Sperm motion characteristics in men with isolated hypogonadotropic hypogonadism treated with gonadotropin

Vol. 51, No.1, January 1989

FERTILITY AND STERILITY

Printed in U.S.A.

Copyright c 1989 The American Fertility Society

Sperm motion characteristics in men with isolated hypogonadotropic hypogonadism treated with gonadotropin David Vantman, M.D.*t:l: George Koukoulis, M.D.:j: Allen S. Burris, M.D.:j:

Steve M. Banks, Ph.D.§ Lisa Dennison, B.A.:j: Richard J. Sherins, M.D.:j: II

Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland

The authors compared curvilinear velocity (Vc) and linearity (L) of sperm from fertile oligospermic men with isolated hypogonadotropic hypogonadism (IHH) to Vc and L of sperm from fertile normal men in order to determine if sperm motion analysis is better than sperm density as an indicator of fertility potential. Nine fertile men with IHH treated with exogenous gonadotropins and 20 fertile normal men were studied. Sperm density was significantly lower in the men with IHH compared with normal men (15.5 ± 4.8 X 106/ml versus 92.4 ± 9.7 X 106/ml; mean± standard error of the mean [SEM]; P < 0.01) as was percent motility (51.4 ± 4.7 versus 73.4 ± 3.1; P < 0.01). While a small but significant difference in Vc was noted between the groups at the 40 p.m/second cumulative distribution point (P < 0.01), no difference in L was found between the two groups. When the men with IHH were subgrouped according to sperm density (> 20 X 106/ml versus :s;20 X 106 /ml, no differences in Vc were found between the subsets, but for L sperm were somewhat less directional for the subgroup with a density :S20 X 106/ml (P = 0.05). Coanalysis using both Vc and L parameters indicated that sperm from IHH patients were distributed similarly to sperm from normal men. However, sperm motion characteristics in men with unexplained infertility were different from values measured in normal men and IHH patients. From our data, we conclude that automated sperm motion analysis is a better indicator of fertility potential than sperm density. Fertil Steril51:162, 1989

Men with isolated hypogonadotropic hypogonadism (IHH) can be successfully treated with exogenous gonadotropins, either human chorionic Received May 13, 1988; revised and accepted October 14, 1988. * Recipient of the Serono Fellowship in Reproductive Endocrinology at the National Institute of Child Health and Human Development. t Present address: Department of Obstetrics and Gynecology, University of Chile, Santiago, Chile. t Developmental Endocrinology Branch, National Institute of Child Health and Human Development. §Office of the Scientific Director, National Institute of Allergy and Infectious Disease, National Institutes of Health. II Reprint requests and present address: Richard J. Sherins, M.D., Director, Division of Andrology, Genetics & IVF Institute, 3020 Javier Road, Fairfax, Virginia 22031.

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Sperm motion in IHH

gonadotropin (hCG) alone or in combination with human menopausal gonadotropin. 1- 6 Subsequent fertility can be readily achieved by men with IHH 7 despite a sperm concentration well below the conventionallower limit of 20 X 106 /ml. 7- 20 These observations indicate that sperm concentration per se is a poor index of fertility potential, suggesting that functional capacity is more important than sperm number. The aim of the present study was to compare the sperm motion characteristics of fertile oligospermic men with IHH to those of fertile normal men in order to determine whether or not automated sperm motion analysis is better than sperm density as an index of fertility potential.

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MATERIALS AND METHODS

Table 1 Sperm Concentration and Percent Motility from Patients with Isolated Hypogonadotropic Hypogonadism and Normal Fertile Men a

Subject Selection and Semen Acquisition

Twenty normal men who had impregnated their partner during the past 2 years, and nine patients with IHH who had fathered a child after treatment with exogenous gonadotropins, were studied. In 5 of 9 men with IHH, the sperm density was <20 X 106 /ml, whereas in 4 men, the sperm density was greater than 20 X 106 /ml. Each subject collected one semen specimen by masturbation after a strict 36 to 48 hours of abstinence, which was examined within 60 minutes after ejaculation.

Concentration (Xl06 /ml)

Motility (%)

Normal men (n = 20)

92.4 ± 9.7

73.4 ± 3.1

IHH patients All subjects (n = 9)

15.5 ± 4.8b

51.4 ± 4.7b

30.0 ± 3.1b

50.1 ± 4.0b

Sperm density ;e,20 X 106 /ml (n= 4) Sperm density <20 X 106 /ml (n = 5)

5.5 ± 1.5 b,c

52.4 ± 7.4b

Sample Processing Values shown as mean ± standard error of the mean. P < 0.01 compared with normal men. c P < 0.01 compared with IHH patients with >20 X 106 sperm/ml. a

After liquefaction, 10 ~l of undiluted semen was placed into a Makler chamber (Sefi-Medical Instruments, Rehovot, Israel) and analyzed by computer-assisted semen analysis (CASA). For sperm count, nine random fields were analyzed per sample. If the initial analysis revealed a sperm concentration higher than 40 X 106 /ml, the sample was diluted with the patient's own cell-free seminal plasma in order to minimize cell collisions which can interfere with the assessment of curvilinear velocity.21 When the semen sample contained large numbers ofnonsperm particles or when sperm density was <20 X 106 /ml, concentration was determined manually using a Makler chamber to avoid overestimation of sperm density. 21 CASASystem

The CellSoft System consists of an Olympus BH-2 phase contrast microscope (Olympus Optical Co., Ltd., Tokyo, Japan) with lOX objective, a Panasonic videocamera (CCTC), two Panasonic videomonitors, a Panasonic videocassette recorder (Panasonic, Denver, CO), an IBM AT computer (Office Automation, Bethesda, MD; International Business Machines, Armonk, NY) and the CellSoft software (CryoResourses Ltd., New York, NY). The software parameters for optimizing the measurement of sperm density and analysis of sperm motion have been reportedpreviously. 21 -23 Sperm Motion Analysis

Fifteen to twenty random microscopic fields were analyzed for each semen sample to assess spe.rm motion characteristics. The frequency distributions for curvilinear velocity (V c) and linear-

Vol. 51, No.1, January 1989

b

ity (L) were determined by the CellSoft program. Because V c and L are not normally distributed, describing Vc and L by their mean values is not valid. 24 Accordingly, the cumulative percent distribution of cells was calculated at increasing inter'-:als of Vc and L. 24 Individual patient values then were compared to the weighted average of the cumulative distributions of Vc and L for normal men as well as to values for men with unexplained infertility previously reported. 24 Statistical Analysis

Sperm concentration and percent motility were analyzed by the Wilcoxon rank sum test, and the cumulative distribution for curvilinear velocity and linearity were analyzed by the Kolmogorov-Smirnov (nonparametric) test. Data were considered statistically significant when P < 0.05. RESULTS Sperm Concentration and Percent Motility

Mean values for sperm concentration and percent motility from normal men and IHH subjects are summarized in Table 1. Mean sperm density and percent motility were significantly different between the normal men and men with IHH (P < 0.001). However, there was no significant difference in percent motility between the subgroup of men with IHH whose sperm density was >20 X 106 /

Vantman et al.

Sperm motion in IHH

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which were located 34.2% of cells of normal men and 51.2% of cells of men with IHH (P = 0.01). By contrast, the cumulative distributions for L were indistinguishable between the two groups (P > 0.2). Among the IHH patients, the cumulative distribution for Vc did not differ (NS) between the subgroup with a sperm density >20 X 106 sperm/ml and those men with <20 X 106 sperm/ml (Fig. 1B). However, for the two subgroups of IHH patients, the cumulative distribution for L was significantly greater (less directional) for patients whose sperm density was <20 X 106 sperm/ml than for patients with more than 20 X 106 sperm/ml; La was the point of maximum difference for linearity between the two subgroups (P = 0.05). Figure 2 shows a coanalysis of the cumulative distribution values at Vc 40 and La for each IHH patient plotted in reference to the weighted average

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Figure 1 The weighted averages for the cumulative distributions of curvilinear velocity (Vc) and linearity (L) for normal fertile men (donors), fertile men with IHH, and a group of men with unexplained infertility. (A) comparison of normal men with IHH patients. (B) comparison of IHH patients with >20 X 106 sperm/ml with IHH patients with <20 X 106/ml. (C) comparison of normal men and IHH patients with men with unexplained infertility. Data are shown as cumulative sperm number (percent) at increasing intervals ofVc and L.

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Figure 1 shows the weighted average for the cumulative distributions of curvilinear velocity (Vc) and linearity (L) for normal fertile men, fertile men with IHH, and a previously reported24 group of men with unexplained infertility (n = 53). Using 15 to 20 random fields of observation, the total number of cells available to assess Vc and L ranged from 44 to 130 (median, 76) for normal men and from 7 to 133 (median, 28) for patients with IHH. For V c, the cumulative distributions for normal fertile men and IHH patients were significantly different between Vc40 to Vc 60 JLm/sec (Kolmogorov-Smirnov, nonparametric test, P = 0.05); 40 JLm/ sec was the point of maximum difference below

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164

Vantman et al.

Sperm motion in IHH

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Figure 2 Distribution of individual patient curvilinear velocity (Vc) and linearity (L) values at Vc40 , L 3 compared with the weighted average values of normal fertile men at that level. (A) data from normal fertile men and IHH patients. (B) data from men with unexplained infertility.

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of V c40 and La for fertile normal men. For convenience, the mean Vc 40 and La values for fertile normal men were reset at 0. Semen specimens from IHH patients with more cells at Vc40 than the mean value for normal fertile men were located to the left of the center line (slower), while specimens with fewer cells at V c40 than the mean for fertile men were located to the right of the center line (faster). Regarding linearity, specimens with lower La values (more linear) were plotted above the center line and specimens with higher La values (less linear) were plotted below the center line. By chi-square analysis, the IHH patients as well as the normal fertile men were evenly distributed in the four quadrants of the graph (Fig. 2A), indicating that sperm from the IHH patients were similar in terms of curvilinear velocity and linearity to those of normal fertile men (P > 0.2). DISCUSSION

Despite numerous studies of sperm count and semen quality, it has been difficult to establish lower boundaries for these parameters among fertile men. 18•19•25 Nevertheless, in many laboratories, a sperm density of 20 X 106 sperm/ml has been set as the lower limit of normal because the incidence of infertility increases as sperm concentrations fall below this level. 8 •14 •15•18•19•25 Accordingly, the term "oligospermia" has come to describe a cause of male infertility, even though studies of fertile men seeking vasectomy 19 and those participating in drug trials of potential contraceptives 16 have demonstrated that impregnation can occur at a sperm density of 5 X 106/ml. Moreover, a recent study from our laboratory showed that more than 90% of men with IHH treated with exogenous gonadotropins can impregnate their spouse despite a sperm concentration well below 20 X 106/ml. 7 Thus, we postulate that it is functional capacity of sperm, rather than the actual sperm number, which is critical to whether or not a man can impregnate his partner. Functional sperm abnormalities might include defects in the ability of sperm to fertilize an ovum, or motility disorders that prevent sperm from reaching the site of fertilization. Objective techniques to assess cell motion characteristics such as CASA now provide a scientifically credible method to measure sperm motility and determine its role in disorders of fertility. Our data reveal that "oligospermic" fertile men with IHH have sperm roo-

Vol. 51, No.1, January 1989

tion characteristics (Vc and L) that are similar to those of normal fertile men. In contrast, we have shown in a previous study that 80% of men with unexplained infertility, have sperm motion characteristics (V c and L) that are significantly slower and less directional than those from normal fertile men. 24 These data support the contention that the sperm motion characteristics of men with IHH are normal, despite markedly reduced sperm density. There are several caveats to be noted in measurements of sperm motion characteristics. Semen must be diluted with cell-free seminal plasma to a concentration below 40 X 106 /ml in order to avoid cell-cell collisions that interfere with assessments of high-velocity sperm. 21 Furthermore, CASA techniques require optimization of the number of tracking points and the equipment settings for accurate cell motion analysis. 21 - 2a Recently, we have also recognized that because sperm movements in semen are not normally distributed, a description of Vc or L as a mean value is not statistically valid. Instead, cumulative distributions of Vc and L more accurately quantitate motion characteristics and more clearly define subpopulations of sperm that are slower and less directional than those of normal men as well as sperm whose motions are indistin· guishable from those of normal men. 24 In comparing sperm motion of IHH patients with that of men with unexplained infertility (Fig. 1C), the cumulative distributions for curvilinear velocity and linearity were significantly different between Vc 20 to Vc60 (P < 0.001) and L 2 to L 5 (P < 0.001); the points of maximum difference were Vc40 and La. The difference in the cumulative distribution at Vc 40 between men with unexplained infertility and IHH patients was similar in magnitude to the difference measured between IHH patients and normal fertile men. Though no attempt has been made to define a rigid boundary for cumulative velocity and linearity between fertile and infertile men, the small increase in proportion of slower sperm among IHH patients provides a reasonable boundary for fertile men for the curvilinear velocity parameter. Acknowledgments. We wish to thank Serono Laboratories, Inc. for their generous support of our research effort in sponsoring a Fellowship in Reproductive Endocrinology, and Mrs. Kathy Shoobridge for her expert preparation of the manuscript. REFERENCES 1. Burger HG, de Kretser DM, Hudson B, Wilson JD: Effects of preceding androgen therapy on testicular response to hu-

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Sperm motion in IHH

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