Malm2018.pdf

ANDROLOGY

ISSN: 2047-2919

ORIGINAL ARTICLE

Correspondence: Gunilla Malm, Reproductive Medicine, Department of Translational Medicine, Lund € , 205 02 Malmo € , Sweden. University, SUS Malmo E-mail: [email protected]

Keywords: fertility, male infertility, reproduction, semen analysis

Association between semen parameters and chance of fatherhood - a long-term follow-up study 1

Received: 12-Mar-2018 Revised: 20-Sep-2018 Accepted: 24-Sep-2018

G. Malm

, 2L. Rylander, 1A. Giwercman

and 3T. B. Haugen

1 Molecular Reproductive Research, Department of Translational Medicine, Lund University, Skane € , Sweden, 2Division of Occupational and Environmental Medicine, Lund University Hospital, Malmo University, Lund, Sweden, 3Faculty of Health Sciences, OsloMet–Oslo Metropolitan University, Oslo, Norway

doi: 10.1111/andr.12558

ABSTRACT Background: Evaluation of male fertility includes standard semen analysis; however, there is uncertainty about the value of sperm parameters in predicting fertility. Objective: To evaluate the association between semen parameters and fatherhood during a long-time period. Materials and methods: Semen parameters (total sperm count, concentration, motility, and morphology) and sperm DNA fragmentation Index (DFI) assessed on samples collected from 195 Norwegian men from the general population in 2001/2002 were matched with information about fatherhood until 2015, obtained from the Medical Birth Register. The parameters were dichotomized as normal vs. abnormal according to the WHO reference values from 1999 and 2010. Cut-offs at 20% and 30% were used for DFI. Results: Among men who had no children before 2003, those with normal progressive sperm motility had more often become fathers (WHO 1999, cut-off ≥50%, adjusted OR 2.8, 95% CI 1.3–6.1 and WHO 2010, cut-off ≥32%; aOR 4.2, 95% CI 1.1–15). Based on the WHO 1999 reference value, men with normal sperm concentration (≥20 9 106/mL) had more often become fathers (aOR 3.1, 95% CI 1.1–8.6). Men with progressive sperm motility ≥50% and concentration ≥20 9 106/mL did more often achieve fatherhood (aOR 8.4, 95% CI 2.1–34). For DFI, there was a borderline significance at cut-off 20% in the group of men who had ever been fathers (OR 2.7, 95% CI 1.0–7.0 p < 0.05). Discussion: The results indicate that sperm progressive motility, sperm concentration, and DFI are associated with fatherhood during a longer time period, with sperm motility being most consistent. Although the sample size is relatively small and our results should be replicated in larger studies, they may be of clinical relevance. Conclusion: Semen parameters may have a diagnostic value not only in a short time frame but also for predicting future fertility potential.

INTRODUCTION In most Western societies, there is a tendency to postpone parenthood, partly due to socioeconomic factors. Both increasing maternal and paternal age are associated with a higher risk of involuntary childlessness (Sartorius & Nieschlag, 2010). Therefore, there is a growing interest and need of offering both men and women assessment and counseling regarding their present and future fertility potential (Birch Petersen et al., 2015). In addition to the clinical examination, the evaluation of male fertility is traditionally based on standard semen analysis, including total sperm count, concentration, motility, and morphology. To standardize and improve the quality of semen analysis, WHO has published laboratory guidelines for the examination of human semen (World Health Organization, 1999, 2010). © 2018 American Society of Andrology and European Academy of Andrology

However, there is uncertainty about the value of standard sperm parameters to predict fertility. Earlier studies have indicated that the probability of conception increases with increasing sperm concentration up to 40–55 9 106/mL (Bonde et al., 1998; Guzick et al., 2001; Slama et al., 2002). Sperm motility has been shown to be of limited predictive value in some studies (Bonde et al., 1998; Slama et al., 2002), whereas other studies have shown sperm motility to be the best predictive factor for fertility (Larsen et al., 2000; Nallella et al., 2006; Jedrzejczak et al., 2008). Sperm morphology is the most controversial parameter of the semen parameters due to the lack of standardization and the fact that scoring criteria and reference values vary across centers. Thus, different methodologies may have prevented a consensus Andrology, 1–6

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on the clinical value of sperm morphology evaluation(Tomlinson et al., 2013). Impairment of sperm DNA integrity has been suggested as an independent predictor of male fertility (Bungum et al., 2007; Giwercman et al., 2010), but due to the lack of standardization and reference values, this parameter has still not been included in routine fertility assessment (Esteves et al., 2017; Rex et al., 2017). In order to evaluate the association between semen parameters and chance of fatherhood during a longer time period, we have performed a follow-up study by relating the semen parameters in a cohort of Norwegian men, collected in 2001–2002 (Malm et al., 2004), with data on children fathered until 2015 by these men from the Norwegian Medical Birth Registry. Furthermore, the participants were asked to fill in a questionnaire about fertility wishes to investigate whether the pattern was similar to the information from the registry. In contrast to previous studies, we have focused on the association between semen parameters and fatherhood during a long-term period (13 years and more) following the semen analysis.

MATERIALS AND METHODS Subjects and study design During the period May 2001 to March 2002, a cohort of Norwegian men, between 19 and 40 years old, were recruited from the general population (Malm et al., 2004). The original aim of the initial study was to evaluate a possible seasonal variation in male reproductive function among the participating men. Of the 221 men at study start, 204 men fulfilled the study criteria and delivered one semen sample in the summer and one in the winter. In the follow-up study in 2015, data if and when these men had become fathers were obtained from the Norwegian Medical Birth Registry for 195 of the 204 men. The remaining nine men were lost to follow-up. These were either dead (1), or emigrated (2), or lacking information of unknown reasons. The cohort was divided into two groups, if they had become fathers before and after January 1st 2003, that is, nine months after the study was completed in March 2002 and if they ever had become fathers. In order to discriminate between voluntary and involuntary childlessness, men from the 2001 to 2002 study were contacted in 2014 to 2015 and asked to complete a new questionnaire online about their present health and fertility status. The subjects were asked if they ever had tried to get a biological child and if they had biological children, yes or no? One hundred and one men (52%) replied. The study is approved by the Regional Committee for Medical and Health Research Ethics, South East, Norway, REC number 2013/604. Semen analysis The semen samples were collected in 2001–2002 and analyzed according to recommendations in the WHO guidelines from 1999 (World Health Organization, 1999). Subsequently, for sperm concentration, total number, percentage motile sperm, and percentage morphologically normal sperm, they were categorized as normal or abnormal according to both the 1999 and 2010 cut-off reference values (shown in Table 1). In the WHO 1999 manual, it was recommended that progressively motile 2

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spermatozoa should be categorized as rapid or slow, whereas in the, WHO 2010 manual, the recommendation is that progressive motile spermatozoa should be in one category (World Health Organization, 2010). The motility categories are denoted grade A (rapid progressive), grade B (slow progressive), and A+B (progressive) in the tables. For morphology, the lower reference limit for normal forms (5th centile; 4%), according to the WHO guidelines from 2010, was used (World Health Organization, 2010). All participants were recommended an abstinence period of 2–3 days before delivering the semen sample, and the length of abstinence was recorded. As previously described, (Malm et al., 2004), the semen volume was determined by weighing and the spermatozoa concentration with a Neubauer hemocytometer after dilution of the ejaculate using positive displacement pipette. For motility assessment, a drop of ejaculate was placed on a slide mounted on a heated stage (37°C). The motility of 200 spermatozoa was scored as rapid (A), slow or sluggish progressive (B), non-progressive (C), and immotile (D). The interobserver coefficient of variation (CV) was calculated based on analysis of the same sample performed by the two technicians and was 9% for the sperm concentration and 5% for the motility assessment (Rylander et al., 2009). Sperm chromatin structure assay All analyses were done using the same flow cytometer which was calibrated using aliquots of the same batch of calibrator for every run. The principles and procedure to measure DNA damage by the flow cytometry-based semen chromatin structure assay, SCSA, are described in detail elsewhere (Evenson & Jost, 2000). In brief, the semen sample was subjected to a short acid treatment that denatures DNA at sites of single–or double-strand breaks. The spermatozoa are stained with a fluorescent DNA dye which differentially stains double (green)- and single (red)stranded DNA. The extent of DNA denaturation quantified by flow cytometry is expressed as DFI, which is the ratio of red to total fluorescence intensity, that is, the level of denatured DNA over the total DNA. Five thousand cells were analyzed by FACSort (Becton Dickinson, San Jose, CA, USA), and the data were analyzed using the SCSASOFT software (SCSA Diagnostics, Brookings, SD, USA). A reference sample from a normal donor ejaculate retrieved from the laboratory repository was used (Evenson & Jost, 2000). The same reference sample was used for the whole study period and run for every fifth sample. The intra-laboratory Table 1 Cut-off reference values for semen characteristics in World Health Organization (WHO) manuals from 1999 to 2010

Total sperm count (106) Sperm concentration (106/mL) Motility, grade A (%) Motility, grade A+B (%) Total motility, grade A+B+C (%) Morphology (% normal forms) Volume (mL)

WHO (1999)

WHO (2010)a

40 20 25 50

39 15

b

4 2.0

32 40 4c 1.5

a

Lower reference limit obtained from lower fifth centile value. bValue not defined, but strict criteria suggested. cStrict (Tygerberg) criteria, Grade A, rapid progressive motility (>25 lm/sec); grade B, slow/sluggish progressive motility (5– 25 lm/sec); grade C, non-progressive motility.

© 2018 American Society of Andrology and European Academy of Andrology

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SEMEN PARAMETERS AND CHANCE OF FATHERHOOD

coefficient of variation (CV) for the DFI analysis was 4.5%. All analyses were conducted at the same laboratory. Statistical analysis The background characteristics are shown as mean (SD) and median (range). For each individual, the mean value of the semen parameters was calculated based on the summer and winter values. By logistic regressions, generating odds ratios (ORs) and 95% confidence intervals (CIs), we investigated the associations between each of the standard semen parameters (volume, total sperm count, concentration, motility [progressive and total], morphology, and DFI) and fatherhood. The outcome ‘fatherhood’ was defined in two ways: had become father for the first time after January 1st 2003 (yes vs. no; i.e. those who had become fathers before this recruitment were excluded in these analyses) and had ever become father (yes vs. no; including all men). The standard semen parameters were dichotomized according to the reference values given by the WHO guidelines in 1999 and 2010, respectively (Table 1). For DFI, we dichotomized at either 20% or 30% which were based on studies showing that the chance of spontaneous pregnancy decreases when DFI exceeds 20–30% and almost no chance if DFI is above 30%. A combined variable based on the parameters sperm motility and sperm concentration was created using the WHO reference values (three categories: both parameters above the cut-off reference values, one parameter above and the other one below the cut-off reference values, and both parameters below the cut-off reference values). In addition to the crude analyses, we also performed analyses where we adjusted for age (as continuous variable) and abstinence time (as continuous variable). Since register data do not discriminate between voluntary and involuntary childlessness, separate analyses among the 101 men who answered the questionnaire were performed. Those who stated that they voluntarily had chosen not to have children were excluded in these analyses. A p-value <0.05 was considered statistically significant. All analyses were performed using SPSS (IBM SPSS Statistics 23, Chicago, IL, USA).

• •

(adjusted OR [aOR] 2.8, 95% CI 1.3–6.1, p = 0.008) (Table 4). The same was seen for men with sperm concentration ≥20 9 106/mL (aOR 3.1, 95% CI 1.1–8.6, p = 0.03). The combination of sperm motility and sperm concentration was also significantly associated with higher chance of fatherhood when both parameters were above these cut-off values (aOR 8.4, 95% CI 2.1–34, p = 0.003). For the whole group of men (had ever become fathers), the result was similar for progressive sperm motility ≥50%; aOR 2.7, 95% CI 1.4–5.5, p = 0.005). For sperm concentration, no significant association was found (≥20 9 106/mL; aOR 2.4, 95% CI 0.9– 5.9), whereas the combination of sperm motility and sperm concentration showed a significant association (aOR 5.6, 95% CI 1.8–18, p = 0.004) (Table 4). Applying WHO cut-off values from 2010 The chance of becoming father for the first time after January 1st 2003 was higher for men with progressive sperm motility ≥32% than below (aOR 4.2, 95% CI 1.1–15, p = 0.03) (Table 4). No significant association was observed for sperm concentration (≥15 9 106/mL). Also, in the whole group, there was a higher chance for becoming a father for those men who had progressive sperm motility ≥32% (aOR 3.4, 95% CI 1.1–10, p = 0.03), whereas for sperm concentration, no significant association was observed (Table 4). Analysis of the association of the combined variable sperm motility and sperm concentration and outcome using the WHO 2010 reference values could not be done due to few men with abnormal values (data not shown). In the whole group of men, there was a borderline significance for the association between fatherhood and DFI at cut-off 20% (OR 2.7, 95% CI 1.0–7.0, p < 0.05). Due to few men with DFI >30%, calculations based on this cut-off level could not be performed. Among the 101 responders of the questionnaire, 72 confirmed child wishes. Neither in the group who had become father for the first time after January 1st 2003 nor among those who ever had become father, significant associations with sperm parameters were found, but the trend was similar to the results based on the registry data (data not shown).

DISCUSSION RESULTS Background characteristics The mean (SD) and median (range) for the semen parameters for respective group are summarized in Table 2. Prior to the start of the study (2001/2002), 30% of the men had fathered at least one child. In 2015, the last year from which the register data were available, 71% were fathers. In Table 3, differences for age, sperm concentration, sperm motility, and registered fathers are shown between responders and nonresponder to the follow-up questionnaire in 2015. Association between semen parameters and fatherhood Applying WHO cut-off values from 1999 Based on the data from the Medical Birth Register, the chance of becoming a father for the first time after January 1st 2003 was higher when progressive sperm motility was ≥50%, than below © 2018 American Society of Andrology and European Academy of Andrology

We found that men with progressive sperm motility in the reference range more often had become fathers as compared to those with values below the limit. Similar results were seen regarding fatherhood after January 1st 2003 and ever fatherhood. Our findings are in agreement with previous results, showing that sperm motility may be is a useful predictive parameter with regard to fertility outcome (Nallella et al., 2006; Jedrzejczak et al., 2008). Sperm motility is considered to be an important indicator of semen quality and fertility potential, an indirect marker for normal development during spermatogenesis and normal composition of seminal plasma and required for transport through the female genital tract and penetration of the corona radiata (Nallella et al., 2006). The chance of pregnancy increases with increasing number of motile sperm in the ejaculate (Tomlinson et al., 2013). Guzick et al. showed a cut-off >63% for sperm motility in fertile men whereas values between 32% and 63% were indicative of borderline fertility (Guzick et al., 2001). Furthermore, Nallella et al. reported that men with Andrology, 1–6

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Table 2 Background characteristics from 2001 to 2002, with mean (standard deviation, SD) and median (range) for men who had become fathers before and after January 1st 2003, men who had ever become fathers, and those who not had become fathers Semen parameters mean(SD)/median (range)

Had become fathers before January 1st 2003 n = 62

Had become fathers after January 1st 2003 n = 78

Had ever become fathersa n = 140

Had never become fathers n = 54

Abstinence time (h) Total count (106 per ejaculate) Concentration (106/mL) Motility A+B (%) (WHO 1999) Motility A+B+C (%) (WHO 2010) Motility A (%) Morphology (%) Semen volume (mL) DFI (%)c

89 (37)/84 (21–216) 281 (198)/251 (15–835) 80 (59)/66 (6–318) 48 (9)/50 (22–63) 33 (4)/34 (17–39) 25 (10)/25 (7–51) 8 (5)/7.0 (0–20)b 3.7 (1.6)/3.2 (1.2–8.2) 11 (6)/10 (3–34)f

76 (24)/72 (18–168) 267 (190)/208 (6–836) 62 (41)/59 (2–222) 48 (8)/50 (11–60) 32 (4)/33 (12–38) 28 (10)/27 (4–48) 7 (4)/6 (0–19)c 4.0 (1.3)/3.9 (1.5–7.5) 11 (7)/10 (2–52)

82 (31)/72 (18–216) 274 (193)/220 (6–836) 72 (50)/62 (2–318) 48 (8)/50 (11–63) 32 (4)/34 (12–39) 27 (10)/26 (4–51) 7 (5)/7 (0–20)d 3.9 (1.4)/3.7 (1.2–8.2) 11 (6)/10 (2–52)g

88 (39)/81 (42–288) 216 (1863/162 (8–849) 58 (42)/54 (5–158) 44 (11)/46 (10–65) 31 (5)/32 (11–39) 24 (12)/25 (3–53) 8 (5)/6 (2–24)e 3.8 (1.7)/3.8 (1.1–8.8) 13 (9)/10 (3–39)h

n, numbers; h, hours; A, rapid progressive motility; B, slow/sluggish progressive motility; C, non-progressive motility. aIncludes fatherhood both before and after the sample collections in 2001–2002. bData from 4 subjects are missing. cData from 8 subjects are missing. dData from 12 subjects are missing. eData from 10 subjects are missing. fData from 3 subjects are missing. gData from 3 subjects are missing. hData from 2 subjects are missing.

Table 3 Age and semen parameters from responders and non-responders to the questionnaire (2015) Semen parameters mean (SD)/median (range)

Responders n = 101

Non-responders n = 103

Age (years)

41 (5)a 40 (32–52) 71 (50) 60 (2–318) 25 (10) 26 (5–50) 46 (8) 47 (11–61) 72 (71%)

40 (5)b 39 (31–51) 64 (46)c 58 (5–222) 26 (11)d 26 (3–53) 47 (10)e 50 (10–65) 73 (71%)

Concentration (106/mL) Motility, A (%) Motility, A+B (%) Registered fathers n (%)

n, numbers; A, rapid progressive motility, B, slow/sluggish progressive motility. Data from 1 subject are missing. bData from 8 subjects are missing. cData from 6 subjects are missing. dData from 6 subjects are missing. eData from 6 subjects are missing. a

proven fertility had a motility ranging between 72.5  16.6, and sperm motility was superior compared to other semen parameters with less overlap between men with proven and non-proven fertility (Nallella et al., 2006). In a study of Norwegian fertile men comparing groups with time to pregnancy (TTP) during the first cycle with those who conceived within 12 cycles, only the total number of sperm with progressive motility was significantly different when analyzed by multiple logistic regression (Haugen et al., 2006). In our study, also sperm concentration was associated with higher probability of becoming a father after sample collection for those with a value of ≥20 9 106/mL, but the whole group, this was a significant finding only before adjusting for age and abstinence time. The threshold of sperm concentration related to fertility has been a matter of discussion. Slama et al. found that a sperm concentration less than 20 9 106/mL was associated with decreased fecundity (Slama et al., 2002). In other studies, sperm concentration or sperm morphology has been shown to be the best predicting semen parameters (Bonde et al., 1998; Zinaman et al., 2000). Bonde et al. followed 430 young couples who did not use contraception and concluded that motility is of limited value and that the probability of conception increases with sperm concentration up to 40 9 106/mL. This study was designed to characterize the relation between semen quality and the probability of 4

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conception in a single menstrual cycle; whereas we have studied long-term fertility. As we only had a single semen sample at each time point (summer, winter), intra-individual variation is a factor that could potentially have had an effect on the results. There is a trend in postponing childbearing and therefore an increasing demand among men and women with no known reproductive problems to obtain individual assessment and information about their fertility status (Hvidman et al., 2015). Earlier studies have focused on establishing threshold values for semen parameters to define subjects at high risk of subfertility (Tomlinson et al., 2013). In our approach, we rather focused on how cut-off levels are associated with the subsequent probability of fatherhood in a long-term perspective. The semen parameters were categorized according to both WHO cut-off reference values from 1999 and 2010, the latter being based on a more welldefined reference population and more standardized methods than the 1999 values (World Health Organization, 1999, 2010). Based on the way of recruitment of participants, the men included were from the general population and relatively young (19–40 years old) at baseline in 2001–2002. Prior to the start of the recruitment, 30% of the men had fathered at least one child, and in 2015, the last year from which the register data were available, 71% were fathers. Only minor differences for age, sperm concentration, sperm motility, and numbers of children were seen between responders and non-responders to the follow-up questionnaire in 2015. During the follow-up time between the sample collection (2001–2002) and 2015, some of the responders to the questionnaire reported having been diagnosed with hypertension, hypercholesterolemia, type I and II diabetes, but none of them declared fertility problems. All of these men who reported child wish had become fathers. Some studies demonstrate that a combination of semen parameters provides the best diagnostic profile that could discriminate fertile from infertile men, since each semen characteristic contributes a different aspect to fertility (Nallella et al., 2006). This was also found in our study, with a higher chance of fatherhood associated with when both sperm motility and sperm concentration were above the cut-off reference values than if one of the parameter was below. In contrast to sperm motility and concentration, sperm morphology was not associated with fatherhood in our study. This is in agreement with some previous studies but not with others, © 2018 American Society of Andrology and European Academy of Andrology

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Table 4 Odds ratios for sperm parameters at cut-off reference values according to World Health Organization (WHO) 1999 and 2010 when comparing those who had become fathers after January 1st 2003 vs. those who had not and those who ever had become fathers vs. those who had not Had become fathers after January 1st 2003 Univariate

DNA fragmentation index (DFI) (%)

Concentration (106/mL) Motility A (%) Motility A+B (%) Morphology (normal forms %) Semen volume (mL) Concentration+ motility (A+B) (106/mL + %)

Adjusteda

Univariate

n yes/no

OR (95%CI)

OR (95%CI)

n yes/no

OR (95%CI)

OR (95%CI)

<20

72/43

2.5 (0.8–7.6)

1.8 (0.6–5.9)

127/43

2.7 (1.0–7.0)

2.3 (0.8–6.3)

6/9

Ref

Ref

10/9

Ref

Ref

<40

6/8

Ref

Ref

10/8

Ref

Ref

≥40 <20

72/46 8/12

2.1 (0.7–6.4) Ref

2.4 (0.7–7.6) Ref

130/46 13/12

2.2 (0.8–6.0) Ref

2.0 (0.7–5.7) Ref

≥20 <25 ≥25 <50 ≥50 <4

70/42 30/25 48/29 39/39 39/15 17/9

2.5 (0.9–6.6) Ref 1.4 (0.7–2.8) Ref 2.6 (1.2–5.5) Ref

3.1 (1.1–8.6) Ref 1.6 (0.8–3.4) Ref 2.8 (1.3–6.1) Ref

127/42 61/25 79/61 72/39 68/15 32/9

2.8 (1.2–6.5) Ref 1.1 (0.6–2.1) Ref 2.5 (1.2–4.9) Ref

2.4 (0.9–5.9) Ref 1.2 (0.6–2.2) Ref 2.7 (1.4–5.5) Ref

≥4 <2 ≥2 <20 + <50

53/35 3/8 75/46 4/9

0.8 (0.3–2.0) Ref 4.3 (1.1–17) Ref

0.8 (0.3–2.0) Ref 6.4 (1.5–26) Ref

96/35 11/8 129/46 8/9

0.8 (0.3–1.8) Ref 2.0 (0.8–5.4) Ref

0.8 (0.3–1.9) Ref 2.6 (0.9–7.2) Ref

<20 + ≥50 or ≥20 + <50 ≥20 + ≥50

39/33 35/12

2.6 (0.8–9.4) 6.6 (1.7–25)

3.0 (0.8–11) 8.4 (2.1–34)

69/33 63/12

2.4 (0.8–6.6) 5.9 (1.9–18)

2.1 (0.7–6.2) 5.6 (1.8–18)

Ref

Ref

Ref

Ref

WHO (1999) Total count (10 per ejaculate)

Adjusteda

Cut-Off value

≥20

6

Had ever become fathers

b

WHO (2010)c Total count (106 per ejaculate) Concentration (106/mL) Motility A+B (%) Morphology (normal forms %)

<39

6/7

9/7

≥39 <15 ≥15 <32 ≥32 <4

72/47 6/7 72/47 4/8 74/46 17/9

1.8 (0.6–5.6) Ref 1.8 (0.6–5.6) Ref 3.2 (0.9–11) Ref

2.1 (0.6–6.8) Ref 2.0 (0.6–6.8) Ref 4.2 (1.1–15) Ref

131/47 10/7 130/47 7/8 133/46 32/9

2.2 (0.8–6.1) Ref 1.9 (0.7–5.4) Ref 3.3 (1.1–9.6) Ref

1.9 (0.7–5.6) Ref 1.5 (0.5–4.3) Ref 3.4 (1.1–10) Ref

≥4

53/35

0.8 (0.3–2.0)

0.8 (0.3–2.0)

96/35

0.8 (0.3–1.8)

0.8 (0.3–1.9)

Bold numbers indicate significant values (p < 0.05). n, numbers of men with and without offsprings; OR, odds ratio; ref, reference; CI, confidence intervals. A, rapid progressive motility; B, slow/sluggish progressive motility. aAdjusted for age and abstinence time. bWHO cut-off reference values from 1999. cWHO cut-off reference values from 2010.

most likely due procedural differences (Tomlinson et al., 2013). Several classification systems for assessment of sperm morphology have been introduced the last decades, thus, making it difficult to compare various studies, and the association between normal sperm morphology evaluated by strict criteria and fertility remains controversial. Slama et al. found an association between the proportion of morphologically normal sperm and TTP both with strict and less strict criteria (Slama et al., 2002), and Bonde et al. found a relation between the proportion of sperm with normal morphology according to WHO 1992 (Special Programme of Research Development and Research Training in Human Reproduction World Health Organization, 1992) criteria and likelihood of pregnancy (Bonde et al., 1998). Our results suggest a better chance for becoming a father when DFI is <20%, although only significant in the group of men who ever had become fathers. This is in agreement with earlier studies; the chance for conception decreases when DFI, as determined by the sperm chromatin structure assay (SCSA), exceeds 20% (Bungum et al., 2011). © 2018 American Society of Andrology and European Academy of Andrology

The strength of the study is the long-term follow-up after semen analysis. A strength is also that the men were recruited from the general population. However, the recruitment may result in a selection toward subfertile males, especially among the oldest men, who might have interest in joining a study concerning fertility. Furthermore, the study group is small, thus, replication of the findings in a larger population is necessary to draw firm conclusions. Another weakness of our study is the lack of data on female fertility as well as the broad age range at recruitment (19–40 years), representing a heterogeneity regarding family planning. Therefore, we also included the group ‘ever become father’. Furthermore, according to the registry data, five of the 195 men had become fathers by in vitro fertilization. Moreover, we performed many comparisons and can therefore not exclude some by chance findings. In conclusion, despite the relatively small sample size in the present study, our results indicate that sperm motility and sperm concentration, both as a single parameter and in combination, as well as DFI can be useful when predicting the chance of ever Andrology, 1–6

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becoming father during a longer time period. Our results also indicate that semen parameters may have a diagnostic value not only in a short time frame and thus may be clinically useful when predicting future fertility potential. Before clinically implementation, larger studies are needed to confirm our findings.

CONFLICT OF INTEREST The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FUNDING This research has been given grants from the Swedish Research Council (Grant 2014_3185), Skane University Hospital Foundation, and Swedish governmental funding for clinical research.

DISCLOSURES None of the authors has any financial or nonfinancial relationship to disclose.

AUTHORS’ CONTRIBUTIONS GM, AG, and TBH are responsible for study conception, design, and collection of data. All authors performed statistical analyses, interpreted data, and approved of the final version to be published.

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