Infertile men with semen parameters above WHO reference limits at first assessment may deserve a second semen analysis: Challenging the guidelines in the real-life scenario

Luca Boeri; Edoardo Pozzi; Paolo Capogrosso; Giuseppe Fallara; Federico Belladelli; Luigi Candela; Nicolò Schifano; Christian Corsini; Walter Cazzaniga; Daniele Cignoli; Eugenio Ventimiglia; Marina Pontillo; Massimo Alfano; Francesco Montorsi; Andrea Salonia

doi:10.1371/journal.pone.0280519

. 2023 Jan 19;18(1):e0280519. doi: 10.1371/journal.pone.0280519

Infertile men with semen parameters above WHO reference limits at first assessment may deserve a second semen analysis: Challenging the guidelines in the real-life scenario

Luca Boeri ¹, Edoardo Pozzi ^2,³, Paolo Capogrosso ⁴, Giuseppe Fallara ^2,³, Federico Belladelli ^2,³, Luigi Candela ^2,³, Nicolò Schifano ^2,³, Christian Corsini ^2,³, Walter Cazzaniga ^2,³, Daniele Cignoli ^2,³, Eugenio Ventimiglia ², Marina Pontillo ⁵, Massimo Alfano ², Francesco Montorsi ^2,³, Andrea Salonia ^2,^3,^*

Editor: Stefan Schlatt⁶

PMCID: PMC9851544 PMID: 36656872

Abstract

Objectives

To investigate which infertile men with semen parameters above WHO reference limits at first semen analysis deserve a second semen test.

Materials and methods

Data from 1358 consecutive infertile men were analysed. Patients underwent two consecutive semen analyses at the same laboratory. Descriptive statistics and logistic regression models tested the association between clinical variables and semen parameters. A new predicting model was identified through logistic regression analysis exploring potential predictors of semen parameters below WHO reference limits after a previously normal one. Diagnostic accuracy of the new model was compared with AUA/ASRM and EAU guidelines. Decision curve analyses (DCA) tested their clinical benefit.

Results

Of 1358, 212 (15.6%) infertile men had semen parameters above WHO reference limits at first analysis. Of 212, 87 (41.0%) had a second semen analysis with results above WHO reference limits. Men with sperm parameters below reference limits at second analysis had higher FSH values, but lower testicular volume (TV) (all p<0.01) compared to men with a second semen analysis above WHO limits. At multivariable logistic regression analysis, lower TV (OR 0.9, p = 0.03), higher FSH (OR 1.2, p<0.01), and lower total sperm count (OR 0.9, p<0.01) were associated with second semen analyses below WHO limits. DCA showed the superior net benefit of using the new model, compared to both AUA/ASRM and EAU guidelines to identify those men with a second semen sample below WHO limits after a previously normal one.

Conclusions

Approximately 60% of infertile men with a first semen analysis above WHO limits have a second analysis with results below limits. The newly identified risk model might be useful to select infertile men with initial semen results above WHO limits who deserve a second semen analysis.

Introduction

Semen analysis is considered the cardinal point throughout the investigation of men presenting for couple’s infertility, along with a comprehensive medical and reproductive history, physical examination and hormonal investigation [1, 2]. Nonetheless, the individual semen parameter provides only partial information of the actual fertility potential. In fact, even having strictly normal sperm parameters according to World Health Organization (WHO) reference values [3, 4] per se does not reliably account for fertility [5]. In this context, a recent case-control study showed that sperm parameters above WHO limits were found in approximately 12% of infertile men and in only 41% of age-matched fertile controls [5]. Moreover, between 20% and 30% of men are infertile despite having semen analysis above limits, normal medical history and normal physical examination, thus configuring the condition of unexplained male infertility [1, 6]. As a whole, semen parameters are highly variable biological measures and may vary substantially from ejaculate to ejaculate. Therefore, the American Urological Association/American Society for Reproductive Medicine (AUA/ASRM) guidelines and WHO references criteria recommend analysing at least two consecutive samples in every infertile man [2, 7]; conversely, the European Association of Urology (EAU) Guidelines on Sexual and Reproductive health suggests that a single test is sufficient in case of normal semen analysis according to WHO reference criteria, whereas a second assessment should be taken into consideration in case of sperm abnormalities [1, 8]. According to physiological spermatogenesis, a second analysis is considered reliable after a 3-month time frame [9]; thereof, if not adequately collected, a second semen analysis could potentially lead to unnecessary costs and delays over the treatment work-up of infertility, and previous studies have investigated the need to collect more than one semen sample in clinical practice [10–13]. As a whole, a number of studies did not find significant differences among semen parameters obtained on consecutive analyses both in fertile and infertile men [10, 11, 13]. Conversely, other reports showed greater intraindividual variations in infertile men compared to sperm donors [12, 14]. Therefore, the overall clinical benefit of performing one versus two consecutive semen analyses in infertile men is still a matter of debate. This is even more true in infertile men with semen parameters above WHO limits at first semen sample, where current scientific guidelines are discordant in suggesting the need for a confirmatory second test [1, 2]. For instance, in their cohort of 2,566 infertile men, Blickenstorfer et al. showed that in cases with semen sample above WHO limits at first assessment, 27% of the second samples were below limits [10]. Thus, a proper identification of those men with semen samples above WHO limits who would truly benefit from a second investigation in the real-life setting is needed.

Thereof, we aimed to investigate: i) the rate of and the predictors of semen analyses below WHO limits at second investigation after a first one above limits in a homogeneous cohort of non-Finnish, white-European men seeking medical attention for primary couple’s infertility; and ii) to build a predictive model for second semen analysis below WHO limits that could be used to identify infertile men that deserve a confirmatory test over the diagnostic work-up.

Materials and methods

Data from a cohort of 1562 consecutive white-European men assessed at a single academic centre for couple’s infertility (non-interracial infertile couples only) between September 2012 and September 2020 were analysed. According to the WHO criteria, infertility was defined as not conceiving a pregnancy after at least 12 months of unprotected intercourses regardless of whether or not a pregnancy ultimately occurs [15]. Patients were only enrolled if they were ≥18 and ≤55 years old and had either pure male factor infertility (MFI) or mixed factor infertility. MFI was defined after a comprehensive diagnostic evaluation of all the female partners by expert Gynaecologists, which included a detailed medical, reproductive and family history as well as a general and gynaecological physical examination. Furthermore, the ovulatory status, ovarian reserve testing, the structure and patency of the female reproductive tract were requested in all cases.

All participants were homogenously assessed by the same expert academic urologist (A.S.), with a thorough medical history and a complete physical examination. Health-significant comorbidities were scored with the Charlson Comorbidity Index (CCI), coded using the International Classification of Diseases, 9^th revision [16, 17]. Likewise, weight and height were measured, calculating body mass index (BMI) for each participant [18]. Testes volume (TV) was assessed in all cases using Prader’s orchidometer estimation [19]; for the specific purpose of this study, we calculated the mean value between the two sides. Varicocele was also clinically assessed in every patient [20]. Venous blood samples were drawn from each patient between 7 AM and 11 AM after an overnight fast. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (tT), prolactin, thyroid-stimulating hormone (TSH) and sex hormone-binding globulin (SHBG) levels were measured for every individual. Sperm DNA fragmentation (SDF) index, was measured by sperm chromatin structure assay (SCSA) [21]. According to our internal diagnostic protocol, chromosomal analysis and genetic testing were performed in every infertile man (i.e., karyotype analysis and Y-chromosome microdeletions and cystic fibrosis mutations tests) [22]. All patients underwent two consecutive semen analyses at least 3 months apart [8]; semen samples were collected by masturbation after a sexual abstinence of 2–7 days and analysed within 60 minutes of ejaculation, in accordance with the WHO criteria.

The improved Neubauer hemocytometer chamber (100-μm-deep; Brand™ Blaubrand™ Neubauer Improved Counting Chambers, Fisher Scientific, Loughborough, UK) was used for the calculation of sperm concentration and total sperm count in the ejaculate. Sperm morphology was assessed through the following steps: preparation of a smear of semen on a slide; fixing and staining the slide (Testsimplets® Prestained Slides, Waldeck GmbH & Co. KG, Münster, Germany); examination with brightfield optics at ×1000 magnification (Nikon Eclipse E 200, Nikon Instruments Europe B.V., Rome, Italy) with oil immersion; and assessment of approximately 200 spermatozoa per replicate for the percentage of normal or abnormal forms. Sperm motility was assessed by mixing twice the sample, using a wet preparation of 20 microM deep for each replicate, by examining the slide with phase-contrast optics at ×200 magnification and by assessing approximately 200 spermatozoa per replicate for the percentage of different motile categories.

In the laboratory for semen analysis a continuous quality assurance programme has been developed and maintained for several years. It relies on a quality manual containing standard operating procedures (SOP) and a detailed set of instructions for the different processes and methods used in the laboratory. Internal quality control (IQC) is implemented with the inclusion of IQC materials in the laboratory’s regular workload and the results for these materials are monitored using quality control charts. External quality control (EQA) is regularly performed through peer comparison and proficiency testing programmes (Italian EQA program). Results are sent to a central facility that assesses the performance of the laboratory. Continuous training and education of the laboratory personnel is also undertaken.

For the specific purposes of this study, we considered semen volume, sperm concentration, progressive sperm motility and morphology. Total motile sperm count was calculated and further categorized according to Hamilton et al [23]. According to WHO reference criteria, oligozoospermia was defined as <15 million sperm per mL; asthenozoospermia as <32% progressive motility, and teratozoospermia as <4% of normal forms. Likewise, oligo-astheno-teratozoospermia (OAT) was defined when all three anomalies occur simultaneously and azoospermia was considered as the complete absence of spermatozoa in semen after centrifugation [8]. The same laboratory was used for analyses of all parameters. We excluded 204 (13.1%) men because they missed one or more of the entry criteria [i.e., abnormal genetic tests (any type) (n = 32; 2.0%); symptoms suggestive for genitourinary infections (n = 21; 1.3%) or positive semen or urine cultures (n = 146; 9.3%); a history of assisted reproductive techniques (ART) (any type) during the preceding year (n = 5; 0.3%)]. A convenient sample of 1358 infertile men was considered for the final statistical analyses. Data collection followed the principles outlined in the Declaration of Helsinki. All men signed an informed consent agreeing to share their own anonymous information for future studies. The study was approved by our Hospital Ethical Committee (Prot. 2014—Pazienti Ambulatoriali).

Statistical methods

Distribution of data was tested with the Shapiro–Wilk test. Data are presented as medians (interquartile range; IQR) or frequencies (proportions). The analyses consisted of several statistical steps. First, we considered only infertile men with a first semen sample with results above WHO references criteria (n = 212, 15.6%). Second, in this cohort, the Mann–Whitney test and the Chi-square test were used to compare baseline clinical and demographics characteristics, hormonal values, and semen parameters between those individuals with a second semen analysis above (Group 1) vs. below (Group 2) WHO limits. Third, univariable and multivariable logistic regression analyses tested the association between clinical, hormonal, and seminal characteristics (i.e., age, CCI, FSH, TV and sperm concentration on the first sample) with second semen analysis below WHO limits after a first test above limits. Receiver Operating Characteristic (ROC) curves were generated to find TV, FSH and sperm concentration threshold values (defined as Youden J Index) to predict a second semen analysis below WHO limits. Fourth, these factors were used to create a new data-driven predictive model for sperm parameters below limits at a second test. Considering 1-point for each positive variable of the model, we calculated the positive predictive value (PPV) for second semen analysis below WHO limits as a function of the number of positive variables. Fifth, decision curve analysis (DCA) was used to assess the clinical benefit of using the newly identified model in respect to EAU [1] and AUA/ASRM guidelines [2]. Lastly, the diagnostic accuracy of the new model was compared with the AUA/ASRM guidelines, using a DeLong test. Statistical analyses were performed using SPSS v.26 (IBM Corp., Armonk, NY, USA) and R (2019), a language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria). All tests were two sided and statistical significance level was determined at p < 0.05.

Results

Of 1358, 212 (15.6%) men had semen parameters above WHO limits at first assessment. Of those, 87 (41.0%) had a second semen analysis above WHO limits, while 80 (37.7%), 35 (16.5%) and 10 (4.7%) men showed 1, 2 and 3 sperm characteristics below limits at second test, respectively. Of note, the ejaculatory abstinence time was similar between the two semen analyses.

Table 1 details descriptive statistics of the entire cohort of men. Moreover, Table 1 also details baseline descriptive statistics of the sub cohort of men (n = 212) with two consecutive semen analyses above limits (Group 1) compared to patients with a first above and a second below WHO limits (Group 2). Of this sub cohort, men in Group 2 had higher CCI and FSH values, but lower TV (all p≤0.04) as compared with than those in Group 1. Despite within normal ranges in both groups, men in Group 1 had greater total sperm number and TMSC even at first assessment compared to patients in Group 2 (p<0.001). For each group, the individual abstinence time between the first and second test was similar [mean (standard deviation) difference: Group1: 0.32 (0.2) days; Group 2: 0.33 (0.2) days]; similarly, abstinence time was not different among groups.

Table 1. Descriptive statistics of the whole cohort of infertile men and according to second semen analysis characteristics.

	Overall	Group 1	Group 2	p-value^*
No. of individuals	1358	87 (41.0%)	125 (59.0%)
Age (years)				0.6
Median (IQR)	37.0 (34–40)	37.0 (34–41)	37.0 (34–41)
Range	18–55	20–54	25–54
Duration of infertility (months)				0.4
Median (IQR)	24 (12–36)	24.0 (12–36)	24.0 (15–36)
Range	12–228	12–135	24–108
BMI (kg/m²)				0.9
Median (IQR)	24.9 (23.2–26.8)	24.6 (23.4–25.9)	24.4 (23.0–26.7)
Range	18.5–41.2	19.8–39.2	18.6–36.8
CCI (score)				0.04
Median (IQR)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)
Mean (SD)	0.1 (0.2)	0.01 (0.1)	0.2 (0.1)
Range	0–8	0–1	0–4
CCI ≥ 1 [No. (%)]	84 (6.2)	1 (1.1)	9 (7.2)	0.04
Current smoking status [No. (%)]	387 (28.5)	20 (23.0)	33 (26.4)	0.6
Mean TV (Prader’s estimation)				<0.001
Median (IQR)	18.0 (14–20)	23.0 (18–25)	18.0 (13–23)
Range	4–25	8–25	8–25
Varicocele [No. (%)]	821 (60.5)	49 (56.3)	69 (55.2)	0.8
tT (ng/mL)				0.9
Median (IQR)	4.7 (3.6–5.9)	4.8 (3.7–6.4)	4.8 (3.7–5.9)
Range	0.9–23.5	1.8–9.9	2.0–13.4
FSH (mUI/mL)				<0.001
Median (IQR)	4.6 (3.0–7.6)	3.3 (2.1–4.6)	4.7 (2.9–6.8)
Range	0.8–45.8	0.8–15.3	1.2–15.0
LH (mUI/mL)				0.3
Median (IQR)	3.8 (2.7–5.2)	3.0 (2.3–4.5)	3.4 (2.5–4.4)
Range	0.9–34.2	0.8–8.7	1.1–12.4
Prolactin (ng/mL)				0.9
Median (IQR)	8.2 (6.2–11.5)	8.1 (6.0–12.4)	8.2 (6.3–11.1)
Range	1.9–34.3	3.4–22.4	2.6–32.3
SHBG (nmol/L)				0.1
Median (IQR)	32.0 (24–41)	35.8 (24–43)	29.0 (22–40)
Range	9.0–85.0	13.0–57.0	9.0–85.0
TSH (mUI/L)				0.5
Median (IQR)	1.6 (1.1–2.3)	1.6 (1.1–2.7)	1.6 (1.2–2.1)
Range	0.5–6.6	0.5–4.2	0.6–5.4
Sexual abstinence (days)				0.7
Median (IQR)	3.0 (2.0–5.0)	3.0 (2.0–5.0)	3.0 (2.0–5.0)
Range	2.0–7.0	2.0–7.0	2.0–7.0
Semen volume (mL)				0.8
Median (IQR)	3.0 (2.0–4.0)	3.0 (2.0–4.0)	3.0 (2.0–4.0)
Range	1.0–11–0	1.0–8.0	1.0–8.0
Sperm concentration (x10⁶/mL)				<0.001
Median (IQR)	18.1 (5.0–42.1)	62.0 (35–94)	34.0 (24–58)
Range	0.1–455.3	16.0–455.3	15.0–198.0
Total sperm number (x10⁶ per ejaculate)				<0.001
Median (IQR)	52.0 (14–142)	173.6 (118–282)	120.0 (80–180)
Range	0.1–992.0	16.5–896.0	18.0–992.0
Progressive sperm motility (%)				0.2
Median (IQR)	25.0 (10–39)	46.5 (40–56)	44.0 (38–53)
Range	0.0–96.0	32.0–76.0	32.0–85.0
Normal sperm morphology (%)				0.9
Median (IQR)	3.0 (1–12)	13.0 (6–36)	12.0 (8–30)
Range	0.0–100.0	4.0–94.0	4–90.0
TMSC (x10⁶)				<0.001
Median (IQR)	10.4 (1.2–40.1)	82.2 (52.4–126.8)	52.8 (30.5–82.7)
Range	0.0–615.1	0.0–492.8	7.2–615.1
TMSC groups [No. (%)]				0.2
< 5 x106	533 (39.2)	--	--
5–20 x106	287 (21.1)	2 (2.3)	7 (5.6)
> 20 x106	538 (39.6)	85 (97.7)	118 (94.4)
SDF (%)				0.8
Median (IQR)	32.8 (19.5–48.6)	26.4 (10–39)	27.5 (14–54)
Range	0.4–97.7	7.0–72.0	5.0–93.0

Open in a new tab

Keys: Group 1: infertile men with two consecutive semen samples with results above WHO limits; Group 2: infertile men with a first semen analysis above WHO limits and a second test below WHO limits.

BMI = body mass index; CCI = Charlson Comorbidity Index; TV = testicular volume; tT = total Testosterone; FSH = follicle-stimulating hormone, LH = luteinizing hormone SHBG = Sex hormone binding globulin; TSH = Thyroid-stimulating hormone; TMSC = Total Motile Sperm Count SDF = Sperm DNA fragmentation Index.

* p value according to the Mann-Whitney test and Chi Square test, as indicated.

Table 2 reports logistic regression models predicting semen analysis below WHO limits after a first test above limits. At multivariable logistic regression analysis, lower TV (OR 0.9, p = 0.03), higher FSH values (OR 1.2, p<0.01) and lower total sperm number at first semen analysis (OR 0.9, p<0.01) were associated with second semen analyses below WHO limits, after accounting for age and CCI.

Table 2. Logistic regression models predicting semen analysis below WHO limits after a first test above limits (No. = 212).

	UVA model			MVA model
	OR	p-value	95% CI	OR	p-value	95% CI
Age	0.97	0.4	0.92–1.03	0.97	0.5	0.90–1.07
CCI	4.25	0.1	0.75–15.82	2.82	0.2	0.51–12.29
TV	0.88	<0.01	0.84–0.94	0.93	0.03	0.86–0.99
FSH	1.31	<0.01	1.13–1.51	1.22	<0.01	1.10–1.47
Total sperm number^*	0.97	<0.001	0.95–0.99	0.97	<0.01	0.94–0.99

Open in a new tab

Keys: UVA = Univariate model; MVA = Multivariate model, CCI = Charlson Comorbidity Index

TV = testicular volume; FSH = follicle-stimulating hormone, LH = luteinizing hormone

*at first semen analysis

ROC curves showed that TV <15 ml, FSH values >6 mUI/ml and total sperm number < 120 x10⁶ had good predictive ability for sperm parameters below WHO limits at second analysis (all AUC >0.7). Thereafter, a risk score for second semen sample results below limits using the three previous variables was developed. Considering 1-point for each of the previous positive variable of the model, PPV of a second semen analysis below WHO limits increased from 38.5% to 74.4%, 77.1% and 100% among patients with risk scores of 0, 1, 2 and 3, respectively (p<0.001). The DeLong’s test confirmed that the new model (AUC 0.73, 95% CI: 0.56–0.71) performed better than the AUA guidelines (AUC 0.64, 95%CI: 0.69–0.77) (p<0.001).

Lastly, DCA (Fig 1) displays the superior net benefit of using the new model in terms of predicting a second semen analysis below WHI limits, compared to the EAU and AUA/ASRM guidelines recommendations.

Discussion

The lack of consensus among international guidelines on the issue prompted us to analyse findings from a relatively large homogeneous cohort of primary infertile men with the specific aim of identifying patients at higher risk of having a semen analysis below WHO limits following a first investigation above limits. Of clinical importance, we found that approximately 60% of infertile men with a first semen analysis above limits depicted a second test below WHO limits [8]. Patients with lower TV, higher FSH values and lower total sperm number at the first semen analysis emerged to be at greater risk of subsequent semen samples below WHO limits, thus deserving a confirmatory analysis. A new risk score based on these associated factors was then compared to the AUA/ASRM guidelines recommendation to test all infertile men with two consecutive semen samples. We showed the net benefit of the new risk score in respect to the AUA/ASRM guidelines in identifying infertile men with a second semen sample with results below limits after a first one above limits. Therefore, the proposed risk score could be used in clinical practice for the management of infertile men with a first semen analysis above WHO limits. It is well known that semen analysis per se is a macroscopic evaluation of sperm characteristics and does not necessarily and reliably distinguish between fertile and infertile men [5]. Moreover, semen parameters are subject to a certain degree of intra-individual variability over time [14]. Consequently, at least two consecutive semen analyses are usually requested for the initial management of infertile couples in clinical practice. While this approach is certainly endorsed in case of sperm parameters with results below WHO limits at baseline investigation, this is not the case for infertile men presenting with a first semen analysis above limits. Indeed, current EAU and AUA/ASRM Guidelines report conflicting recommendations regarding the need for a confirmatory semen analysis in infertile men with a first test above WHO limits [1, 2]. This criticism is of primary clinical importance because unnecessary additional samples from men with a first semen analysis above WHO limits could lead to increasing costs and frustration for the couple; on the contrary, missing a second semen samples with results below limits could delay further diagnostics tests and a tailored treatment. Likewise, exclusively relying on a single semen analysis during the diagnostic work-up of infertile men may also increase the risk of referring couples to ART even without receiving an adequate baseline andrological evaluation [24]. That said, it is becoming worldwide endorsed the idea that reference ranges and reference limits for semen parameters are useful for research purposes, to guide diagnostic testing and treatment choice, but decision limits based on clinical and statistical considerations should be used in clinical practice for the management of infertile men [25].

Previous studies have investigated the rate of concordance between consecutive semen analyses and the need for a second test in healthy sperm donors and infertile men. A population based-study with 998 volunteers aged 20–60 years, analysed data from 332 men who provided single semen samples and 666 men who provided two samples [11]. Authors found that semen parameters were similar between the one-sample group and the two-sample group; moreover, the difference in semen parameters between first and second samples were relatively small. Despite some intra-individual variations of sperm quality, one ejaculate was deemed as a sufficient indicator of semen quality in epidemiological studies [11]. Leushuis et al. [12], for instance, described the within-individual variability of semen analyses in male partners of subfertile couples in a large retrospective cohort. They showed a high within-subject variability for sperm motility and morphology, thus questioning the classification of man based on a single semen measurement. The Environment and Reproductive Health (EARTH) Study investigated the long-term variability in semen parameters in a cohort of 329 infertile men who provided 768 semen samples [13]. Authors showed no significant differences between mean values of first samples and the means of the remaining samples. However, when semen parameters were dichotomized, a second sample was necessary to estimate the prevalence of pathological results according to WHO reference criteria [13]. Of note, 51% of men with sperm parameters above WHO limits at first assessment had at least one semen parameter below the WHO reference limits in their second sample [13]. More recently, Blickenstorfer et al. [10] analysed data from 2,566 infertile men who underwent at least two consecutive semen analyses and showed that 51% of the second analyses confirmed the initial findings; conversely, 27% of men who presented with normozoospermia in the first semen sample had a second sample with results below limits. Finally, they reported a limited discriminating capacity of each semen parameter to distinguish between men with a second semen analysis above vs. below WHO limits. Overall, our results confirm the latter mentioned findings, since we showed that 60% of infertile men with a first semen analysis above WHO limits had a second test below limits. In addition, we found that lower TV, lower total sperm number and higher FSH values were significantly associated with the risk of having a semen test below limits. The clinical implication of our study is several-fold. First, this is the first study to establish a novel risk model for the selection of infertile men presenting with semen analysis above WHO limits at first assessment that would deserve a confirmatory test because at higher risk of a second semen analysis below limits. Of clinical importance, the novel score is user-friendly since it is based on clinical, hormonal and sperm parameters that are essential components of the baseline diagnostic work up of every infertile man. As compared with previous data, current findings are even more clinically relevant since we had investigated a relatively large cohort of same ethnicity, non-Finnish white-European primary infertile men with an identical thorough clinical, hormonal and semen evaluation. Conversely, most of the previous studies had investigated the association and predicted ability of consecutive semen parameters while ignoring clinical and laboratory features of their cohorts [10, 13], thus potentially limiting the clinical validity of their findings in the real-life setting. Likewise, we showed the net benefit of applying a personalised approach to identify those men with a second semen test below WHO limits, based on the specific newly developed risk score as compared with the AUA/ASRM Guidelines model of testing all. The implementation of our risk score in clinical practice might spare unnecessary test and avoid delaying in treatment for a specific cohort of infertile men with semen analysis above WHO limits. As a whole, our results strongly outline the compulsory need that every infertile man gets a comprehensive evaluation by specialists in male reproduction [1, 2, 24], including those with a first semen analysis above WHO limits. In fact, on the one hand, approximately 60% of them will show semen parameters below WHO limits at a second test, and on the other, semen analysis per se cannot be relied on to rule out a male factor cause of infertility. Our study is not devoid of limitations. First, even though we analysed a relatively large, homogeneous, same-ethnicity cohort of infertile men, this was a single centre-based study, raising the possibility of selection biases; thereof, larger studies across different centres and cohorts are needed to externally validate our findings. Second, despite all semen analyses have been performed at the same laboratory according to WHO reference criteria [8], it is not possible to completely exclude a partial inter-observers variability, which surely cannot be considered sufficient to explain such an impressive 60% of discrepancy between consecutive semen samples in the same patient.

Conclusions

In this cross-sectional study we found that approximately 60% of infertile men with a first semen analysis above WHO limits depicted a second test with results below limits. Lower testicular volume, higher FSH values and lower total sperm number at first analysis were independently associated with a second semen analysis below limits. Thereof, we proposed a novel and highly performing risk score to identify infertile men at greater risk of having a second semen sample with results below WHO limits after a first one above limits. This score could be used in clinical practice to better tailor the management of men presenting for couple’s infertility despite having initial semen samples above WHO limits, while avoiding unnecessary examination and delays in treatment.

Supporting information

S1 Dataset

(XLSX)

Click here for additional data file.^{(357KB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Minhas S, Bettocchi C, Boeri L, Capogrosso P, Carvalho J, Cilesiz NC, et al. European Association of Urology Guidelines on Male Sexual and Reproductive Health: 2021 Update on Male Infertility. Eur Urol 2021;S0302–2838(21)01982-5. doi: 10.1016/j.eururo.2021.08.014 [DOI] [PubMed] [Google Scholar]
2.Schlegel PN, Sigman M, Collura B, De Jonge CJ, Eisenberg ML, Lamb DJ, et al. Diagnosis and treatment of infertility in men: AUA/ASRM guideline part I. Fertil Steril 2021;115(1):54–61. doi: 10.1016/j.fertnstert.2020.11.015 [DOI] [PubMed] [Google Scholar]
3.World Health Organization. Infertility definitions and terminology. Accessible at: https://www.who.int/reproductivehealth/topics/infertility/definitions/en/. Accessed March 28, 2020. [Google Scholar]
4.Boeri L, Fallara G, Pozzi E, Belladelli F, Corsini C, Raffo M, et al. The impact of different WHO reference criteria for semen analysis in clinical practice: Who will benefit from the new 2021 thresholds for normal semen parameters? Andrology 2022. doi: 10.1111/andr.13213 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Boeri L, Belladelli F, Capogrosso P, Cazzaniga W, Candela L, Pozzi E, et al. Normal sperm parameters per se do not reliably account for fertility: A case-control study in the real-life setting. Andrologia 2021;53(1):e13861. doi: 10.1111/and.13861 [DOI] [PubMed] [Google Scholar]
6.Hamada A, Esteves SC, Nizza M, Agarwal A. Unexplained male infertility: diagnosis and management. Int Braz J Urol 2012;38(5):576–94. doi: 10.1590/s1677-55382012000500002 [DOI] [PubMed] [Google Scholar]
7.WHO, laboratory manual for the examination and processing of human semen. 2010. [PubMed] [Google Scholar]
8.Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HWG, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update 2010;16(3):231–45. doi: 10.1093/humupd/dmp048 [DOI] [PubMed] [Google Scholar]
9.Heller CG, Clermont Y. Spermatogenesis in man: an estimate of its duration. Science 1963;140(3563):184–6. doi: 10.1126/science.140.3563.184 [DOI] [PubMed] [Google Scholar]
10.Blickenstorfer K, Voelkle M, Xie M, Fröhlich A, Imthurn B, Leeners B. Are WHO Recommendations to Perform 2 Consecutive Semen Analyses for Reliable Diagnosis of Male Infertility Still Valid? J Urol 2019;201(4):783–91. doi: 10.1016/j.juro.2018.11.001 [DOI] [PubMed] [Google Scholar]
11.Zhu Q-X, Gao E-S, Pathak N, Wu J-Q, Zhou W-J. Single or double semen samples: the dilemma in epidemiological studies on semen quality. Hum Reprod 2016;31(3):511–7. doi: 10.1093/humrep/dev326 [DOI] [PubMed] [Google Scholar]
12.Leushuis E, van der Steeg JW, Steures P, Repping S, Bossuyt PMM, Blankenstein MA, et al. Reproducibility and reliability of repeated semen analyses in male partners of subfertile couples. Fertil Steril 2010;94(7):2631–5. doi: 10.1016/j.fertnstert.2010.03.021 [DOI] [PubMed] [Google Scholar]
13.Chiu Y-H, Edifor R, Rosner BA, Nassan FL, Gaskins AJ, Mínguez-Alarcón L, et al. What Does a Single Semen Sample Tell You? Implications for Male Factor Infertility Research. Am J Epidemiol 2017;186(8):918–26. doi: 10.1093/aje/kwx169 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Keel BA. Within- and between-subject variation in semen parameters in infertile men and normal semen donors. Fertil Steril 2006;85(1):128–34. doi: 10.1016/j.fertnstert.2005.06.048 [DOI] [PubMed] [Google Scholar]
15.World Health Organization web chapter on couple’s infertility. 2017. http://www.who.int/reproductivehealth/topics/infertility/definitions. Accessed May 2, 2019.
16.Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 1987;40(5):373–83. doi: 10.1016/0021-9681(87)90171-8 [DOI] [PubMed] [Google Scholar]
17.Salonia A, Matloob R, Gallina A, Abdollah F, Saccà A, Briganti A, et al. Are infertile men less healthy than fertile men? Results of a prospective case-control survey. Eur Urol 2009;56(6):1025–31. doi: 10.1016/j.eururo.2009.03.001 [DOI] [PubMed] [Google Scholar]
18.Ventimiglia E, Capogrosso P, Colicchia M, Boeri L, Serino A, Castagna G, et al. Metabolic syndrome in white European men presenting for primary couple’s infertility: investigation of the clinical and reproductive burden. Andrology 2016;4(5):944–51. doi: 10.1111/andr.12232 [DOI] [PubMed] [Google Scholar]
19.Boeri L, Capogrosso P, Ventimiglia E, Cazzaniga W, Pozzi E, Belladelli F, et al. Testicular volume in infertile versus fertile white-European men: a case-control investigation in the real-life setting. Asian J Androl 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Boeri L, Capogrosso P, Ortensi I, Miacola C, Cai T, Verze P, et al. Diagnostic and therapeutic workup of male infertility: results from a Delphi consensus panel. Int J Impot Res 2021. doi: 10.1038/s41443-021-00511-x [DOI] [PubMed] [Google Scholar]
21.Evenson DP. Sperm chromatin structure assay (SCSA®). Methods Mol Biol 2013;927:147–64. [DOI] [PubMed] [Google Scholar]
22.Ventimiglia E, Capogrosso P, Boeri L, Pederzoli F, Cazzaniga W, Scano R, et al. When to Perform Karyotype Analysis in Infertile Men? Validation of the European Association of Urology Guidelines with the Proposal of a New Predictive Model. Eur Urol 2016;70(6):920–3. [DOI] [PubMed] [Google Scholar]
23.Hamilton J a. M, Cissen M, Brandes M, Smeenk JMJ, de Bruin JP, Kremer J a. M, et al. Total motile sperm count: a better indicator for the severity of male factor infertility than the WHO sperm classification system. Hum Reprod 2015;30(5):1110–21. doi: 10.1093/humrep/dev058 [DOI] [PubMed] [Google Scholar]
24.Pozzi E, Boeri L, Candela L, Capogrosso P, Cazzaniga W, Fallara G, et al. Infertile couples still undergo assisted reproductive treatments without initial andrological evaluation in the real-life setting: A failure to adhere to guidelines? Andrology 2021. doi: 10.1111/andr.13071 [DOI] [PubMed] [Google Scholar]
25.WHO laboratory manual for the examination and processing of human semen. Sixth edition. Available at: https://www.who.int/publications/i/item/9789240030787.

PLoS One. doi: 10.1371/journal.pone.0280519.r001

Decision Letter 0

Stefan Schlatt

17 Jun 2022

PONE-D-22-12621Infertile men with normal semen parameters at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenarioPLOS ONE

Dear Dr. Salonia,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 24 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Stefan Schlatt

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

3. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

Additional Editor Comments:

This study aims to describe differences between a primary and secondary semen analysis for evlautaion of infertility. The authors assess a second sample from subjects who had shown normal values in accordance to current guidelines. Depending on the likelihood of an abnormal second semen

analyses the authors propose a new predictive model to better identify subjects with high risk of pathological changes despite a normal first ejaculate score. The authors describe limitations of their work and the paper can add to discussions on improvement of guidelines and recommendations. The referee made several important comments which affect the validity of the findings. The authors need to address these comments and should revise thier manuscript accordingly.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: There is no comprehensive description of how the basic semen analysis was performed. Data on variability and compliance with e.g. demand for comparison of replicate assessments, use of equipment able to provide realiable results, interntal and external quality control is essential for this study.

Furthermore, although it is true that there is definition of infertility based on TTP=< 12 months, but this has been challenged not the least based on older comprehensive studies (Tietze 1968 e.g.) and recent studied showing huge differences between short time to pregnancy and WHO "reference limits" - and those "reference limits" have also been challenged in the most recent WHO manual (2022).

Authors claim semen assessments were done "within two hours". This is clearly NOT in compliance with WHO 2010: page 21, 2.5 Sperm Motility second paragraph: "Sperm motility within semen should be assessed as soon as possible after liquefaction of the sample, preferably at 30 minutes, but in any case within 1 hour, following ejaculation, to limit the deleterious effects of dehydration, pH or changes in temperature on motily."

This calls in question to what extent reliable laboratory methods have been used. Lack of compliance with WHO recommendations can indicate that the assessments are influenced by significant technical variability - and that the reported change from "normal" to "abnormal" to a significant degree depend on technical /laboratory/ errors.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Jan 19;18(1):e0280519. doi: 10.1371/journal.pone.0280519.r002

Author response to Decision Letter 0

29 Jul 2022

Dr. Emily Chenette, PhD

Editor-in-Chief, PLOS ONE

Dr. Stefan Schlatt

Academic Editor, PLOS ONE

Milan, July 14th, 2022

Dear Dr Chenette,

dear Dr. Schlatt,

please find enclosed the revised version of the manuscript titled “Infertile men with normal semen parameters at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenario” (PONE-D-22-12621- Authors: Luca Boeri, et al.) to be considered for publication in PLOS ONE.

We are very grateful to the Reviewers for their insightful comments to our paper.

List of the changes made in the manuscript:

REVIEWER #1

COMMENT#1.

There is no comprehensive description of how the basic semen analysis was performed. Data on variability and compliance with e.g. demand for comparison of replicate assessments, use of equipment able to provide reliable results, internal and external quality control is essential for this study.

A1. We thank the Reviewer#1 for this insightful comment. We have included a detailed description of basic semen analysis and quality control methods in the Methods section of the manuscript.

COMMENT#2.

A2. We thank the Reviewer#1 for this comment. It is well known that infertility is not related to the mere sperm quality, but we relied on the WHO definition of infertility, which is the one used by most of the international scientific societies (e.g., European Association of Urology Guidelines; American Society for Reproductive Medicine).

COMMENT#3.

A3. We thank the Reviewer#1 for this critical comment. During the revision of our manuscript, we have double checked once more the whole procedure with our laboratory biologists and indeed we may confirm that all samples have been and are rigorously analysed within 60 min. from collection. The text has been revised accordingly.

COMMENT#4.

A4. As comprehensively detailed before, our laboratory analyzed all semen samples strictly according to WHO indications. We have also included internal and external quality control throughout the Methods section of the manuscript.

We hope that the paper is now suitable to be considered for publication in the Original Articles section of PLOS ONE.

Sincerely yours,

Andrea Salonia, on behalf of all the authors

Andrea Salonia, MD, PhD, FECSM

University Vita-Salute San Raffaele

Division of Experimental Oncology/Unit of Urology, URI-Urological Research Institute

IRCCS Ospedale San Raffaele

Email: salonia.andrea@hsr.it

Attachment

Submitted filename: Response to Reviewers - 14 07 2022.doc

Click here for additional data file.^{(46KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0280519.r003

Decision Letter 1

Stefan Schlatt

30 Aug 2022

PONE-D-22-12621R1Infertile men with normal semen parameters at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenarioPLOS ONE

Dear Dr. Salonia,

Please submit your revised manuscript by Oct 14 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Stefan Schlatt

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The reviewer requests raises a few concerns primarily in regard to the interpretation of the data. The authors may address these concerns in a revised version of the manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: I thank the authors for clarifying a number of matters.

Still I think there are unclear items to work with.

1. Authors only work with sperm concentration, but declares sperm numbers were calculated. It was clear already 2010 that the WHO manual recommends sperm number rather than concentration that depends on both sperm number and dilution by fluid from other sources.

2. Variation in ejaculatory abstinence time can be an important source for variation. There is not even a discussion about this and no data available, only in M&M that abstinence time was 2-7 days. Could there be individual variability due to variation in abstinence time (or even ejaculatory frequency) before the two semen analyses?

3. Have the authors considered that limits above the lower 5th percentile "limit" byt the WHO 2010 and 2021 could be better to distinguish between men with semen abnormalities and men without? Looking at e.g. Keihani et al 2021 indicating that men in couples with short time to initiation of pregnancy have much higher semen analysis results compared to men in couples with 5-12 months.

Thus, I do agree with the authors that the so called WHO limits (which should not be mistaken for limits between fertile and subfertile men) do mean that there is a risk to miss men with fertility problems. What I miss (or have missed) is a discussion whether higher DECISION LIMITS could be an alternative solution to repeating semen analysis

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

PLoS One. 2023 Jan 19;18(1):e0280519. doi: 10.1371/journal.pone.0280519.r004

Author response to Decision Letter 1

18 Sep 2022

Dr. Emily Chenette, PhD

Editor-in-Chief, PLOS ONE

Dr. Stefan Schlatt

Academic Editor, PLOS ONE

Milan, September 18th, 2022

Dear Dr Chenette,

dear Dr. Schlatt,

please find enclosed the revised version of the manuscript titled “Infertile men with normal semen parameters at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenario” (PONE-D-22-12621R1- Authors: Luca Boeri, et al.) to be considered for publication in PLOS ONE.

We are very grateful to the Reviewers for their insightful comments to our paper.

List of the changes made in the manuscript:

Journal Requirements

A1. The whole Reference list has been reviewed carefully; no papers have been retracted.

REVIEWER #1

COMMENT#1.

Authors only work with sperm concentration, but declares sperm numbers were calculated. It was clear already 2010 that the WHO manual recommends sperm number rather than concentration that depends on both sperm number and dilution by fluid from other sources.

A1. We thank the Reviewer#1 for this critical comment. Accordingly, total sperm number data has been included in the Results section of the manuscript. Thus, we have included total sperm number instead of sperm concentration in the analyses and previous findings were confirmed. The text has been revised accordingly.

COMMENT#2.

Variation in ejaculatory abstinence time can be an important source for variation. There is not even a discussion about this and no data available, only in M&M that abstinence time was 2-7 days. Could there be individual variability due to variation in abstinence time (or even ejaculatory frequency) before the two semen analyses?

A2. Ejaculatory abstinence time was similar between the two semen analyses [Median (IQR) 3 (1-4) days]. The Results section of the manuscript has been revised, accordingly.

COMMENT#3.

Have the authors considered that limits above the lower 5th percentile "limit" byt the WHO 2010 and 2021 could be better to distinguish between men with semen abnormalities and men without? Looking at e.g. Keihani et al 2021 indicating that men in couples with short time to initiation of pregnancy have much higher semen analysis results compared to men in couples with 5-12 months. Thus, I do agree with the authors that the so called WHO limits (which should not be mistaken for limits between fertile and subfertile men) do mean that there is a risk to miss men with fertility problems. What I miss (or have missed) is a discussion whether higher DECISION LIMITS could be an alternative solution to repeating semen analysis

A3. We thank the Reviewer#1 for this insightful comment. We agree with the Reviewer#1 and the WHO2021 suggesting that semen parameters per se cannot distinguish between fertile vs. infertile men. However, here we included infertile men only as for WHO definition (i.e., not conceiving after at least 12 months of unprotected intercourses regardless of whether or not a pregnancy ultimately occurs) which is actually independent from semen analysis parameters. Thus, semen parameters are used in clinical practice to guide diagnosis testing and the choice of treatment options for infertile men. Therefore, we aimed to challenge international guidelines concerning the need for a single or confirmative semen analysis. The need to use decision limits in clinical practice has been included in the Discussion section of the manuscript.

We hope that the paper is now suitable to be considered for publication in the Original Articles section of PLOS ONE.

Sincerely yours,

Andrea Salonia, on behalf of all the authors

Andrea Salonia, MD, PhD, FECSM

Vita-Salute San Raffaele University

Division of Experimental Oncology/Unit of Urology, URI-Urological Research Institute

IRCCS Ospedale San Raffaele

Email: salonia.andrea@hsr.it

Attachment

Submitted filename: Response to Reviewers - Sept 18 2022.doc

Click here for additional data file.^{(48KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0280519.r005

Decision Letter 2

Stefan Schlatt

2 Nov 2022

PONE-D-22-12621R2Infertile men with normal semen parameters at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenarioPLOS ONE

Dear Dr. Salonia,

Please submit your revised manuscript by Dec 17 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Stefan Schlatt

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

The reviewer and I are positive about the content of the paper and considered the revisions useful. However, the refree still raises a few minor concerns which in my view are relevant and indeed improve the papers validity. The data need to be presented in a format that can easily be readable and contains all labels. I ask the authors to once more go through the suggestions and fix the errors listed by the reviewer.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

**********

6. Review Comments to the Author

Reviewer #1: I do believe this study merits to be published but there are still a number of aspects that if considered could make this a landmark publication. There are also flaws that need to be corrected.

1. The supporting information is not sufficiently transparent.

a. Many labels and contents are in Italian - must be in English

b. Many cells have only an Error indication

c. A complete lack of explanations of column labels, how the reader should be able to analyse the data (e.g. compare first and second sample

2. The systematic use of terms like "normal semen" and "abnormal semen" although even WHO has pointed out that data should not be interpreted in that way: reference limits in WHO 2010 and 2021 are NOT limits between fertility and infertility. It would be better to refer to "men with semen results below/above WHO reference limts"

WHO 2021 even points to the fact that male factor infertility have different causes, and therefore it is not very likely that there exists ONE true limit concerning semen analysis results for all causes - decision limits are more likely to support the clinical work with male reproductive disorders.

3. In table one, authors show the range of a number semen analysis parameters. It is strange that among men with "normal" first and second sample, low sperm numbers, concentrations, volumes etc are found!

4. Concerning the abstinence time - there are only results on group level available. What would be of interest is the comparison of each individuals abstinence time for the two samples (average difference and variability).

5. A very interesting aspect that has not been approached (as I can see it) is that the WHO reference limits 2010 and 2021 are too low! Againg thinking about e.g. Keihani 2021 this is not unlikely at all. The data the authors have could carry important information about this: could it be that using higher cut-offs would make a majority of men with first "normal" result to be above the WHO 2010/2021 level but under a tentative, higher cut-off? A graph showing the results of "normal-abnormal" men's first results in relation to cut-off level and compared to "normal-normal" men's

Of course, this would mean that the heavily promoted need to for a second semen analysis based on the often misunderstood WHO 2010/2021 reference limits becomes of less interest. But the matter deserves serious scientific attention not the least since the WHO 2021 points to the weaknesses of the presented reference limits.

6. Authors repeatevly use the term"infertile men", but the definition is not sufficiently described. How where female factors ruled out?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

PLoS One. 2023 Jan 19;18(1):e0280519. doi: 10.1371/journal.pone.0280519.r006

Author response to Decision Letter 2

13 Nov 2022

Dr. Emily Chenette, PhD

Editor-in-Chief, PLOS ONE

Dr. Stefan Schlatt

Academic Editor, PLOS ONE

Milan, November 06th, 2022

Dear Dr Chenette,

dear Dr. Schlatt,

please find enclosed the revised version of the manuscript titled “Infertile men with semen parameters above WHO reference limits at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenario” (PONE-D-22-12621R2- Authors: Luca Boeri, et al.) to be considered for publication in PLOS ONE.

We are very grateful to the Reviewers for their insightful comments to our paper.

List of the changes made in the manuscript:

REVIEWER #1

COMMENT#1.

The supporting information is not sufficiently transparent.

a. Many labels and contents are in Italian - must be in English

b. Many cells have only an Error indication

c. A complete lack of explanations of column labels, how the reader should be able to analyse the data (e.g. compare first and second sample).

A1. We thank the Reviewer#1 for this comment. The supporting information file has been revised.

COMMENT#2.

The systematic use of terms like "normal semen" and "abnormal semen" although even WHO has pointed out that data should not be interpreted in that way: reference limits in WHO 2010 and 2021 are NOT limits between fertility and infertility. It would be better to refer to "men with semen results below/above WHO reference limts"

A2. We thank the Reviewer#1 for this comment. The entire text has been revised accordingly.

COMMENT#3.

In table one, authors show the range of a number semen analysis parameters. It is strange that among men with "normal" first and second sample, low sperm numbers, concentrations, volumes etc are found!

A3. Table 1 reports baseline descriptive statistics of men according to their second semen analysis. In details Group 1 includes men with two consecutive semen analyses above WHO limits and Group 2 includes infertile men with a first semen sample above and a consecutive second one below limits. Of note, sperm parameters included in table 1 are baseline values (meaning at first assessment). The primary aim of table 1 is showing baseline characteristics of men to identify potential discrepancies within the two groups. Moreover, sperm concentration and volume were note lower in Group 1 compared to group 2.

COMMENT#4.

Concerning the abstinence time - there are only results on group level available. What would be of interest is the comparison of each individuals abstinence time for the two samples (average difference and variability).

A4. We thank the Reviewer#1 for this comment. We performed a Wilcoxon Signed Rank Test to assess potential difference in each individual abstinence time for the two samples. No statistical difference was noted. Difference in abstinence time for Group 1: average 0.32 days; variability 0.2; difference in abstinence time for Group 2: average 0.33 days; variability 0.2. The text has been revised accordingly.

COMMENT#5.

A very interesting aspect that has not been approached (as I can see it) is that the WHO reference limits 2010 and 2021 are too low! Againg thinking about e.g. Keihani 2021 this is not unlikely at all. The data the authors have could carry important information about this: could it be that using higher cut-offs would make a majority of men with first "normal" result to be above the WHO 2010/2021 level but under a tentative, higher cut-off? A graph showing the results of "normal-abnormal" men's first results in relation to cut-off level and compared to "normal-normal" men's

A5. We thank the Reviewer#1 for this comment. We totally agree that reference limits should be taken with caution in clinical practice. Particularly due to the fact that they derived from the 5th percentile of distribution from a cohort of fertile men. However, the WHO reference criteria are commonly used in clinical practice and the proposal of new cut-offs are beyond the aim of our study. In this study we aimed to identify baseline characteristics of men with semen parameters above the WHO limits (currently used worldwide) to dictate a second test. We believe that our study has a strong implication and relevance in the everyday clinical practice. We will conduct a new study in the near future thanks to your suggestion to depict different scenarios with higher limits.

COMMENT#6.

Authors repeatevly use the term"infertile men", but the definition is not sufficiently described. How where female factors ruled out?

A6. As reported in the Methods section, couple’s infertility was defined by WHO definition (not conceiving a pregnancy after at least 12 months of unprotected intercourses regardless of whether or not a pregnancy ultimately occurs). Moreover, each men underwent a comprehensive clinical, physical, hormonal, genetic and seminal investigation to identify the cause of male infertility. Male factor infertility was defined after a comprehensive diagnostic evaluation of all the female partners by expert Gynaecologists, which included a detailed medical, reproductive and family history as well as a general and gynaecological physical examination. Furthermore, the ovulatory status, ovarian reserve testing, the structure and patency of the female reproductive tract were requested in all cases. The text has been revised accordingly.

We hope that the paper is now suitable to be considered for publication in the Original Articles section of PLOS ONE.

Sincerely yours,

Andrea Salonia, on behalf of all the authors

Andrea Salonia, MD, PhD, FECSM

Vita-Salute San Raffaele University

Division of Experimental Oncology/Unit of Urology, URI-Urological Research Institute

IRCCS Ospedale San Raffaele

Email: salonia.andrea@hsr.it

Attachment

Submitted filename: Response to Reviewers - November 06 2022.doc

Click here for additional data file.^{(51.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0280519.r007

Decision Letter 3

Stefan Schlatt

2 Jan 2023

Infertile men with semen parameters above WHO reference limits at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenario

PONE-D-22-12621R3

Dear Dr. Salonia,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stefan Schlatt

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have responsed adequately and the paper is now acceptable for publication.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0280519.r008

Acceptance letter

Stefan Schlatt

8 Jan 2023

PONE-D-22-12621R3

Infertile men with semen parameters above WHO reference limits at first assessment may deserve a second semen analysis: challenging the guidelines in the real-life scenario

Dear Dr. Salonia:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stefan Schlatt

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Dataset

(XLSX)

Click here for additional data file.^{(357KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers - 14 07 2022.doc

Click here for additional data file.^{(46KB, doc)}

Attachment

Submitted filename: Response to Reviewers - Sept 18 2022.doc

Click here for additional data file.^{(48KB, doc)}

Attachment

Submitted filename: Response to Reviewers - November 06 2022.doc

Click here for additional data file.^{(51.5KB, doc)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0280519.ref001] 1.Minhas S, Bettocchi C, Boeri L, Capogrosso P, Carvalho J, Cilesiz NC, et al. European Association of Urology Guidelines on Male Sexual and Reproductive Health: 2021 Update on Male Infertility. Eur Urol 2021;S0302–2838(21)01982-5. doi: 10.1016/j.eururo.2021.08.014 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref002] 2.Schlegel PN, Sigman M, Collura B, De Jonge CJ, Eisenberg ML, Lamb DJ, et al. Diagnosis and treatment of infertility in men: AUA/ASRM guideline part I. Fertil Steril 2021;115(1):54–61. doi: 10.1016/j.fertnstert.2020.11.015 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref003] 3.World Health Organization. Infertility definitions and terminology. Accessible at: https://www.who.int/reproductivehealth/topics/infertility/definitions/en/. Accessed March 28, 2020. [Google Scholar]

[pone.0280519.ref004] 4.Boeri L, Fallara G, Pozzi E, Belladelli F, Corsini C, Raffo M, et al. The impact of different WHO reference criteria for semen analysis in clinical practice: Who will benefit from the new 2021 thresholds for normal semen parameters? Andrology 2022. doi: 10.1111/andr.13213 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0280519.ref005] 5.Boeri L, Belladelli F, Capogrosso P, Cazzaniga W, Candela L, Pozzi E, et al. Normal sperm parameters per se do not reliably account for fertility: A case-control study in the real-life setting. Andrologia 2021;53(1):e13861. doi: 10.1111/and.13861 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref006] 6.Hamada A, Esteves SC, Nizza M, Agarwal A. Unexplained male infertility: diagnosis and management. Int Braz J Urol 2012;38(5):576–94. doi: 10.1590/s1677-55382012000500002 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref007] 7.WHO, laboratory manual for the examination and processing of human semen. 2010. [PubMed] [Google Scholar]

[pone.0280519.ref008] 8.Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HWG, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update 2010;16(3):231–45. doi: 10.1093/humupd/dmp048 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref009] 9.Heller CG, Clermont Y. Spermatogenesis in man: an estimate of its duration. Science 1963;140(3563):184–6. doi: 10.1126/science.140.3563.184 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref010] 10.Blickenstorfer K, Voelkle M, Xie M, Fröhlich A, Imthurn B, Leeners B. Are WHO Recommendations to Perform 2 Consecutive Semen Analyses for Reliable Diagnosis of Male Infertility Still Valid? J Urol 2019;201(4):783–91. doi: 10.1016/j.juro.2018.11.001 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref011] 11.Zhu Q-X, Gao E-S, Pathak N, Wu J-Q, Zhou W-J. Single or double semen samples: the dilemma in epidemiological studies on semen quality. Hum Reprod 2016;31(3):511–7. doi: 10.1093/humrep/dev326 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref012] 12.Leushuis E, van der Steeg JW, Steures P, Repping S, Bossuyt PMM, Blankenstein MA, et al. Reproducibility and reliability of repeated semen analyses in male partners of subfertile couples. Fertil Steril 2010;94(7):2631–5. doi: 10.1016/j.fertnstert.2010.03.021 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref013] 13.Chiu Y-H, Edifor R, Rosner BA, Nassan FL, Gaskins AJ, Mínguez-Alarcón L, et al. What Does a Single Semen Sample Tell You? Implications for Male Factor Infertility Research. Am J Epidemiol 2017;186(8):918–26. doi: 10.1093/aje/kwx169 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0280519.ref014] 14.Keel BA. Within- and between-subject variation in semen parameters in infertile men and normal semen donors. Fertil Steril 2006;85(1):128–34. doi: 10.1016/j.fertnstert.2005.06.048 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref015] 15.World Health Organization web chapter on couple’s infertility. 2017. http://www.who.int/reproductivehealth/topics/infertility/definitions. Accessed May 2, 2019.

[pone.0280519.ref016] 16.Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 1987;40(5):373–83. doi: 10.1016/0021-9681(87)90171-8 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref017] 17.Salonia A, Matloob R, Gallina A, Abdollah F, Saccà A, Briganti A, et al. Are infertile men less healthy than fertile men? Results of a prospective case-control survey. Eur Urol 2009;56(6):1025–31. doi: 10.1016/j.eururo.2009.03.001 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref018] 18.Ventimiglia E, Capogrosso P, Colicchia M, Boeri L, Serino A, Castagna G, et al. Metabolic syndrome in white European men presenting for primary couple’s infertility: investigation of the clinical and reproductive burden. Andrology 2016;4(5):944–51. doi: 10.1111/andr.12232 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref019] 19.Boeri L, Capogrosso P, Ventimiglia E, Cazzaniga W, Pozzi E, Belladelli F, et al. Testicular volume in infertile versus fertile white-European men: a case-control investigation in the real-life setting. Asian J Androl 2021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0280519.ref020] 20.Boeri L, Capogrosso P, Ortensi I, Miacola C, Cai T, Verze P, et al. Diagnostic and therapeutic workup of male infertility: results from a Delphi consensus panel. Int J Impot Res 2021. doi: 10.1038/s41443-021-00511-x [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref021] 21.Evenson DP. Sperm chromatin structure assay (SCSA®). Methods Mol Biol 2013;927:147–64. [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref022] 22.Ventimiglia E, Capogrosso P, Boeri L, Pederzoli F, Cazzaniga W, Scano R, et al. When to Perform Karyotype Analysis in Infertile Men? Validation of the European Association of Urology Guidelines with the Proposal of a New Predictive Model. Eur Urol 2016;70(6):920–3. [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref023] 23.Hamilton J a. M, Cissen M, Brandes M, Smeenk JMJ, de Bruin JP, Kremer J a. M, et al. Total motile sperm count: a better indicator for the severity of male factor infertility than the WHO sperm classification system. Hum Reprod 2015;30(5):1110–21. doi: 10.1093/humrep/dev058 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref024] 24.Pozzi E, Boeri L, Candela L, Capogrosso P, Cazzaniga W, Fallara G, et al. Infertile couples still undergo assisted reproductive treatments without initial andrological evaluation in the real-life setting: A failure to adhere to guidelines? Andrology 2021. doi: 10.1111/andr.13071 [DOI] [PubMed] [Google Scholar]

[pone.0280519.ref025] 25.WHO laboratory manual for the examination and processing of human semen. Sixth edition. Available at: https://www.who.int/publications/i/item/9789240030787.

PERMALINK

Infertile men with semen parameters above WHO reference limits at first assessment may deserve a second semen analysis: Challenging the guidelines in the real-life scenario

Luca Boeri

Edoardo Pozzi

Paolo Capogrosso

Giuseppe Fallara

Federico Belladelli

Luigi Candela

Nicolò Schifano

Christian Corsini

Walter Cazzaniga

Daniele Cignoli

Eugenio Ventimiglia

Marina Pontillo

Massimo Alfano

Francesco Montorsi

Andrea Salonia

Roles

Abstract

Objectives

Materials and methods

Results

Conclusions

Introduction

Materials and methods

Statistical methods

Results

Table 1. Descriptive statistics of the whole cohort of infertile men and according to second semen analysis characteristics.

Table 2. Logistic regression models predicting semen analysis below WHO limits after a first test above limits (No. = 212).

Fig 1. Decision curve analysis.

Discussion

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Stefan Schlatt

Roles

Author response to Decision Letter 0

Decision Letter 1

Stefan Schlatt

Roles

Author response to Decision Letter 1

Decision Letter 2

Stefan Schlatt

Roles

Author response to Decision Letter 2

Decision Letter 3

Stefan Schlatt

Roles

Acceptance letter

Stefan Schlatt

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases