Clinical and laboratory differences between chromosomal and undefined causes of non-obstructive azoospermia: A retrospective study

ABSTRACT BACKGROUND: Knowledge of clinical and laboratory differences between chromosomal and undefined causes aids etiological research on non-obstructive azoospermia. OBJECTIVE: Compare clinical and laboratory differences between men with non-obstructive azoospermia due to chromosomal anomalies versus undefined causes DESIGN AND SETTING: A cross-sectional retrospective study conducted at a public university hospital in Campinas (Brazil) METHODS: All men aged 20–40 years with non-obstructive azoospermia were included in the analysis. RESULTS: The 107 cases included 14 with Klinefelter syndrome (KS) (13%), 1 with mosaic KS, 4 with sex development disorders (2 testicular XX, 1 NR5A1 gene mutation, and 1 mild androgen insensitivity syndrome) (4%), 9 with other non-obstructive azoospermia etiologies (8%), and 79 with undefined causes. The 22 chromosomal anomaly cases (14 KS, 1 mosaic KS, 2 testicular XX, 4 sex chromosome anomalies, and 1 autosomal anomaly) were compared with the 79 undefined cause cases. The KS group had lower average testicular volume, shorter penile length, and lower total testosterone levels but greater height, arm span, serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and gynecomastia frequency (absent in the undefined group and affecting more than half of the KS group). Patients with testicular XX DSD had LH, FSH, and penile length data intermediate between the KS and undefined cause groups, testicular volume similar to the KS group, and other data similar to the undefined group. CONCLUSION: Clinical and laboratory data differentiate men with non-obstructive azoospermia and chromosomal anomalies, particularly KS and testicular XX, from those with undefined causes or other chromosomal anomalies.


INTRODUCTION
Male infertility is defined as the biological inability of a man to induce pregnancy in a fertile woman after unprotected sexual intercourse for at least one year. 1 A study of a North American population revealed that 12% of male individuals aged 15-44 years are infertile. 2 The main factors related to infertility include obesity, infection, neoplasms, cryptorchidism, smoking, varicocele, chromosomal anomalies, sperm duct defects, scrotal exposure to high temperatures, hormonal imbalances, celiac disease, medications, heavy metal poisoning, and exposure to ionizing radiation. [3][4][5] Regarding the genetic causes of male infertility, approximately 15-20% of men with severe non-obstructive azoospermia or oligospermia have microdeletions on the long arm of the Y chromosome (AZFa, b, or c regions) where the spermatogenesis genes are located. 6,7 Some cases of male infertility may also be related to disorders of sex development (DSD), such as Klinefelter syndrome (KS), testicular 46,XX DSD, and disorders related to the synthesis or action of testicular hormones. 8 Currently available DSD cohorts in the literature mostly include pediatric patients, with genital ambiguity being the main reason for referral. 9,10 In contrast, studies of genetic causes of male infertility have mainly focused on chromosomal anomalies and Yq microdeletions. 11,12 In these studies, as well as in the guidelines on male infertility, 3 DSD are not specifically considered a cause.
A recent study by our group of 84 men with non-obstructive infertility ( 13 Considering that about one-fourth of men with severe infertility have chromosomal anomalies and that the clinical and laboratory characteristics of these men, compared to those without chromosomal anomalies, have been insufficiently studied in the literature, it is necessary to verify whether these data can be useful for distinguishing men with chromosomal anomalies from those without a clear cause of their non-obstructive azoospermia.

OBJECTIVE
To compare clinical and laboratory data of men with nonobstructive azoospermia due to chromosomal anomalies versus undefined causes.

Population data
This observational cross-sectional retrospective study was based on a medical record analysis. All included patients received med-

Clinical data
The following clinical data were obtained from the patients'  14 arm span (cm) and ratio to height, body mass index (BMI) in kg/m 2 , penile length (in cm), 15 and testicular volume (in mL).

Laboratory and genetic data
In all cases, a conventional G-banding karyotype study was performed with 400-band resolution and a minimum count of 20 metaphases. In those with normal karyotypes, analysis of a Yq microdeletion was performed using the polymerase chain reaction multiplex technique and 28 molecular markers that mapped the three regions considered the azoospermia locus (AZFa, AZFb, and AZFc).
The following laboratory data were also obtained from the medical records: LH (IU/L), FSH (IU/L), and total testosterone (ng/mL) serum concentrations, karyotype data, Yq microdeletion data, and other cytogenetic or molecular tests.

Statistical analysis
The statistical analysis was performed using SPSS (Statistical Package for the Social Sciences), version 20.0, Chicago, USA applying absolute and relative frequency data using the Mann-Whitney and Fisher tests with a level of significance of P < 0.05.

RESULTS
A total of 150 cases were evaluated, but only 107 met the inclu- Due to the small number of cases in some of these groups, we decided to compare only the data between the 14 patients with KS and 79 patients in the undefined cause group.
There was no statistically significant difference (Mann-Whitney test) between the KS group (n = 14) and the undefined group (n = 79) in parental height (P = 0.37), arm span and height ratio (P = 0.98), or BMI (P = 0.15) ( Table 1). However, a statistically significant difference was observed in mean testicular volume (P < 0.0001), penile length (P < 0.001), and total testosterone level (P < 0.001), all of which were lower in the KS group, as were stature (P < 0.001), difference between the patient's height and mean parental height (P < 0.0001), arm span (P < 0.01), LH serum concentrations (P < 0.0001), and FSH serum concentrations (P < 0.0001), which were all higher in the KS group ( Table 2). A significant difference was observed in the presence of gynecomastia between the KS and undefined groups; it was absent in the undefined cause group and present in more than half of the KS patients ( The data for the remaining cases (other sex chromosomes or autosomal anomalies) did not differ significantly from those of the undefined group.

DISCUSSION
This study determined that approximately 20% of non-obstructive azoospermia cases were associated with chromosomal anomalies or DSD. These results correspond with the percentage reported in the current literature (approximately 15%). 16,17 KS is the most frequent genetic and chromosomal cause of non-obstructive azoospermia. According to Abramsky and Chapple 18 (1997), approximately 3% of male infertility cases are   19,20 Individuals with testicular XX can also be identified among patients with non-obstructive azoospermia. For the most part, these individuals have the SRY gene translocated on one of the X chromosomes, and their phenotype is virtually identical to that of individuals with KS. In such cases, the lack of sperm production is due to the absence of other genes on the Y chromosome. 21 Other autosomal or sex chromosome anomalies can also occur but at a much lower frequency, as observed in the present study.
Cases of XY partial gonadal dysgenesis, combined gonadal dysgenesis, and ovotesticular DSD can also be found among individuals with typical or highly virilized male genitalia, which is only detected during adulthood infertility investigations. 22 Other 46,XY DSD that are not associated with gonadal differentiation disorders may also go undiagnosed during childhood and have infertility as their main complaint. This is what often happens in cases of hypogonadotropic hypogonadism, milder forms of testosterone synthesis defects, and also in androgen receptor mutations with a male phenotype (MAIS) or barely noticeable genital ambiguity, in which there is a reduction in sperm production due to defects in androgenic activity. 23,24 Individuals with 5-alpha-reductase type 2 deficiency may also have more pronounced androgenization of the external genitalia which goes undiagnosed in childhood but present as adulthood infertility due to underdevelopment of the prostate and seminal vesicles. 25 In the present study, apart from the KS and testicular XX cases, 1 case of XY partial gonadal dysgenesis due to the NR5A1 gene mutation and 1 case of MAIS with the AR gene mutation were diagnosed. Therefore, the results of the present study show that observation of clinical and laboratory data is very important for the differentiation of cases of non-obstructive azoospermia with chromosomal anomalies or DSD of other etiologies.
Except for the KS and testicular XX cases, the other sex chromosomes and autosomal anomalies did not exhibit clinical or laboratory differences from cases of undefined cause. Few studies in the literature have shown these results. 26,27 However, compared to undefined cases, KS patients presented with smaller testicular volume, shorter penis length, taller stature, greater arm span, higher serum LH and FSH concentrations, and lower testosterone levels.
Gynecomastia was absent in the undefined cause group and was present in more than half of the patients with KS. In the literature, testicular volumes lower than 6 mL were present in more than 95% of KS cases, 28 the same being true for increased serum gonadotropins. [28][29][30] Decreased serum testosterone levels were present in 63-85% of KS cases and occurred more frequently in the older age group. [28][29][30] Gynecomastia is reportedly present in 38-75% of affected adolescents and adults. 26,28,29 Increased stature occurs in approximately 30% of children and adults with KS. 26,28,31 Smaller penile size is present in 10-25% of children and adults with KS. 26,28 Therefore, although the findings described in the present study are frequent in KS, our data confirm that they are an important tool for differentiating patients with KS from those with other non-chromosomal cases of non-obstructive azoospermia.
The patient with mosaic KS had a laboratory profile similar to that of the undefined group but had clinical features similar to KS with the exception of normal testicular volume. Previous studies demonstrated that it is difficult to pinpoint a specific clinical feature in cases of sex chromosome mosaicism without actually knowing the percentage of each lineage in various tissues, including gonads. 32 Patients with testicular XX DSD (n = 2) exhibited clinical features similar to those of the undefined group except for decreased testicular volume and hypergonadotropic hypogonadism, but these were not as severe as the results seen in the KS group. Similar data have been described in the literature. 21,33,34

CONCLUSION
Despite the fact that this study has all the limitations of retrospective data collection, it provides important clinical information that supports medical investigations in men with non-obstructive infertility from a significant sample evaluated homogeneously by a single clinical service over a 10-year period with karyotyping performed in all cases and Yq microdeletions investigated in all patients with a normal karyotype.
Therefore, two main conclusions may be drawn from this study: first, chromosomal anomalies were the cause of approximately 20% of non-obstructive azoospermia cases. Second, clinical and laboratory differences existed among different non-obstructive azoospermia etiologies, especially undefined, KS, and testicular XX cases. For this reason, the results of this study provide an important information resource that will be very useful for physicians and other healthcare professionals during investigations and requests for complementary tests during the etiological evaluation of non-obstructive azoospermia.