	62.1 ± 9.9	65.3 ± 8.3	62.3 ± 11.2
Mean linear index (%)	76.5 ± 13.6	87.4 ± 6.7	82.4 ± 7.1	86.3 ± 4.0	80.1 ± 13.8
Mean lateral head displacement (µm)	2.5 ± 1.4	2.1 ± 0.5	2.4 ± 0.6	2.6 ± 0.4	3.1 ± 3.0
Mean head oscillation frequency (Hz)	14.1 ± 3.1	14.4 ± 2.3	15.2 ± 1.4	14.7 ± 2.0	12.0 ± 5.1

Comparison between stationary control, rotation control and clinostat rotation.

Values given as mean ± standard deviation. Differences between three groups were not statistically significant for all parameters.

2. Comparison of stationary control and rotation control

At low‐speed rotation, all parameters except for mean path velocity, mean track speed and mean lateral head displacement were higher for the rotation control. At high‐speed rotation, all parameters were higher for the rotation control. However, no significant differences were detected between the stationary control and the rotation control (Table 1).

3. Comparison of stationary control and clinostat rotation

At low‐speed rotation, mean path velocity, mean track speed, mean progressive velocity and mean head oscillation frequency were slightly reduced, but the other parameters were slightly increased in clinostat rotation. At high‐speed rotation, many parameters were increased, but mean path velocity, mean track speed and mean progressive velocity were decreased; many parameters showed higher values with clinostat rotation, but some were decreased. However, no significant differences were detected between the stationary control and the clinostat rotation in any of the parameters (Table 1).

Parabolic flight experiments

Compared to the control (1G), at µG there was a significant decrease in motility from 45.33 ± 23.98 to 26.85 ± 21.60% (P = 0.004), mean progressive motility from 32.15 ± 23.35 to 18.53 ± 17.34% (P = 0.007), mean linearity from 68.54 ± 17.14 to 57.50 ± 27.62% (P = 0.036) and mean linear index from 83.08 ± 13.04 to 68.95 ± 31.85% (P = 0.033). Although the differences were not significant, a decrease was also observed in the other parameters, from 51.79 ± 14.78 to 48.35 ± 26.72 (µm s⁻¹) for mean path velocity, 64.29 ± 16.19 to 59.53 ± 34.31 (µm s⁻¹) for mean track speed, 43.67 ± 16.45 to 39.75 ± 23.64 (µm s⁻¹) for mean progressive velocity, and from 11.44 ± 6.37 to 8.15 ± 5.66 (Hz) for mean head oscillation frequency. These findings indicate that the percentage motility of sperm and the forward movement were significantly decreased.

Only motility was significantly decreased at 2G as compared to µG, from 45.33 ± 23.98 to 30.95 ± 24.49% (P = 0.014). Motility was lower at µG than at 2G (Fig. 4a–d). Furthermore, at µG, among the motile sperm the ratio of rapid sperm was significantly decreased, from 37.02 ± 23.16 to 20.81 ± 18.16% (P = 0.04) as compared with the control, medium sperm from 8.32 ± 12.75 to 6.01 ± 9.64%, slow sperm from 3.46 ± 7.50 to 2.21 ± 3.87%, with a decrease in the total percentage of actively moving sperm. These findings showed that only the active sperm decreased significantly. From these results, the proportion of static sperm significantly increased from 51.19 ± 26.21 to 70.90 ± 22.49% (P = 0.04) (Fig. 5).

Figure 4.

Motility (%, ▪) and mean progressive motility (%, □) were measured at 1G, µG, and 2G during parabolic flight. The measurement temperature was at 30°C in the motility analyzer. Motility was significantly lower at µG (*III) and 2G (*V) than at 1G (*I) (*I vs. *III: P = 0.004, *I vs *V: P = 0.014, paired t‐test). Mean progressive motility was significantly lower at µG (IV*) than at 1G (*II) (*II vs *IV: P = 0.007, paired t‐test) (Fig. 4a). However, there was no particular change in mean path velocity (µm s⁻¹, ▪), mean track speed (µm s⁻¹, □), or mean progressive velocity (µm s⁻¹, ), which are parameters of speed (Fig. 4b). All parameters are not statistically significant (mean SD) in Fig. 4b. Mean linearity (%, ▪) and mean linear index (%, □), which are parameters of progression, were significantly lower at µG than at 1G (*I vs *III: P = 0.036, *II vs *IV: P = 0.033, paired t‐test), but showed no particular change at 2G (Fig. 4c). No particular change was seen in mean lateral head displacement (µm, ▪) or mean head oscillation frequency (Hz, □) (Fig. 4d). All parameters are not statistically significant (mean SD) in Fig. 4d.

Figure 5.

The ratio of rapid sperm with a motility rate (%) of 25 µm s⁻¹ or more was significantly lower at µG (□) than at 1G (▪) during parabolic flight. The percentage of static sperm was significantly increased (chi‐square test). The measurement temperature was at 30°C in the motility analyzer. Rapid, >25; medium, ≥10 ≤25; slow, <10; static, 0 (µm s⁻¹).

DISCUSSION

THE EFFECT OF an outer space environment on reproduction must be considered from a number of viewpoints. We can anticipate some effects of a vacuum environment on fetal growth, the effects of temperature and humidity on the fetal growth and reproduction, the possibility of genetic damage by solar, cosmic rays and electromagnetic fields, and the effects of stress on fetal growth and hormone balances. There are also major concerns related to the inhibition of spermatogenesis as a result of decreased testicular blood flow caused by the movement of body fluid to the head in males, ¹⁸ , ¹⁹ , ²⁰ as well as decreased testosterone levels under µG ²¹ and their effects on fertilization and embryogenesis. As we enter a cosmic age, the effect of µG on reproduction is a major topic.

In the present study, we examined human sperm motility in a µG environment. In clinostat experiments, the effect of a µG environment on sperm motility tended to decrease, although the decrease was not significant. There was also no significant difference in sperm motility between the stationary control and rotation control at various r.p.m. speeds. There was no signification difference between the stationary control and clinostat rotation. In 1992, Engelmann et al. used frozen sperm to examine sperm motility using a sperm motility analyzer (TEXUS, Technologishe Experimente Unter Schwerelosigkeit) under µG and normal gravity. They reported that the motility of the more rapid sperm increased significantly under µG. ¹⁴

The clinostat has been used to study gravitropism in plants for a long time. Even now, when studies in space have become possible, it is still used as a means of producing a µG environment on earth, along with various methods of analysis. A 3‐D clinostat, in which the specimen is placed on a revolving stand that rotates around two orthogonal axes, has been used to experimentally simulate a µG environment in cosmic life science studies. ²² , ²³ Although it is not certain that the clinostat can fully reproduce the µG environment in space, it can be used to study the effects of gravity. In clinostat experiments, the specimen is placed in a microtube; by rotation at a certain angular velocity, momentum is transported to the center from the wall of the rotating container because of the viscosity of the fluid, and gradually the flow in the container stops. Hamazaki et al. reported, however, that when a density gradient occurs in the fluid, or when the fluid contains granules or specimen with a density different from the fluid, a constant flow is induced even when rotated at a constant angular velocity. ²⁴

The observed increase in sperm motility was thought to have possibly been caused by hyperactivation ²⁵ seen immediately before fertilization as a result of problems caused by the solvent and the rotation stimulus. To eliminate the problem of ‘flow’ of the solvent because of rotation and the problem of ‘hyperactivation’, we carried out experiments using a rotation control and a clinostat rotation, and found that sperm motility tended to decrease. However, in parabolic flight experiments, the motility rate and progressive velocity were significantly lowered under µG. These findings therefore suggest that sperm motility is reduced in a µG environment. The reason why no significant difference was observed in the clinostat experiments was thought to be because the measurements were made at 1G after stopping the clinostat. Furthermore, as motility tended to be reduced in parabolic flight experiments, we consider human sperm motility to be lowered by microgravity. One reason for the decline in sperm motility might be a chemical change in the intracellular environment. For example, when lymphocytes were cultivated under simulated microgravity in a rapidly rotating clinostat and in space flight, reduced proliferation was observed. ²⁶ , ²⁷ , ²⁸ It may be that microgravity affects the expression of cytokines and the intracellular signaling mechanism at the single‐cell level. ²⁸ , ²⁹ , ³⁰

In a cooperative study with the National Space Development Agency of Japan (NASDA), we confirmed that the egg fertilizing rate of mice sperm was not affected by the clinostat. ³¹ Furthermore, as Wogemuch et al. found no differences in morphological abnormalities in fertilization and egg division, ³² decreased sperm motility is considered to be an adaptive strategy to the change in gravity. In either case, sperm motility has been confirmed to decrease in µG conditions. There is considered to be an effect on the male reproductive system in mammals. Further study is needed, not only into the effect of gravity on the various processes of reproduction, for example fertilization, early embryogenesis, gestation and delivery, but also the problems of cosmic rays and stress in space.