Contemporary Diagnostic Work-Up for Male Infertility: Emphasizing Comprehensive Baseline Assessment.

Data collection followed the principles outlined in the Declaration of Helsinki; all patients had signed an informed consent agreeing to deliver their own anonymous information along with blood and seminal fluid. The study was approved by the IRCCS San Raffaele Hospital Ethical Committee, Milan, Italy (Prot. 2014 – Pazienti Ambulatoriali). Supporting evidence: our research and clinical experience have consistently underscored the significance of differentiating between primary and secondary infertility as the baseline step when assessing a couple seeking first medical help for reproductive purposes. This classification not only allows for a more precise diagnosis but also paves the way for more tailored treatment ( e.g. , according to patient’s partner age and diminished ovarian reserve [ 9 ]) and preventive strategies plans [ 2 10 11 ]. Implications: adopting a differentiated approach based on whether infertility is primary or secondary may: i) enhance the awareness of the physician and the couple before the difficulties of secondary couple’s infertility; and enhance treatment outcomes and improve overall male patients’ health. Here, typical example was the finding of an association between a higher prevalence of metabolic syndrome in this specific setting and worsening of semen parameters as a whole, i.e., semen volume, sperm concentration, and normal sperm morphology [ 10 ]. Recommendation: we recommend the adoption of a holistic and segregated approach for primary and secondary infertility in clinical practice. Supporting evidence: the importance of a contemporary evaluation of both partners already over the first steps of the infertility management path is well-supported by numerous studies [ 11 12 13 ]. Evidence shows that a woman's age significantly affects her fertility potential, mostly because of a significant quantitative and qualitative depletion of the ovarian reserve over time [ 9 14 15 16 ]. In this context, anti-Müllerian hormone (AMH) (0.77–14.5 ng/mL) and antral follicle count (AFC) have been identified as a reliable marker of ovarian reserve both in spontaneous and assisted pregnancies [ 9 17 18 ], aiding in the prediction of a woman's response to fertility treatment [ 19 ]. These findings demonstrate the importance of an individualized couple’s infertility management work-up, comprehensively tailored in terms of woman's age, ovarian reserve, and both partners’ overall health status [ 12 ]. However, it is essential to recognize that solely focusing on the female partner should not overlook potential male factors contributing to infertility. Indeed, the unique aspects of the female reproductive system, thus including the biological clock and conditions like polycystic ovary syndrome and endometriosis, certainly warrant a thorough assessment of each female partner, but they also strongly outline not to skip a thorough investigation of male partner factors, since this could lead to unsuccessful reproductive outcomes [ 12 ]. Implications: a comprehensive assessment of the female partner throughout infertility management can lead to more effective and individualized treatments, improving the chances of successful conception [ 14 20 ]. Moreover, it can empower couples with knowledge and understanding, enabling them to make informed decisions about their fertility journey [ 12 ], potentially even increasing success rates in infertility treatment and contributing to overall improvements in couple’s reproductive health. Recommendation: we fully support what the guidelines suggest [ 1 5 ], we recommend a contemporary comprehensive assessment of the female partner, with a particular focus on factors such as age, AMH levels and AFC, general health, lifestyle, and medical history, for the successful tailored infertility treatments. Supporting evidence: medical history should evaluate any risk factors that could negatively impact on male’s fertility, focusing on lifestyle, family history (including testicular cancer), comorbidities [ 21 ], previous testicular surgery and excluding any potential known gonadotoxin exposure [ 22 ]. To this aim, the medical history of infertile patients should evaluate any congenital or acquired condition affecting testicles’ integrity or function, such as cryptorchidism (uni- or bilateral), history of testicular torsion and/or trauma. Likewise, potential iatrogenic etiologies, including gonadotoxic medications ( e.g. , anabolic drugs, chemotherapeutic agents, etc.), use of illicit drugs ( e.g. , marijuana, cocaine, opioids), previous urogenital/pelvic surgery and exposure to radiation, and environmental exposure ( e.g. , air pollution [ 23 ]) should be considered [ 8 24 25 26 ]. In addition, compelling evidence has accumulated in regard to the close relationship between overall health status and infertility [ 21 27 ]. Consequently, it appears that comorbid conditions known to have adverse effects on men's overall health are often linked to impaired reproductive functioning, irrespective of patients' age. This observation suggests that certain medical conditions, such as diabetes, hypertension, obesity, and other non-communicable chronic diseases (NCDs), can significantly impact toward male fertility regardless of age, thus making it imperative to address and manage these health concerns when managing infertility in men. Moreover, infertility itself may increase the risk of developing additional comorbidities compared to the general population [ 3 28 ]. Additionally, the observed trend of European males delaying fatherhood over the last two decades, coupled with the rising incidence of health-relevant comorbidities, can further impact men's fertility [ 29 ]. Based on historical World Health Organization (WHO) recommendations [ 30 ] along with the most updated scientific societies guidelines [ 1 5 ], we emphasize the importance of systematically collecting a detailed medical history, as it plays a crucial role in patient management. Practically speaking, our experience has shown that patients with poorer general health exhibit lower sperm concentration, decreased total testosterone (tT) levels (2.48–8.36 ng/mL), and elevated follicle-stimulating hormone (FSH) values (1.4–18.1 mUI/mL) [ 21 ]. Moreover, those with abnormal semen parameters are at higher risk of cancer, including melanoma, testicular, prostate, and colorectal cancer [ 31 32 33 ]. Furthermore, growing analyses has explored the connection between male infertility and noncancerous disorders, revealing the detrimental impact of conditions like urogenital and systemic infections, autoimmune diseases, endocrinopathies (hypo- or hyperthyroidism), metabolic disorders (type 2 diabetes mellitus, obesity, metabolic syndrome), chronic kidney disease, and liver diseases (chronic hepatitis) on fertility [ 28 34 35 36 37 38 ]. Implications: a comprehensive medical history taking throughout each couple’s infertility diagnostic work-up is crucial. Collecting a detailed medical history helps identify oncologic and non-oncologic NCDs linked to poor male general health, eventually affecting overall reproductive function. An accurate medical history collection allows detection of life- or health-threatening conditions underlying male infertility. MFI can serve as an early marker of the possible developing oncologic and non-oncologic NCDs, thus emphasizing the importance of secondary prevention for overall men's health protection. Recommendations: we recommend a comprehensive diagnostic work-up that includes a detailed medical history assessment in every infertile man. This should encompass an evaluation of lifestyle risk factors, family history ( e.g. , including testicular cancer), comorbidities, and any potential known gonadotoxin exposure. It is crucial to address and manage comorbid conditions known to adversely affect overall men's health as these can significantly impact toward male fertility, irrespective of age. We further recommend that medical history collection should also be focused on identifying congenital or acquired conditions that affect testicular integrity or function— e.g. , cryptorchidism, history of testicular torsion and/or trauma. Potential iatrogenic etiologies, including gonadotoxic medications, use of illicit drugs, previous urogenital/pelvic surgery, and radiation exposure, should be also considered. Of clinical significance, MFI can serve as a surrogate marker of early developing of oncologic and non-oncologic chronic NCDs. Thereof, we emphasize the importance of secondary prevention strategies in the management of infertile men. Supporting evidence: a respectful but meticulous physical examination is a prerequisite in the evaluation of MFI. First, this includes the observation of secondary sexual characteristics [ 39 ]. In instances of congenital testicular deficiency, as seen in Klinefelter syndrome, the clinical presentation is more frequently explicit. Delayed puberty, along with an overall eunuchoid phenotype characterized by scant body hair, high-pitched voice, and small testes, penis, and prostate, are commonly observed clinical manifestations during the pre- or peri-pubertal period [ 39 ]. Second, it’s important to note the intrinsic link between obesity, hypogonadism (primarily functional), and MFI [ 38 40 ]. Consequently, measurement of body mass index (BMI) and waist circumference is mandatory in all individuals [ 2 41 42 ]. Third, a thorough urological physical examination of the external genitalia represents the mandatory keystone step in a clinical evaluation of MFI. Although assessing the scrotum can be challenging in some cases ( e.g. , obese patients), it is imperative to evaluate testes' size, texture, and consistency [ 43 ]. In routine clinical practice, testicular volume (TV) is gauged using Prader’s orchidometer, a reliable and cost-effective proxy for ultrasonography-measured TV [ 44 45 ]. However, owing to the diverse demographics studied (such as geographic location, diet, ethnicity, and environmental factors), uniform reference values for Prader’s orchidometer-derived TV are yet to be established [ 46 47 ]. In this context, our data showed that infertile men have an eight-fold higher risk of diminished TV, with a positive correlation identified between TV and tT levels, while a negative correlation between TV and FSH/luteinizing hormone (LH) (1.7–8.6 mUI/mL) [ 43 ]. Along with TV measurement, testes consistency should be evaluated, with a focus on palpable abnormalities of the epididymis and of the vas deferens. Potential testicular masses, which may hint at cancer as a linkage between male infertility and testicular cancer is well-established [ 48 49 50 ]. Specifically, patients with testicular germinal cell tumors, prior to cancer treatment, have reported Leydig cell dysfunction, resulting in diminished sperm quality [ 48 49 ]. Given its profound link with MFI [ 8 ], Reproductive Medicine experts must explore the presence of asymptomatic varicocele. Although the diagnosis is primarily made through physical examination [ 47 ], we strongly consider the need to investigate each man presenting for couple’s infertility with color Doppler ultrasonography for a more accurate diagnosis of varicocele, and the consequent therapeutic approach [ 1 51 52 53 ]. Consistent with previous studies [ 30 54 55 56 ], our findings revealed that up to 37.3% of patients seeking initial medical assistance for MFI had clinically diagnosed varicocele [ 57 ]. In addition to this, a careful evaluation regarding the absence of the vas deferens is essential, especially among those seeking first medical help for azoospermia [ 8 ]. In this context, approximately 26% to 75% of men affected by unilateral absence of the vas deferens harbor ipsilateral renal anomalies including agenesis [ 58 59 ]. Consequently, abdominal imaging should be offered to men with vas deferens agenesis regardless of the cystic fibrosis transmembrane conductance regulator (CFTR) status to allow for optimal patient counseling. A thorough physical examination of infertile men should also consider the assessment of the penis. Evaluating its size and detecting potential physical alterations are integral to this path. Indeed, an accurate assessment of the penis can identify common abnormalities such as phimosis, short frenulum, fibrotic nodules, epispadias, and hypospadias, all of which can contribute to male fertility impairment [ 11 ]. Additionally, a digital rectal examination may be useful to rule out prostate abnormalities before initiating any form of testosterone therapy in hypogonadal men who eventually have ended the reproductive path. This is particularly important given the established link between MFI and an increased risk of prostate cancer [ 60 61 62 63 ]. In this context, we found that a higher proportion of infertile men under 40 years of age exhibited a prostate-specific antigen (PSA) value of >1 ng/mL compared to their age-comparable fertile counterpart. A significant correlation was observed between PSA concentration and impaired semen parameters among infertile patients [ 63 ]. Implications: a thorough physical examination in the evaluation of MFI includes the assessment of secondary sexual characteristics, BMI, urological examination, as well as the scrutiny of the penis (and prostate, if indicated and after explicit explanation to the patient). Furthermore, it is crucial to acknowledge the observed correlation between TV and tT levels, the established link between obesity and hypogonadism, the notable prevalence of varicocele and testicular abnormalities ( e.g. , testis masses, testis cancer) among infertile men, the need for abdominal imaging in cases of vas deferens agenesis, and the heightened risk of prostate cancer in men with MFI. Recommendations: we recommend a comprehensive physical examination as a prerequisite over the evaluation of MFI. This examination should include assessing secondary sexual characteristics, measuring BMI and waist circumference, conducting a thorough genital examination, and scrutinizing the prostate when indicated (mostly in men with a family history of prostate cancer and in those with PSA levels higher than expected per age [ 64 65 ]). Additionally, it is essential to consider the correlation between TV and tT levels, recognize the link between obesity and hypogonadism, be aware of the prevalence of varicocele and testicular abnormalities among infertile men, and conduct abdominal imaging when encountering vas deferens agenesis. Moreover, due attention should be given to the increased risk of prostate cancer in individuals with MFI. Supporting evidence: semen analysis plays a pivotal role over the diagnostic assessment of MFI, providing valuable information about macroscopic sperm quality as well as giving useful insights regarding the need for additional testing ( e.g. , genetic analysis, sperm DNA fragmentation [SDF], etc.) [ 1 66 ]. However, it is important to note that semen parameters do not stand alone, and cannot predict fertility or infertility outcomes per se [ 6 ]. Even if this holds true, to ensure consistency and accuracy, the analysis should adhere to the WHO criteria reported on the Laboratory Manual for the Examination and Processing of Human Semen [ 66 ]. Our data indicate that the recent implementation of new reference criteria for semen parameters by WHO-21 has led to a shift in the severity categorization of semen abnormalities, compared to previous editions [ 67 ]. More in details, one in three infertile men demonstrated a deterioration in semen categorization according to WHO-21 vs. WHO-10. These men displayed more severe clinical, hormonal, and SDF index characteristics [ 67 ]. Additionally, pregnancy outcomes via assisted reproductive techniques were lower for men with worsening sperm abnormalities per WHO-21 [ 67 ]. Consequently, WHO-21 criteria clearly appear to identify a subset of patients (with abnormal semen parameters) more accurately, who were previously considered “normal” according to WHO-10 parameters. Implications: the introduction of WHO-21 reference criteria highlights how semen quality should be perceived as a continuum, urging caution in the binary division between “normal” and “abnormal”. This artificial dichotomy should be used judiciously, serving only as a broad guideline in clinical practice. The implications can lead to a more accurate diagnosis and better-informed treatment strategies [ 67 ]. Recommendation: we recommend the adoption of WHO-21 reference criteria in clinical practice for semen analysis to enhance diagnostic accuracy and patient treatment plans, thus following the indications and reference criteria outlined in the WHO Laboratory Manual for the Examination and Processing of Human Semen (6th edn.), or alternatively rigorously adhering to the previous version (5th edn.). Supporting evidence: the clinical need of repeating a semen analysis in the setting of MFI evaluation is still debated. The American Urological Association (AUA) and the American Society for Reproductive Medicine (ASRM) guidelines recommend to perform at least two consecutive semen analyses in every infertile man despite having a normal one at first evaluation [ 5 68 ]. In contrast, the recently published EAU Guidelines on sexual and reproductive health keep proposing that one test is sufficient if it aligns with WHO reference criteria and no sperm abnormalities are detected [ 11 ]. This discrepancy fuels the debate on the clinical benefit of one versus two or more consecutive semen analyses over the MFI diagnostic work-up. Our data suggests that many infertile men who pass initial semen analysis per WHO standards may experience a decline in subsequent tests [ 69 ]. Factors such as lower TV, higher FSH values, and lower total sperm count at the initial test were found to be closely associated with this decline [ 69 ]. This implies that a single semen analysis may not always accurately represent a man’s fertility status even with macroscopically normal sperm parameters, hence the need for a second investigation in selected cases. These findings advocate for a risk score system to be incorporated into routine clinical practice, potentially improving diagnosis and treatment outcomes in male infertility. Implications: the implications of these findings suggest that even if normal according to WHO-2021 reference criteria, a single semen analysis may not be sufficient to accurately assess a man's fertility status, leading to potential misdiagnosis and misinterpretation. The results also underscore the potential for unnecessary examinations and delays in treatment if a second semen analysis is not performed. Recommendation: we recommend conducting a second semen analysis in every man presenting for couple’s infertility. This holds even more true for patients exhibiting any of the following conditions: reduced TV, elevated FSH values, or a decreased total sperm count at first evaluation. Supporting evidence: the current everyday clinical practice of assessing MFI gives significant importance to semen culture, as it offers insights into potentially reversible factors related to urogenital tract infection (UTI) [ 11 ]. These infections, as prevalent as in 15% to 20% of infertile men, can lead to defects in sperm function as well as impaired spermatogenesis [ 11 ]. However, the existing guidelines lack clarity regarding the timing, settings for semen culture, and specific germs to target. Specifically, according to strict dictates of science deriving from the most rigorous literature, the EAU guidelines suggest performing a semen culture when leukocytospermia is present, which is indicated by >10 6 peroxidase-positive white blood cells per milliliter of ejaculate, possibly indicating an active “infection-driven” inflammation [ 11 ]. Likewise, the AUA/ASRM guidelines, suggest that routine semen cultures have not been prospectively demonstrated to benefit infertile couples, therefore, screening for infection is not needed unless pyospermia is present [ 5 68 ]. Our data, involving 523 white-European infertile men revealed that high leukocyte levels in semen did not always indicate an underlying bacterial infection [ 70 ]. Consequently, when validating the EAU guidelines, it led to an 80% failure rate in detecting infected semen cultures and conducting 120 unnecessary examinations [ 70 ]. Lastly, according to our data, the application of a logistic regression model failed to identify any informative baseline parameter except for serum neutrophil-to-lymphocyte ratio (NLR), which marginally improved the prediction of a positive semen culture [ 70 ]. Implications: no available tool to date is accurate in predicting bacterial infections in infertile men, leading to a high rate of misdiagnosis and unnecessary examinations. In this context, it remains a clinical challenge to identify those who would benefit the most from a semen culture investigation. Recommendation: as a baseline prerequisite, we recommend to incorporating at least serum NLR as a predictive parameter in the everyday clinical practice. Supporting evidence: asymptomatic semen infection is a common occurrence among men seeking medical assistance for primary infertility, with one in five men being affected irrespective of leukocyte counts [ 71 ]. The frequently isolated pathogens include Ureaplasma urealyticum , Enterobacteriaceae spp, human papilloma virus (HPV) (any), Mycoplasma hominis , and Chlamydia trachomatis , many of which are not typically detected through standard semen cultures [ 11 ]. Our data has indicated that the presence of a positive semen culture is intrinsically linked to impaired sperm concentration and reduced progressive sperm motility, especially in cases involving Ureaplasma, Mycoplasma, and HPV [ 71 72 ]. Moreover, high-risk HPV genotypes were found to have a higher impact in terms of semen parameters worsening [ 72 ]. Implications: the aforementioned findings highlight the significant role of a thorough investigation of specific germs in semen over the diagnostic work-up of MFI. The presence of HPV DNA in the semen of primary infertile men, particularly those with exclusively high-risk genotypes, is associated with compromised sperm progressive motility and higher SDF values. These findings not only highlight the potential negative impact of the presence of HPV in semen in terms of male fertility outcomes, but also underscore its implications on overall men's health. Recommendation: we recommend the inclusion of a thorough semen infection testing approach, which encompasses the identification of specific pathogens such as HPV, as well as intracellular organisms including Mycoplasma hominis , Mycoplasma genitalium , Ureaplasma urealyticum , Ureaplasma parvum , Trichomonas vaginalis , and Chlamydia trachomatis . Supporting evidence: semen parameters per se offer “only” a partial view of a man's fertility potential. Unexplained infertility is observed in men with normal semen analysis, medical history, and physical examination, suggesting the involvement of other factors such as genetic alterations, anti-sperm antibodies, oxidative stress imbalance, and sperm dysfunction [ 7 ]. Our data show that only 12% of infertile men and 41% of fertile men have normal sperm parameters [ 6 ]. Implications: these findings imply that the evaluation of MFI must extend beyond simple semen analysis as normal sperm parameters do not perfectly mirror male fertility. Recommendation: we recommend expanding the diagnostic approach for MFI beyond simple semen analysis. Supporting evidence: since 41% of fertile men and 12% of infertile men exhibit normal macroscopic/conventional sperm parameters, conventional semen analysis on its own may not provide sufficient accuracy in the setting of male infertility diagnostic workup [ 6 ]. Likewise, known infertility risk factors (higher levels of systemic inflammation and signs of metabolic diseases) have been shown to be correlated with higher SDF levels [ 38 73 74 ]. However, even with the implementation of guidelines set forth by EAU for sexual and reproductive health, there are limitations in identifying primary infertile men with possible pathological SDF levels [ 75 ]. Implications: conventional semen analysis proves inadequacy in accurately assessing male infertility in a number of cases, while the use of SDF has shown to be promising in reducing the number of idiopathic infertility diagnoses; nevertheless, there remains a dearth of appropriate guidelines for its application. Recommendation: we recommend incorporating SDF over the assessment of infertility patients. SDF is a measure of the damage to the DNA of sperm cells, and it has been shown to be a predictor of male infertility. Men presenting with unexplained infertility, history of poly-abortions, Prader <15, age ≥38 years and a total motile sperm count <20×10 6 have an increased risk of presenting with a pathological SDF. Moreover, our experience has been mostly with SCSA (sperm chromatin structure assay) which emerged as a valuable tool for assessing DNA damage in sperm. The standardized protocol of SCSA offers a significant advantage, as it minimizes inter-laboratory variation, ensuring consistent and reliable results across different facilities. Moreover, the clinical threshold for normalcy of SDF <30% at SCSA provides a clear benchmark for normal samples, allowing laboratories to identify potential issues with DNA damage in sperm, even when up to 30% of cells show damage [ 76 77 ]. Supporting evidence: DNA damage in sperm, primarily attributed to oxidative stress caused by reactive oxygen species (ROS), stands as a prominent factor contributing to impaired sperm function [ 78 ]. This damage is largely associated with modifiable lifestyle factors and it has been linked to a variety of negative effects on the fertility function [ 73 ]. While various strategies exist to mitigate the effects of DNA damage and utilize them for potential treatments, there is still a lack of consensus regarding the appropriate selection criteria for ROS testing and the standardization of assay methods [ 79 80 ]. Implications: oxidative stress-induced fertility impairment is a significant contributor to male infertility, associated with modifiable lifestyle factors and negative effects on fertility, yet the lack of consensus on selection criteria and assay standardization hinders routine ROS testing and treatment strategies. Recommendation: we do not recommend routine ROS testing in infertile men. The impact of oxidative stress-induced DNA damage on sperm functionality can be more easily assessed via SDF measurement and an accurate data collection regarding possible lifestyle factors influencing ROS production. Supporting evidence: current EAU guidelines suggest offering cryopreservation among men with MFI and severe oligozoospermia (<4 million sperm/mL) [ 11 ]. Moreover, poor data are actually available in terms of fertility preservation in both young men of reproductive age and aging men with prostate, bladder, or kidney cancer. This is a significant area of concern, as men are increasingly experiencing fatherhood in late adulthood. Furthermore, widely available screening programs and heightened public awareness about urological cancers have resulted in a larger proportion of young men with newly diagnosed neoplasms seeking ablative treatment, potentially leading to de novo male infertility. Our data suggest that about 32% of interviewed urological patients expressed willingness to bank sperm before undergoing potentially fertility-damaging therapy, with cancer being an independent predictor positively associated with the desire for sperm cryopreservation [ 81 82 ]. Given the increasing number of newly diagnosed patients with prostate, bladder, or kidney cancer, and the potential desire for future fatherhood, these findings should prompt physicians to inform their patients about the possible fertility implications of most surgical or non-surgical procedures. Implications: the implications of these findings extend to the physician's role in patient care. Often, physicians do not discuss treatment-related male infertility or the options for fertility preservation, such as cryopreservation, with their patients. This lack of communication could deprive the patients of the opportunity to preserve their fertility, even when they have severe oligozoospermia. Consequently, it's essential for physicians to recognize the importance of this technique and its potential to improve the quality of life for cancer patients. Recommendation: we recommend offering cryopreservation to all men with severe oligozoospermia (<4 million sperm/mL). Moreover, a comprehensive information about fertility preservation approaches should be provided as early as possible during cancer treatment planning (especially to those patients in which oncological treatments will have a detrimental effect on their fertility potential). Supporting evidence: male hypogonadism is frequent in infertile men, with several distinct phenotypes, each reflecting an underlying disease characteristic [ 1 39 ]. Infertile men may present with either secondary or primary hypogonadism. According to the circulating levels of tT, FSH, and LH men can be classified into different types of hypogonadism [ 39 ]. Notably, primary hypogonadal men face a significantly heightened risk of azoospermia (24-fold increase) and small TV (13-fold increase) compared to eugonadal men, thus portraying the most unfavorable clinical scenario in terms of impaired fertility [ 83 ]. Moreover, it has been observed that median levels of sex-hormone-binding globulin (SHBG), which influence the availability of testosterone, tend to rise across age quartiles while decreasing in concurrence with increases in BMI [ 84 ]. Implications: male hypogonadism is a frequently encountered condition among infertile men, with various subtypes indicating distinct underlying diseases. By evaluating the levels of sex hormones, men can be effectively categorized into different groups. Furthermore, age and BMI have been found to influence SHBG, which have implications on free testosterone levels. Recommendation: we recommend the inclusion of a comprehensive evaluation of sex hormones levels, over the diagnostic work-up of MFI— i.e. , tT, FSH, LH, SHBG, and albumin. Given the significant impact of age and BMI on SHBG levels and the subsequent availability of testosterone, it is crucial to consider these factors when formulating a management strategy for infertile men with hypogonadal conditions. Supporting evidence: limited research has been conducted on hormones other than gonadotropins and tT in the context of MFI. It has been observed that unexplained high levels of alpha-fetoprotein (AFP) contribute to nearly 2% of cases in primary infertile patients without any prior history of associated disorders. Elevated serum AFP levels have been associated with abnormal sperm counts, advanced age, obesity, and a higher prevalence of comorbid conditions [ 85 ]. Recent advancements in specific inhibin assays have unveiled that circulating inhibin B can have a role over MFI diagnostic work-up. Inhibin B is exclusively produced by the testis, primarily by Sertoli cells in prepubertal testes, while its production in adults remains controversial [ 86 ]. Inhibin B regulates FSH secretion through negative feedback [ 86 ]. During life, inhibin B production changes, with peaks after birth and during puberty, influenced by FSH and spermatogenic status. In general, serum inhibin B levels are positively correlated with TV and sperm counts, and they decrease in infertile patients [ 86 87 88 89 ]. Inhibin B levels per se are not a reliable predictor of sperm presence in testicular biopsy samples [ 89 90 ]. Circulating AMH levels have a role in the infertile man diagnostic work-up. In fact, in the context of men with idiopathic non-obstructive azoospermia (iNOA) undergoing microdissection testicular sperm extraction (mTESE), while circulating hormones associated with primary hypogonadism do not predict sperm retrieval, AMH levels and the AMH-to-tT (AMH/tT) ratio emerged to achieve independent predictor status for sperm retrieval at surgery. Positive sperm retrieval can be predicted using AMH cutoff values of 4.62 ng/ml and AMH/tT cutoff values of 1.02, respectively [ 91 92 ]. Currently, the lack of other clinically reliable biomarkers makes predicting sperm retrieval results at TESE in iNOA very difficult, leading to up to 50% of unnecessary surgical operations [ 93 94 ]. Implications: limited research exists on non-sex hormones in male infertility. Although inhibin B is a valuable marker of spermatogenesis, its measurement's clinical relevance for individual infertile patients is still limited. Similarly, AMH levels and AMH/tT ratio independently predict sperm retrieval during mTESE. Unexplained high levels of AFP are observed in nearly 2% of primary infertile patients without associated disorders [ 85 ]. As a whole, although inhibin B and AMH levels play a role in diagnosing male infertility, data suggest that both hormones cannot act as stand alone biomarkers of male fertility status over the diagnostic work-up. Recommendation: we recommend that the assessment of a few other analytes, particularly AFP, AMH, and inhibin B, to be incorporated into the comprehensive diagnostic work-up of MFI. This is based on the recent findings that elevated AFP levels are observed in a significant number of primary infertile patients without any associated disorders (unexplained infertility). Furthermore, we recommend dosing AMH levels and calculating the AMH/tT ratio, as these have shown predictive capabilities for sperm retrieval during mTESE in iNOA men. Lastly, we recommend utilizing inhibin B as a valuable marker for assessing spermatogenesis in males, especially in conjunction with FSH levels, TV, and sperm counts. However, it's important to acknowledge that the clinical relevance of AMH and inhibin B levels for individual patients is still somewhat limited. Supporting evidence: current EAU guidelines recommend that men with sperm count alterations (3-19%) undergo a karyotype analysis (KA) when azoospermia or oligozoospermia (spermatozoa < 10 million/mL) is detected. These guidelines also recommend KA if family history suggests repeated spontaneous abortions, malformations, or intellectual disability. In addition, the AUA/ASRM guidelines, suggest that KA and Y-chromosome microdeletion analysis should be only recommended for men with primary infertility and azoospermia or severe oligozoospermia (<5 million sperm/mL) with elevated FSH or testicular atrophy or a presumed diagnosis of impaired sperm production as the cause of azoospermia [ 5 68 ]. However, KA is not always sensitive enough when applied to such sub-category of MFI men. To address this limitation, we proposed a novel nomogram based on LH values, mean TV, and sperm concentration. This nomogram can be used to improve the detection of chromosomal abnormalities at KA [ 95 ]. Implications: despite having a clear specific cut-off may help clinician in everyday clinical practice, the straightforward adherence to EAU recommendations may result in missed diagnoses or unnecessary treatment (80% sensitivity, 37% specificity, and 59% discrimination). Our nomogram, with a 2% probability cut-off, allows for a more careful detection of KA alterations (sensitivity 94%, specificity 33%) with a predictive accuracy of 72% (p=0.02 compared to EAU guidelines). Recommendations: we recommend a more comprehensive evaluation of clinical factors, not just sperm count, using our nomogram to avoid a 20% diagnostic loss of possible KA in infertile men caused by the application of the EAU guidelines, despite not specifically reducing the risk of overtreatment. We recognize that using a nomogram may be more difficult than using standardized cutoffs, but we are confident that greater diagnostic efforts are crucial, as up to 40% of male infertility cases remain unexplained and are therefore not properly diagnosed and treated. Supporting evidence: population-specific variability in genetic and epigenetic heritages among countries continue to fuel debate regarding the clinical value of testing for Y chromosome microdeletion analysis [ 96 ]. Therefore, while an absolute threshold for clinical testing cannot be universally given, current EAU guidelines suggest testing men for Y-chromosome microdeletion if sperm counts are <5 million sperm/mL and recommend a mandatory analysis only if <1 million sperm/mL are found [ 11 ]. Likewise, the AUA/ASMR guidelines suggest testing for Y microdeletion in azoospermic men or severe with severe oligozoospermia (<5 million sperm/mL). On the other hand, CFTR testing is suggested by EAU guidelines, in any patient with unilateral or bilateral absence of the vas deferens or with documented seminal vesicle agenesis [ 11 ]. Instead, the AUA/ASRM suggest CFTR testing in men with vasal agenesis or with any idiopathic obstructive azoospermia [ 5 68 ]. Implications: the spectrum of potential causes of MFI has changed over time, with more men having more severe cases and many more unexplained or idiopathic cases [ 57 ]. Incorrect timing in genetic assessment may delay couple's fertility management but also may lead to even worse fertility outcomes. For instance, we have shown that the duration of MFI is negatively associated with semen parameters, with a higher risk of oligozoospermia and a higher prevalence of NOA [ 97 ]. We have also demonstrated that normal sperm parameters per se are not an adequate and sufficient surrogate marker for male fertility [ 6 ]. Recommendations: in line with current EAU and AUA/ASRM guidelines, we recommend a comprehensive genetic assessment with Y-chromosome microdeletion when oligozoospermia (any severity) or NOA is detected. Moreover, we recommend CFTR testing in men with unilateral or bilateral absence of the vas deferens and/or documented seminal vesicle agenesis and/or idiopathic obstructive azoospermia. This is because potential genetic abnormalities are known to be eventually associated with these conditions, and early diagnosis can help couples to receive the most appropriate counseling and a timely adequate management workup [ 69 ]. Supporting evidence: any effort for a more comprehensive characterization is obviously important throughout everyday clinical practice to manage males with primary infertility for whom a cause is not identified and a suitable therapy is unavailable. Thus, men with idiopathic or unexplained infertility require special consideration. In a large same-ethnicity cohort of primary infertile men, 20% and 5% had clinical criteria for idiopathic or unexplained infertility, respectively. Surprisingly, only minimal clinical differences were found in these two groups compared to their fertile counterparts [ 7 ], thus suggesting that other than physical and hormonal characteristics are the leading cause of these two clinical scenarios. In this context, rare monogenic forms of infertility are not routinely evaluated. Thereof, we have recently investigated the unknown potential genetic causes in 99 couples with pure idiopathic MFI by applying variant prioritization to whole exome sequencing [ 98 ]. The ad-hoc manually curated gene library prioritizes genes already known to be associated with more common and rare syndromic and non-syndromic male infertility forms. Hence, twelve monogenic cases (12.1%) were identified in the whole cohort of patients. Of these, three patients had variants related to mild androgen insensitivity syndrome, two in genes related to hypogonadotropic hypogonadism, and six in genes related to spermatogenic failure, while one patient is mutant in PKD1. Implications: there are at least three critical reasons why an accurate assessment of the underlying genetic alterations is essential: I) identification of the cause of infertility can reduce the psychological distress associated with a missed diagnosis; II) in the context of clinical syndromes, potentially serious health problems may go undiagnosed [ 21 ]; and, III) genetic counseling could provide an evaluation of the likelihood of the transmission of the alteration to the progeny. Moreover, recent findings suggest that next-generation sequencing combined with a manually curated pipeline for variant prioritization and classification can uncover a considerable number of Mendelian causes of infertility, with potentially relevant outcomes in men with idiopathic or unexplained infertility cases. Recommendation: MFI is a heterogenous condition, with genetic causes—mostly still to be discovered and with many rare o minor mutated variants to be assigned in terms of significance; thereof, a more refined and extended genetic assessment should be considered specifically in patients with either idiopathic or unexplained infertility since undiscovered alterations may subtend a possible explanation of this conditions [ 98 ]. Indeed, by undertaking a more complete and comprehensive diagnostic work-up, it is possible to detect at least one underlying cause of MFI in four out of five of these individuals. In this respect, conventional diagnostic tests would have identified this group of males as having idiopathic infertility [ 8 ]. Overall, genome sequencing and a more comprehensive investigation is becoming increasingly clinically relevant for genetic diagnosis of idiopathic infertility, despite many infertile patients carry variants of uncertain significance [ 98 99 100 101 102 ]. Supporting evidence: the use of scrotal color Doppler ultrasound (US) has been shown to provide valuable supporting evidence for the clinical management of varicocele. While physical examination remains a primary method, there are situations where it may be unreliable in assessing venous reflux and diameter. Studies have demonstrated that scrotal Doppler US is particularly useful in such cases, allowing for more accurate and reliable assessments [ 103 104 ]. The ultrasound can effectively detect the presence of venous reflux and measure the venous diameter, providing definitive evidence that aids in the decision-making process for treatment [ 103 ]. Moreover, scrotal Doppler US plays a crucial role in the evaluation of testicular masses, particularly in infertile men with an increased risk of testicular cancer. Studies have shown that infertility may be associated with an elevated risk of testicular malignancies, making the accurate assessment of testicular masses of paramount importance [ 3 50 62 ]. Scrotal Doppler US offers a non-invasive and highly sensitive method to detect and characterize testicular lesions, enabling early identification and differentiation of benign masses from potentially malignant ones [ 50 ]. Implications: based on current literature, scrotal Doppler US has critical clinical implications, aiding in more accurate assessments for varicocele management and early detection of testicular masses in infertile men at higher risk of cancer. It allows targeted interventions and improved treatment outcomes. Recommendation: we recommend the widespread adoption of scrotal US as an essential tool over the clinical management of each infertile man, mostly in the setting of diagnosing varicocele presence and severity and the evaluation of potential testicular masses. The supporting evidence demonstrates its value in providing more accurate assessments and definitive evidence for treatment decisions [ 103 104 ]. Moreover, for infertile men at increased risk of testicular cancer, scrotal US offers a non-invasive and highly sensitive method for early detection and differentiation of benign and potentially malignant masses, potentially leading to improved treatment outcomes and fertility preservation. Supporting evidence: for patients manifesting a low seminal volume, acidic pH, and severe oligozoospermia or azoospermia, medical practitioners often suspect obstruction [ 11 ]. In these cases, scrotal US and transrectal US have proven clinically valuable for detecting absence of the vas deferens. More in details, transrectal US is particularly useful to assess the presence or absence of the vas deferens and/or seminal vesicles [ 46 ]. Implications: transrectal US plays a significant role in assessing obstructive azoospermia secondary to vas deference absence or anomalies related to the obstruction of the ejaculatory ducts. This includes conditions such as ejaculatory duct cysts, seminal vesicular dilatation, or hypoplasia/atrophy. Recommendation: we recommend the use of transrectal US when obstructive azoospermia and related anomalies are suspected.

Infertility affects a significant portion of couples during their reproductive years, with nearly 15% experiencing difficulties in conceiving despite regular and unprotected sexual intercourse [ 1 ]. Male factor infertility (MFI) accounts for approximately 20% of cases, while a combination of male and female factors contributes to 30% of infertility cases [ 1 ]. With up to 50% of infertility cases associated with male factors, clearly emerges how critical is to establish an accurate diagnostic path even in men and implement the most appropriate and tailored treatments [ 2 ]. Likewise, compelling evidence have highlighted the need for a more comprehensive assessment of both pure and mixed MFI, enabling personalized management strategies over routine clinical settings [ 1 3 4 5 6 ]. Additionally, data keep outlining that a substantial proportion of men seeking primary couple's infertility care due to pure male factors present clinical criteria suggestive of either idiopathic or unexplained infertility, thus significantly limiting subsequent therapeutic options [ 7 ]. These findings unveil the urgent need for comprehensive investigations that can bridge the existing knowledge gap and facilitate the development of rational therapeutic protocols specifically tailored to the underlying etiologic factors in the context of MFI in the everyday clinical practice. Indeed, our data suggest that with a more thorough diagnostic work-up, it is possible to identify at least one underlying cause of MFI in 4 out of 5 infertile men [ 8 ]; as a consequence, a more suitable therapeutic approach could be developed on an individual basis. However, men with a less severe phenotype pose a clinical challenge in terms of establishing a potential etiologic factor. In the light of these considerations, this position paper aims to highlight the diagnostic management perspective of the Andrology team at the IRCCS Ospedale San Raffaele and advocates for the adoption of a comprehensive and individualized approach to MFI work-up, while addressing the evolving challenges in the real-life everyday clinical practice ( Fig. 1 ).

Despite existing European and American guidelines which adequately and effectively guide physicians in the everyday clinical approach to couple’s infertility, MFI remains a significant issue for clinicians in the everyday clinical practice. Recent studies emphasize the need for detailed assessments of men with MFI to develop personalized treatment plans. The high rates of unexplained infertility among MFI men points to the urgent need for thorough investigations to detect specific causes and address better tailored and more effective treatment plans. This position paper from the Andrology team at IRCCS Ospedale San Raffaele proposes a comprehensive and personalized approach to MFI, aiming to address ongoing challenges in clinical practice, bridge knowledge gaps, and customize diagnostic workups to improve patient outcomes in the context of MFI (we would humbly think above all of the most complex and severe ones).