Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age

Abstract

Sperm parameter values have been shown to decline with age, according to conventional sperm analysis. However, the effect of age on sperm kinematic parameters has been rarely studied, especially in young fertile men. Here, we studied Computer-Assisted Sperm Analysis (CASA) parameters in a large cohort of men with proven fertility, in order to determine if there is a decline with age in this young fertile population. This retrospective analysis of CASA parameters was conducted on all donors included in the sperm donor programme in the Assisted Reproductive Techniques (ART) Centre of the University Hospital of Nantes between 2006 and 2009. Sperm concentration, motility, and kinetic parameters were recorded by a HTM-Ceros system and compared in 3 groups of sperm donors according to their age: <35 years, 36–40 years, and 41–44 years. A total of 362 ejaculates from 138 donors were analyzed. Values for ALH, VCL, LIN, and STR significantly decreased with age. Sperm concentration, motile sperm proportion, and other kinetic parameters did not differ significantly among the groups. The use of CASA allowed the identification of ALH, VCL, LIN, and STR age-related decrease in young men with proven fertility.

Keywords:

• age
• CASA
• motility parameters
• sperm donor

Introduction

Semen analysis remains the most widely used test to evaluate male fertility. Some studies conducted in the general population or in men attending andrology clinics have shown that semen quality declined with age [Cardona et al. 2009; Sloter et al. 2006]. However, conventional semen analysis has some limitations and is not adequate to diagnose with certainty male infertility [Guzick et al. 2001; van der Steeg et al. 2011]. That is why complementary tests have been developed to improve the predictive power of semen analysis, such as Computer-Aided (also called Computer-Assisted) Semen Analysis (CASA) [Shibahara et al. 2004], DNA fragmentation studies [Cohen-Bacrie et al. 2009], and seminal Reactive Oxygen Species (ROS) measurement [Koppers et al. 2008]. Moreover, semen analysis suffers from variability and subjectivity, especially in estimating motility, even if efforts have been made to standardize technical aspects and improve quality control [WHO 2010]. Therefore it might not be possible to identify potential age-related modifications of sperm parameters. CASA is an automated reproducible method that can provide accurate and objective information on sperm parameters, especially on motility values, providing that it follows regular quality control and that it has been calibrated with manual standard semen analysis [Agarwal and Sharma 2007; Keel et al. 2000]. CASA parameters should not replace World Health Organization (WHO) semen analysis categories [WHO 2010], but they may offer additional insights into sperm movement [De Geyter et al. 1998; Johnson et al. 1996; Togni et al. 1995; Yeung et al. 1997]. The purpose of this work was to study CASA parameters in a large cohort of men with proven fertility, i.e., sperm donors, and to determine if there is a decline with age in this fertile population.

Results

A total of 362 ejaculates from 138 donors was analyzed. The mean number of ejaculates per donor was 2.6 (range 2–6). Mean age was 35.1 ± 5.1 years. Donors had 2.2 children on average, and were recruited approximately 4 years after the birth of their last child. Sperm donors were classified into 3 age categories: <35 years, 36–40 years, and 41–44 years. Mean abstinence time was comparable in the three groups (data not shown). The values of CASA parameters according to age subgroups are summarized in Table 1. All the kinematic parameters were distributed normally, whereas concentration and motility were not. All motility parameters tended to decrease with age, but statistical analysis (ANOVA) showed that only ALH, VCL, LIN, and STR significantly decreased with age among age groups (Table 1). Kruskal Wallis test and Student's t test showed that differences existed among 36–40 and >40 years age groups for ALH and VCL (Table 1). Sperm concentration and total sperm number per ejaculate did not differ significantly among age groups. CASA analyses did not detect an age-related modification in VSL, VAP, rapid progressive motility (grade a), or total motility (grade a + b + c). Using regression analysis, we did not show any significant decrease of kinematic parameters with age in the overall population. However, we found a significant negative association between age and motile or progressively motile sperm percentages (P = 0.05 and P = 0.03, respectively).

Table 1. CASA parameters in sperm donors according to age. Motile sperm percentages are presented according to WHO [1999] guidelines. Results are presented as means (standard deviation).

	<35 years (n = 182 ejaculates)	36–40 years (n = 116 ejaculates)	41–44 years (n = 64 ejaculates)	ANOVA
total sperm concentration (millions/ml)	62.3 (51.8)	63.8 (42.2)	62.92 (39.1)	NS
total sperm count per ejaculate (millions)	195.4 (150)	201 (122.9)	195.5 (100.2)	NS
motile sperm proportion (grade a + b + c) (%)	59.9 (15.9)	59.36 (16.8)	54.92 (20.4)	NS
progressive motile sperm proportion (grade a) (%)	35.2 (12.2)	33.3 (11.7)	32.5 (13.2)	NS
ALH (µm)	3.36 (0.6)	3.38 (0.7)^a	3.19 (0.7)^a	P < 0.001
VSL (µm/s)	46.8 (9.1)	46.6 (10.2)	46.2 (8.3)	NS
VCL (µm/s)	79.7 (13.7)	80.1 (15.5)^a	76 (14.2)^a	P = 0.01
VAP (µm/s)	55.6 (10)	55.6 (11)	54 (9.6)	NS
LIN (VSL/VCL)	60.1 (6.1)	57.9 (6.2)	61.5 (5.7)	P = 0.002
STR (VSL/VAP)	83.3 (3.8)	82 (4.2)	82.3 (4)	P = 0.003

^aP < 0.05 (Kruskal-Wallis test or Student's t test)

Discussion

To our knowledge, this work is the first CASA-based study aiming at identifying age-related sperm motion modifications in young men with proven fertility. We report a significant decline with age of ALH, VCL, LIN, and STR, whereas VSL, VAP, rapid progressive motility (grade a), and total motility (grade a + b + c) were not statistically different among age groups.

This population of sperm donors with proven fertility neither represents the general population nor the population of men attending andrology clinics. However, we took inspiration from the last version of WHO manual for semen analysis [WHO 2010], which stated that the best reference population consists in young healthy fathers whose partner had a time to pregnancy of 12 months or less [Cooper et al. 2010]. Here in our population, time to pregnancy was not recorded during the sperm donor's interview. However, our population was homogeneous, and all men recruited had relatively recently conceived (4 years on average) and did not report any reproductive problem. Therefore, this population of young healthy donors with proven fertility can be considered as a reference fertile population, and could thus be used as a preliminary basis for the publication of CASA reference values. We chose to study all ejaculates separately, rather than the median of values for each donor in order to reduce the effect of intra-individual variations. This can be discussed from a statistical point of view. However, even if within-subject variation of CASA parameter values in sperm donors has been shown to be lower (<25%) than that in infertile patients, some authors have suggested that it could be more useful to consider all ejaculates separately and to increase the number of subjects [Wijchman et al. 2001]. Another interesting approach based on a longitudinal follow up of patients could be of interest. Krause and Habermann [2000] did not find a significant modification with age in sperm parameter values measured by CASA in infertile patients. However, this study was conducted in infertile patients, not in fertile sperm donors, and the interval between analyses was 3 years on average. This raises the question of intra-individual variation and of optimal time interval necessary to detect a significant alteration in sperm motility. Moreover, the population of infertile men studied here was probably very heterogeneous in terms of medical, urological, and surgical history and thus too limited in terms of number to allow accurate detection of fine modification in sperm motion parameters. Many CASA systems are commercially available, based on various detection systems, and therefore potentially yielding slightly different results. However, Holt et al. [1994] showed that ‘within system’ variability is greater than that ‘between systems’. This underlines that differences in sample handling and operator expertise is a more significant source of variation than the CASA systems themselves [Holt et al. 1994]. Our data were obtained with a Hamilton-Thorn system (HTM-CEROS), and should be extrapolated to other systems with caution.

A study using the HTM-Ceros CASA system was published in 2006 by Sloter et al. [Sloter et al. 2006], who compared CASA parameters in a non-clinical cohort of 97 healthy men without reproductive problems. However, contrary to our work, no information was given on their fertility status, as children recently born, and the donor age range was very large (22–80 years). The authors found a significant decrease of motility, LIN, VAP, and VSL with age, whereas we did not find any significant decrease with age for motility, VAP, and VSL. On the contrary, we found a significant decrease of ALH and VCL with age. First, the difference of recruitment could explain at least in part the different conclusions of this study. Our study was conducted in men with proven fertility, i.e., men who became fathers spontaneously, whereas being a father was not a prerequisite in the study by Sloter et al. [2006]. Secondly, sample handling differed significantly. Indeed, men had to provide a semen sample by masturbation at home and then to deliver the container inside an insulated container to a drop box in the study by Sloter et al. [2006], whereas semen was collected directly at the laboratory in our study. Moreover, CASA analyses were conducted on raw semen samples in our laboratory, whereas a dilution was systematically performed in the study by Sloter et al. [2006]. However, both studies can provide interesting and complementary results. Given our results, the hypothesis of age-related decrease of motility and LIN but maintaining ALH leading to fewer spermatozoa reaching the oocyte in older men can be debated. In our study, advancing age was correlated with the decline in sperm ability to reach oocyte, as reflected by lower VCL, STR, and LIN, which are associated with a lower penetration and fusion ability, as reflected by decreased ALH.

Several mechanisms have been raised to explain age-related sperm parameter modifications. Declining sperm motility observed in men over 50 years of age might be due to changes in epididymal and accessory sex gland function [Elzanaty 2007]. Correlation between seminal reactive oxygen species and decreased sperm quality have also been demonstrated [Agarwal et al. 2006]. In a recent study conducted in fertile men, men over 40 years had higher ROS levels than those of younger men [Cocuzza et al. 2008]. Moreover, in the same study, the correlation between ROS levels and age was positive, and significantly negative between ROS levels and sperm concentration and motility [Cocuzza et al. 2008]. These age-related modifications of sperm motion parameters could account, at least in part, for increased time to pregnancy in wives of men over 40 years of age [Kidd et al. 2001]. Whether a decrease in sperm kinematic parameters influences fertilizing ability of spermatozoa remains to be addressed. Indeed, few studies have reported an association between CASA and fertilization in vitro and/or in vivo. An interesting study by Hirano et al. in 2001 reported significant differences in CASA before and after swim up among patients categorized by fertilization rate [Hirano et al. 2001]. Another study by Liu et al. [1991] demonstrated that some kinematic parameters in semen were significantly correlated with in vitro fertilization rate. De Geyter et al. [1998] found that the sensitivity of CASA for the prediction of fertilization was high (74%), whereas the specificity was low (40%). However, these authors concluded that a definite threshold level could not be identified for any of the motion parameters to distinguish the motion characteristics of fertilizing and non-fertilizing spermatozoa [De Geyter et al. 1998]. It might be hazardous to draw conclusions from these studies, conducted with various devices and protocols. However, one might speculate that the association between kinematic parameters and fertilization on one hand, and between kinematic parameters and age on the other hand could account, at least in part, in decreased fertilizing ability of spermatozoa with advancing age. The underlying physiologic mechanisms remain to be clearly identified. All donors in this study had a normal Body Mass Index (data not shown). This is of importance as it has already been shown that obese men are more at risk of semen alterations [Du Plessis et al. 2010]. We did not have reliable information on the smoking status and alcohol or caffeine consumption of our donors. As these factors could have a deleterious impact on spermatogenesis [Martini et al. 2004], they should be included in future studies.

We did not compare conventional sperm analysis with CASA results in this population of sperm donors. In order to improve the efficiency of our andrology laboratory, CASA was used in the first instance for men thought to have normal sperm parameter values, such as sperm donors. CASA can be used as a supplement to manual semen analysis, and theoretically allows objective, reproducible, and large-scale analysis, provided it has been calibrated with manual standard semen analysis according to WHO references. Indeed, quality control of CASA systems is critical in its performance [Krause and Viethen 1999]. Such a study was conducted in our laboratory before CASA routine use, but as previously shown in different studies, the large inter and intra-specimen variability, especially of motility in low concentration samples, did not allow us to replace manual analysis by CASA alone for all men attending the andrology laboratory [Krause 1995; Vantman et al. 1988]. However, despite these limits in conventional semen analysis, CASA provides useful information on sperm motion characteristics, and can bring new insights into sperm fertilization capacity. Even if some variations in the conditions of the experiment can affect CASA motility results [Davis and Boyers 1992], we performed CASA motility assessment with constant machine settings, counting chamber, and time interval between sample collection and CASA assay in order to standardize our results as much as possible. We did not study sperm morphology, as we could not use an automated system at the time of the study. As manual morphology assessment is largely subject to variability [Auger et al. 2000], we did not choose to include this parameter in the present analysis. Further studies including automated morphology assessment should be conducted in order to identify a potential age-related decline in the proportion of morphologically normal forms.

In conclusion, this study is the first to evaluate age-related decrease in sperm CASA parameters in a population of young men with proven fertility. The use of CASA, an interesting tool in terms of objectivity and reproducibility, allowed us to show that ALH and VCL significantly decreased with age, thus bringing new insights into age-related male infertility and supporting the need for further studies aimed at identifying underlying mechanisms. These data highlight the accuracy of CASA in depicting fine sperm motion modifications, and should lead to better medical information aimed at men who choose to delay fatherhood on the risk of age-related alteration of sperm fertilization ability.

Materials and Methods

We analyzed the data of consecutive anonymous sperm donors. These data had been collected prospectively and recorded in a registered database between January 2006 and December 2009 in our fertility center in Nantes, France. All the patients gave written informed consent for the procedures and for digital recording and use of the data related to their history. All ejaculates from sperm donors recruited during this period were retrospectively analyzed. Sperm collection was performed at the laboratory after 2-5 days of sexual abstinence. Appointment was cancelled when the donor reported fever >38°C in the past 3 months. The screening of anonymous sperm donors followed all current recommendations for sperm donors in France: donors were all healthy anonymous volunteers, without any reproductive problem history, unpaid for their participation in the donor program, strictly under 45 years old, who had tested negatively for sexually transmitted diseases (HIV, hepatitis C and B, syphilis) and whose partners had already conceived at least one healthy child.

Sperm analysis was performed according to WHO guidelines, 4^th edition [1999]. After 30 min liquefaction at 37°C, total sperm concentration, motile sperm percentages, progressive motile sperm percentages (grade a), and kinematic parameters, i.e., average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head displacement (ALH) were recorded for each ejaculate with Hamilton-Thorne system (HTM-CEROS, version 12.2L, Beverly, MA, USA) connected to an Olympus microscope (100x total magnification) according to the manufacturer's recommendations. Linearity (LIN) and straightness (STR) were also calculated (LIN = VSL/VCLx100 and STR = VSL/VAPx100). A 7 µL sample of semen was loaded into a pre-warmed disposable analysis chamber with a depth of 20 µm (Leja Products, Nieuw-Vennep, The Netherlands). It was then placed for analysis on the warmed plate (37°C) of the HTM-CEROS. A minimum number of 200 cells was counted for each assay. Cells with an average sperm head's lateral displacement (ALH) > 2.5 µm, path velocity (VAP) > 5 µm/s and straight velocity (VSL) > 11 µm/s were classified as motile cells, regrouping rapid progressive motile cells (‘a’ category), slow motile cells (‘b’ category), and static motile cells (‘c’ category). Among these motile cells, those with an average path velocity (VAP) > 25 µm/s and straight velocity (VSL) > 100 µm/s were classified as rapid progressive motile cells (‘a’ category).

Statistical analysis was performed with a standardized computer program (Medcalc, version 7.0.3.1, Mariakerke, Belgium). ANOVA was used to test for differences in CASA parameters among the 3 age categories. Kruskal-Wallis test (H-test) was used to test the hypothesis that a number of unpaired samples originate from the same population. If the Kruskal-Wallis test is positive (P < 0.05), i.e., the hypothesis that the samples originate from the same population is rejected, we performed a test for pairwise comparison of subgroups according to Conover [1999]. Linear regression analyses were also made. A value of P < 0.05 was considered to be statistically significant.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.