Chromosome studies in 496 infertile males with a sperm count below 10 million/ml

Summary Chromosome studies were performed on 106 men with azoospermia and 390 men with oligozoospermia (consistant sperm count below 10 million/ml). Constitutional chromosome abnormalities were found in 14.1% of the azoospermia group and in 5.1% of the oligozoospermia group. An overall incidence of 7.1% constitutional abnormalities indicates that this criterion of selection may be advisable for routine chromosome analysis of infertile men. A reduction of 25% in the workload increases the yield of chromosome abnormalities in the group of infertile men to 10–14 times above that expected in the normal population.     

A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules.

For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children. Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection. This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions. To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous tubules was conducted. Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells. A cell suspension was obtained by enzymatic digestion, and cells were cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (interference-contrast microscopy). For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 microm, and observed unstained by Nomarski optics. This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis. In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules. Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.     

Feasibility of global gene expression analysis in testicular biopsies from infertile men

Numerous studies have documented the use of microarray analysis to identify patterns of global gene expression that distinguish normal development from that of the diseased state. Yet, there are no reports that compare global gene expression in the fertile and infertile human testis. Here, we report an initial study of global gene expression in testicular biopsies from several men with different infertility phenotypes. We found that microarray analysis of small biopsy samples was suitable for profiling expression of genes known to function in germ cell development and also identified expression of novel genes. Since it is now common for infertile men with spermatogenic failure to use intracytoplasmic sperm injection (ICSI) to achieve biological paternity, we hypothesize that molecular screening of testicular biopsies with microarrays may be suitable: (1) to categorize the molecular phenoytpes of infertile testes in a manner similar to standard morphologic analysis and (2) to initiate larger studies of gene expression in the infertile testes that may identify genetic signatures from biopsies that allow prediction of outcomes.     

Screening for carcinoma in situ of the contralateral testis in patients with germinal testicular cancer.

Two hundred and fifty biopsy specimens from the contralateral testis in patients with unilateral germinal testicular cancer were analysed by light microscopy for carcinoma-in-situ changes. Changes were found in 13 (5.2%) patients. One-third of patients with an atrophic contralateral testis (volume less than or equal to 12 ml) and one-fifth of patients with a history of cryptorchidism had changes in the remaining testis. In the present series 85% of cases with carcinoma-in-situ changes would have been diagnosed if the one-fifth of the patients having an atrophic testis or a history of cryptorchidism or both had been screened. Since the natural course of carcinoma in situ in the contralateral testis of patients with germinal testicular cancer has not been established, the patients are being re-evaluated frequently. To date two patients with carcinoma in situ have developed a second cancer.     

Cryptorchidie et température testiculaire

The testis migrates to a scrotal location before birth. This physiological descent is associated with a reduction in the temperature of the testicular environment since the temperature of the scrotal cavity is lower than that of the body one. This leads to the etablishment of a themperature gradient between the testis and the body which already exists in prepubertal boys. In cases of testicular maldescent (cryptorchidism), the temperature of the testis in its cryptorchid location is much higher than that of the normally descended contrlateral testis. However, there are no data obtained from human studies to establish wether the increased temperature of a cryptorchid testis is responsible for the spermatogenic perturbations typically observed. Nor do we know wether the relocation of a cryptorchid testis to the scrotum permits re-establishment of a normal testicular temperature. Adult men with a history of cryptorchidism constitute about 10% of infertile men, and among these previously cryptorchid infertile men 45% have an abnormally elevated scrotal temperature. This abnormal increase in scrotal temperature is a negative risk factor for fertility: these men have smaller testicular volumes, a more severely impaired spermatogenesis and a higher prevalence of primary infertility than previously cryptorchid infertile than previously cryptorchid infertile men with normal scrotal temperature. However, data provided until now do not allow to know whether elevated temperature is due to the decreased testicular size (hypotrophy) or is a consequence of cryptorchidism per se.     

Immunologically reactive albumin-like protein in human testis and seminal plasma

An immunologically reactive albumin-like protein (albumin) was localized, by an immunostaining technique, in the testis of infertile men (normal spermatogenesis, obstructive azoospermia) at the level of the Sertoli cells and in some cells of the germinal epithelium (secondary spermatocytes and early spermatids). No positive reaction was detectable in prepubertal testis. In vasectomized men, mean seminal albumin values were drastically reduced (by about 80%) in comparison to fertile controls, indicating a probable testicular origin. Mean seminal albumin values were also decreased in patients affected by azoospermia due to a seminiferous tubular lesion (about 40%) and in oligozoospermic patients (about 30%). In the same seminal samples transferrin, an index of Sertoli cell function, was also measured. Albumin and transferrin results were well correlated in the seminal plasma of each group (with the exception of vasectomized subjects), including a group of men with abnormally high concentrations of seminal transferrin. A weak correlation was found between seminal albumin and sperm count. We suggest that the presence of albumin in the human adult testis and in seminal plasma could be related to its ability to transport androgens.     

Intérêt de la ponction épididymaire et de la biopsie testiculaire systématique dans la prise en charge de l’azoospermie obstructive

Introduction

In obstructive azoospermia (OA), even if spermatozoa recovery rate are high, pregnancy rates could be lower as expected. When almost surgeons stop if they could find motile spermatozoa in the epididymis after microsurgical epididymal sperm aspiration (MESA), in our center, we add systematically a testicular biopsy with testicular sperm extraction (TESE). What are our sperm extraction rates in MESA or TESE? Are pregnancy and miscarriage rates different regarding the sperm origin?

Material and methods

A retrospective study including 48 infertile couples with ICSI because of OA. Between 2003 and 2011, each patient had a complete aetiological exploration and a surgery with the association of MESA and TESE. ICSI were asynchronous. Each time it was possible, ICSI was realized first with epididymal spermatozoa.

Results

For 48 couples, 99 ICSI were realized. Fifteen couples had 24 ICSI-TESE because no spermatozoon was found in MESA. Eleven couples had 20 ICSI-TESE because of bad quality of sperm recovered with MESA. Twenty-two couples had 22 ICSI-MESA in first intention. If failed, 11 couples had continued with 12 ICSI-MESA and 10 with 20 ICSI-TESE. Although the number of injected oocytes (7,1±4,1 vs 6,9 ±3,6 P: 0,8) and embryos (4,5±3,0 vs 4,7±2,7; P: 0,7) were not significantly different in the two ICSI groups, the number of top quality embryos (2,4±1,9 vs 3,6±2,0 P: 0,005) and frozen embryos (0,9±1,8 vs 1,7±1,9 P: 0,04) were higher in the ICSI-TESE group. Pregnancy rate per punction (58,5% vs 26,5%, P: 0,002) was higher when testicular spermatozoa were used.

Conclusion

Our approach is original with the systematic association of MESA and TESE for each OA man, when others stop surgery when they can find spermatozoa with MESA. We found that more than the half of epididymal explorations were not useful because negative or of bad quality. Embryo quality and per punction pregnancy rate were better with testicular spermatozoa. Association of MESA and TESE could improve the management of these infertile men without exposing them to an over surgical risk.     

An ADP-ribosyltransferase 3 (ART3) variant is associated with reduced sperm counts in Czech males: case/control association study replicating results from the Japanese population

OBJECTIVES: In about 50% of male infertility the underlying pathogenesis remains unknown. A recent Japanese study provided evidence that the rs6836703: G>A single-nucleotide polymorphism (SNP) from the ADP-ribosyltransferase 3 (ART3) gene is significantly associated with non-obstructive azoospermia. However, the functional significance of this association is unknown and replication studies in unrelated populations are thus necessary. Design: In this study, 257 fertile Czech controls of proven paternity and 98 sub-/infertile patients selected according to stringent exclusion / inclusion criteria were genotyped by High Resolution Melting (HRM) of small amplicons. Setting: This study was performed at University Hospital Motol - Laboratory of reproductive genetics using routinely analyzed cases. Results: Significant differences in allele distribution between fertile and sub-/infertile men were found (OR=1.78, 95% CI: 1.17-2.70; p=0.007). Following sub-stratification of cases according to their sperm counts we found that observed differences in allele distributions were increased in oligozoospermic men with sperm counts of <15 million sperm/mL (OR=1.98, 95% CI: 1.28-3.07; p=0.002). This difference was also reflected in genotype distributions between fertile and sub-/infertile men (p=0.008), and fertile versus oligozoospermic men (p=0.004). Conclusions: Our study serves as a first replication of the original Japanese report and opens new avenues of research. Compared to the Japanese patient cohort, we provided evidence that the analyzed ART3 variant is associated with quantitative impairment of spermatogenesis.     

DAZL 260A > G and MTHFR 677C > T variants in sperm DNA of infertile Indian men

DAZL (deleted in azoospermia-like) 260A > G and MTHFR (methylene tetrahydrofolate reductase) 677C > T are two important autosomal variants associated with impaired spermatogenesis. In this study, we investigated DAZL 260A > G and MTHFR 677C > T variants in sperm DNA and their frequency in oligozoospermic infertile men of Indian origin. The study on sperm DNA was performed, since it is more prone to oxidative stress-induced damage and mutation. One hundred oligozoopsermic infertile men having normal chromosomal complement with intact Y chromosome and 100 age- and ethnically-matched fertile controls were investigated for these variants in their sperm genome. Spermatozoa were separated by gradient centrifugation and DNA was isolated and analyzed for the single nucleotide polymorphisms (SNPs) by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed no significant differences in the frequency of DAZL AG (P = 0.58) and MTHFR CT (P = 0.44) between oligozoospermic infertile men and controls. However, 8% (8/100) oligozoospermic infertile men harbored both the variants and showed significantly (P < 0.0001) lower sperm count (3.28 +/- 1.1 vs 12.50 +/- 4.09) compared to infertile men with either of the single variant. None of the fertile controls showed the presence of the both variants. In conclusion, the combined effect of both DAZL 260A > G and MTHFR 677C > T variants may have role in compromised sperm count. However, further studies are required to find the pathological role of these combined variants in male infertility.     

The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men

We evaluated the effects of combined conventional treatment, oral antioxidants (N-acetyl-cysteine or vitamins A plus E) and essential fatty acids (FA) on sperm biology in an open prospective study including 27 infertile men. The evaluation included sperm characteristics, seminal reactive oxygen species (ROS), FA of sperm membrane phospholipids, sperm oxidized DNA (8-OH-dG), and induced acrosome reaction (AR). Treatment did not improve sperm motility and morphology, nor decrease the concentration of round cells and white blood cells in semen. Sperm concentration increased in oligozoospermic men (7.4+/-1.3 to 12.5+/-1.9 million/ml). Treatment significantly reduced ROS (mean+/-SEM) (775.3+/-372.2 to 150.3+/-105.2 x 10(3)counts/10 second) and 8-OH-dG (45.3+/-10.4 to 16. 8+/-3.3 fmol/microg DNA). Treatment increased the AR (55.1+/-2.2 to 71.6+/-2.2%), the proportion of polyunsaturated FA of the phospholipids, and sperm membrane fluidity. The overall pregnancy rate was 4.5% in 134 months. The per month pregnancy rate tended to be higher in partners of (ex)-smokers (7.15%, n=14,70 months) than in never-smokers (1.6%, n=13,64 months) (OR:4.57, 95% Cl:0.55-38.1).     

Carcinoma in situ of the testis: aneuploid cells in semen

The content of cellular DNA in ejaculates from eight patients with carcinoma in situ of the testis and 26 controls without evidence of testicular neoplasia was studied by flow cytometry. An aneuploid cell population with a ploidy value similar to that found for carcinoma in situ cells was detected in seminal fluid from four of the eight men with carcinoma in situ but in none of the controls. One year after orchidectomy or local irradiation in these four men no aneuploid cells were found in the semen.These findings show that a detectable proportion of malignant germ cells may be released into the seminal fluid of patients with carcinoma in situ of the testis. Analysis of seminal fluid may therefore aid in screening for early neoplasia of the testis.     

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.     

Qualité du gamète testiculaire

The combination of testicular sperm extraction (TESE) and ICSI has constituted a breakthrough in the treatment of men with nonobstructive azoospermia. However, impaired spermatogenesis raises the question of the quality of testicular spermatozoa obtained by testicular biopsy, when considering the male gamete as the product of a sequential and programmed spermatogenic process. The aim of this paper is: 1) To briefly recall the successive events of sperm protein synthesis; 2) To define the characteristics of sperm cells retrieved from the testis and 3) To evaluate the effect of nuclear anomalies on the outcome of ICSI.     

Risk of testicular cancer in subfertile men: case-control study

ObjectiveTo evaluate the association between subfertility in men and the subsequent risk of testicular cancer.DesignPopulation based case-control study.SettingThe Danish population.ParticipantsCases were identified in the Danish Cancer Registry; controls were randomly selected from the Danish population with the computerised Danish Central Population Register. Men were interviewed by telephone; 514 men with cancer and 720 controls participated.ResultsA reduced risk of testicular cancer was associated with paternity (relative risk 0.63; 95% confidence interval 0.47 to 0.85). In men who before the diagnosis of testicular cancer had a lower number of children than expected on the basis of their age, the relative risk was 1.98 (1.43 to 2.75). There was no corresponding protective effect associated with a higher number of children than expected. The associations were similar for seminoma and non-seminoma and were not influenced by adjustment for potential confounding factors.ConclusionThese data are consistent with the hypothesis that male subfertility and testicular cancer share important aetiological factors.

Key messages

• The incidence of testicular cancer has increased in the past 50 years, and there is some evidence to suggest that sperm quality has decreased in the same period
• It has been hypothesised that common aetiological factors may exist for testicular cancer and for male subfertility
• The association between male subfertility and subsequent risk of testicular cancer is strong and consistent with the hypothesis of a common aetiology
• The association is similar for seminoma and non-seminoma, and it persists when several potentially confounding factors are taken into account

     

Specific expression of VCY2 in human male germ cells and its involvement in the pathogenesis of male infertility

Abnormal spermatogenesis in men with Y-chromosome microdeletions suggests that genes important for spermatogenesis have been removed from these individuals. VCY2 is a testis-specific gene that locates in the most frequently deleted azoospermia factor c region in the Y chromosome. We have raised an antiserum to VCY2 and used it to characterize the localization of VCY2 in human testis. Using Western blot analysis, the affinity-purified polyclonal VCY2 antibody gave a single specific band of approximately 14 kDa in size, corresponding to the expected size of VCY2 in all the collected human testicular biopsy specimens with normal spermatogenesis. Immunohistochemical analyses showed that VCY2 localized to the nuclei of spermatogonia, spermatocytes, and round spermatids, except elongated spermatids. At the ultrastructural level, VCY2 expression was found in the nucleus of human ejaculated spermatozoa. To determine the possible relationship of VCY2 with the pathogenesis of male infertility, we examined a group of infertile men with and without Y-chromosome microdeletions and with known testicular pathology using VCY2 antibody. VCY2 was weakly expressed at the spermatogonia and immunonegative in spermatocytes and round spermatids in testicular biopsy specimens with maturation arrest or hypospermatogenesis. The specific localization of the protein in germ cell nuclei indicates that VCY2 is likely to function in male germ cell development. The impaired expression of VCY2 in infertile men suggests its involvement in the pathogenesis of male infertility.     

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: oligozoospermia

Recently, intracytoplasmic sperm injection (ICSI) has been extremely successful for the treatment of male infertility. However, transmission of cytogenetic defects to offspring is a great concern. There are two types of cytogenetic problems in patients seeking ICSI; one is the transmission of genetic defects from patients with constitutional chromosomal abnormalities and the second is the generation of de novo defects in infertile men. Generally about 5.1% of infertile men have chromosomal abnormalities. Among such infertile men, men with severe spermatogenesis defects, including oligozoospermia and azoospermia, are subjects for ICSI. Therefore it is very important to obtain cytogenetic information in these infertile patients. Furthermore, oligozoospermic men with a normal somatic karyotype also have increased frequencies of sperm chromosome abnormalities. Oligozoospermia is usually associated with other sperm alterations, for example oligoasthenozoospermia, oligoteratozoospemia and oligoasthenoteratozoospermia. In this review, the relationship between sperm concentration and sperm aneuploidy frequencies has been analyzed. The inverse correlation between the frequency of sperm aneuploidy and concentration has been reported in extensive studies. Especially in severe oligozoospermia, a significantly higher frequency of sex chromosome aneuploidy has been observed and this has been corroborated in recent clinical outcome data of ICSI.     

Screening of Y chromosome microdeletion which contains AZF regions in 71 Turkish azoospermic men

Screening of Y chromosome microdeletion which contains AZF regions in 71 turkish azoospermic men: In 71 Turkish men Y chromosome microdeletions have been studied before intracytoplasmic sperm injection (ICSI). DNA samples were amplified with 18 STS primers of the azoospermia factor (AZF) region on the Y chromosome by using multiplex polymerase chain reaction (PCR). Microdeletions were detected in 4 azoospermic men (5.6 %); one with a deletion in the AZFb region, while the 3 others had a large deletion extending over multiple chromosomal regions (AZFb+c+d and AZFa+b+c+d). In the patients with microdeletion, no spermatogenetic activity could be detected in testis biopsies. This result confirms the idea that Y chromosome microdeletion analysis is important in investigating the possibility of finding sperm in testicular sperm extraction (TESE). Therefore, we point out the importance of genetic testing and counselling regarding Y chromosome microdeletion for couples requesting ICSI.     

Experimental mild increase in testicular temperature has drastic,but reversible,effect on sperm aneuploidy in men: A pilot study

In mammals testicular and epididymal temperature increase impairs spermatogenesis. This experimental study investigates the effects of a mild testis temperature increase (i.e. testis temperature remains below core body temperature) on sperm aneuploidy in men. In 5 fertile volunteers a testicular temperature increase was induced by maintaining the testes at suprascrotal position using specially designed underwear for 15 ± 1 h daily for 120 consecutive days. After heating men were followed for next 180 days. A control group (27 men) was recruited. Semen samples were collected before, during and after heating period and analyzed for chromosomes X, Y and 18 for aneuploidy using FISH. A total of 234,038 spermatozoa were studied by FISH. At day 34 of heating, mean sperm aneuploidy values were not modified. From day 34 of heating until day 45 post heating, FISH evaluation was not possible due to the drastic fall of sperm count. At day 45 post-heating total sperm aneuploidy percentage was twice higher than before heating whereas. Sex disomy (sperm XY18), sex chromosome nullisomy (sperm 18) were significantly higher than controls. These effects were completely reversed at 180 days post heat exposure. Conclusion: A mild rise in testicular temperature significantly increases sperm aneuploidies, reflecting an effect on the meiosis stage of spermatogenesis. The effect of heating was reversible and suggests that recovery of aneuploidy to normal values requires at least two cycles of spermatogenesis. Nonetheless, the low number of volunteers was a limitation of this pilot study and warrants further research on larger population.     

Sperm fatty acid composition in subfertile men

INTRODUCTION: The lipid composition of spermatozoa plays an important role for successful fertilization. PATIENTS AND METHODS: In the present study, we analyzed the fatty acid (FA) composition of spermatozoa of normozoospermic, asthenozoospermic, oligozoospermic and oligoasthenozoospermic men. RESULTS: Spermatozoa from asthenozoospermic (P<0.01), oligozoospermic (P<0.05) and oligoasthenozoospermic men (P<0.05) had lower levels of docosahexaenoic acid (22:6w3, DHA) than those from normozoospermic men. In oligozoospermic and asthenozoospermic men, spermatozoa 18:0 content was higher than that of normozoospermics (P<0.01 and P<0.001, respectively). 18:1w9 was higher in oligoasthenozoospermic and oligozoospermic samples when compared with normozoospermic samples (P<0.05 for both). While from the point of view of total w6 FAs there was no significant difference among the groups, the w6/w3 ratio was significantly higher in asthenozoospermic samples than in normozoospermic samples (P<0.05). Monounsaturated fatty acids (MFA) were higher in oligozoospermic samples (P<0.05) than in normozoospermic samples, polyunsaturated fatty acids (PUFA) were lower in asthenozoospermic (P<0.01), oligoasthenozoospermic (P<0.05) and oligozoospermic samples (P<0.05) than in normozoospermic samples. Saturated fatty acids (SFA) were significantly higher in asthenozoospermic (P<0.01) and oligozoospermic samples (P<0.05) compared with normozoospermic samples. In correlation analysis, there were significant positive correlations between DHA with sperm motility (r=0.53), sperm concentration (r=0.36) and normal sperm morphology (r=0.30). In addition, there were significant correlations between PUFA with sperm motility (r=0.50), sperm concentration (r=0.35), and normal sperm morphology (r=0.28), and between w6/w3 with sperm motility (r=-0.47), sperm concentration (r=-0.27), and normal sperm morphology(r=-0.24). DISCUSSION: These suggest that decreased DHA and PUFA, and increased w6/w3 in spermatozoa may be related to infertility in oligo- and/or asthenozoospermic men.