Partial-AZFc deletions in Chilean men with primary spermatogenic impairment: gene dosage and Y-chromosome haplogroups.

PURPOSE: To investigate the association of partial-AZFc deletions in Chilean men with primary spermatogenic failure and their testicular histopathological phenotypes, analyzing the contribution of DAZ dosage, CDY1 copies, and Y-chromosome haplogroups. SUBJECTS AND METHODS: We studied 479 Chilean men: 334 infertile patients with histological examination (233 cases with spermatogenic defects and 101 normal spermatogenesis, obstructive controls, OC), and 145 normozoospermic controls (NC). AZFc subdeletions were detected by single-tagged sequences and single nucleotide variants analysis. DAZ-copy number was quantified by real-time qPCR. Y-chromosome haplogroups (Y-hg) were hierarchically genotyped through 16 biallelic-markers. RESULTS: The prevalence of AZFc-partial deletions was increased in cases (6%) compared with NC (1.4%) (P = 0.035). There was no difference between 143 Sertoli-cell only syndrome, 35 maturation arrest, or 35 mix atrophy patients and controls. However, gr/gr deletions were more frequent in 16 subjects with hypospermatogenesis compared with NC (P = 0.003) and OC (P = 0.013). Y-hg R was the most prevalent (~ 50%), but decreased among gr/gr deletions (21%, P = 0.03). The prevalence of Y-hg M increased in cases versus controls, both in total and non-deleted men (3.9 and 3.7% versus 0.4%, P = 0.009 and P = 0.016, respectively). Among gr/gr deletions, Y-hg H increased compared with non-deleted men (14.3% versus 0.4%, P = 0.0047). CONCLUSION: Partial-AZFc deletions in a Chilean admixed population are associated with secretory azo/oligozoospermia and might have a role in the development of hypospermatogenesis. Low represented haplogroups, Y-hg M and Y-hg H, show an association with the occurrence of spermatogenic failure and gr/gr deletions respectively; however, additional studies are required.

Efficacy of MLPA for detection of Y-chromosome microdeletions in infertile Brazilian patients.

PURPOSE: Worldwide publications follow the gold standard method-the polymerase chain reaction (PCR)-for detecting Y-chromosome microdeletions; however, markers are frequently variable between the studies. Can we detect the deletions by another molecular method with more genomic coverage? The Y chromosome harbors several different genes responsible for testicular development and spermatogenesis, and its repetitive conformation predisposes it to complex rearrangements that have clinical impact. Our aim was to evaluate a molecular diagnostic method, the Multiplex Ligand Probe-dependent Amplification (MLPA), which is also a valuable ancillary method for the identification of deletions, duplications, and rearrangements in a single and faster reaction, leading to a better comprehension of patients' phenotypes, and should be considered a useful tool for detection of Y chromosome deletions. METHODS: This is a study of diagnostic accuracy (transversal prospective study) conducted to investigate Y-chromosome deletions in 84 individuals through PCR and MLPA methods. Forty-three infertile men (azoospermic and oligozoospermic) and 41 controls (40 fertile men and 1 normal karyotyped woman) were analyzed by PCR and MLPA techniques. RESULTS: We diagnosed seven (7) deletions (16.2%) by PCR and 9 with MLPA (21%). In addition, we found five (5) duplications and a suggestive mosaic. CONCLUSION: Our results demonstrate that MLPA technique is valuable in the investigation of microdeletions and microduplications. Besides deletions, duplications can cause instability of chromosome genes, possibly leading to infertility. Both studied techniques provide an advantageous diagnostic strategy, thus enabling a better genetic counseling.

Are specialized sperm function tests clinically useful in planning assisted reproductive technology?

CASE STUDY 40-year-old male patient and 32-year-old female partner, with a history of primary infertility of two years duration. The workup revealed idiopathic mild oligoasthenotheratozoospermia, and no apparent female infertility factors. The couple has failed three intrauterine insemination (IUI) cycles, planning more IUI cycles but also considering in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).

Correlation between Y chromosome microdeletion and male infertility.

Dyszoospermia due to genetic factors is the leading cause of male infertility. To explore the correlation between azoospermia factor (AZF) microdeletion of the Y chromosome and male infertility, we evaluated AZF microdeletion on the long arm of the Y chromosome in 166 infertile males and 50 fertile males using multiplex polymerase chain reactions amplification and gel electrophoresis. The results demonstrated that 28 individuals had varying degrees of microdeletion in the AZF region (16.90%); 12 out of the 76 males with azoospermia and 16 out of the 90 males with oligospermia had AZF microdeletion. AZF microdeletion was not observed in any of the healthy controls. In addition, 53.60% of the AZF microdeletions occurred in the AZFc region. It can be concluded that AZF microdeletion on the long arm of the Y chromosome can result in male spermatogenesis dysfunction. Detection of AZF microdeletion can provide a theoretical basis for genetic counseling, as well as improve the diagnosis and treatment of this disease.

Associations of single nucleotide polymorphisms in the Pygo2 coding sequence with idiopathic oligospermia and azoospermia.

Male infertility is often associated with a decreased sperm count. The Pygo2 gene is expressed in the elongating spermatid during chromatin remodeling; thus impairment in PYGO2 function might lead to spermatogenic arrest, sperm count reduction, and subsequent infertility. The aim of this study was to identify mutations in Pygo2 that might lead to idiopathic oligospermia and azoospermia. DNA was isolated from venous blood from 77 men with normal fertility and 195 men with idiopathic oligospermia or azoospermia. Polymerase chain reaction-sequencing analysis was performed for the three Pygo2 coding regions. Non-synonymous single nucleotide polymorphisms (SNPs) were detected and analyzed using SIFT, Polyphen-2, and Mutation Taster softwares to identify possible changes in protein structure that could affect phenotype. Pygo2 sequencing was successful for 178 patients (30 with mild or moderate oligospermia, 57 with severe oligospermia, and 91 with azoospermia). Three previously reported non-synonymous SNPs were identified in patients with azoospermia or severe oligospermic but not in those with mild or moderate oligozoopermia or normozoospermia. SNPs rs61758740 (M141I) and rs141722381 (N240I) cause the replacement of one hydrophobic or hydrophilic amino acid, respectively, with another, and SNP rs61758741 (K261E) causes the replacement of a basic amino acid with an acidic one. The software predictions demonstrated that SNP rsl41722381 would likely result in disrupted tertiary protein structure and thus could be involved in disease pathogenesis. Overall, this study demonstrated that SNPs in the coding region of Pygo2 might be one of the causative factors in idiopathic oligospermia and azoospermia, resulting in male infertility.

Varicocele and male infertility in Northeast China: Y chromosome microdeletion as an underlying cause.

The prevalence of Y chromosome microdeletions among azoospermic, severe oligozoospermic, moderate oligozoospermic, and mild oligozoospermic patients with varicocele-related and idiopathic infertility shows conflicting data in Asian countries. We aimed to detect this frequency in Northeast China, and investigated spermatogenic defects whether associated with varicocele or Y chromosome microdeletions. All samples underwent a thorough physical examination, semen analysis, and PCR analyses for Y chromosome microdeletions. We randomly selected 150 infertile non-obstructive azoospermic patients with left varicocele (Group 1), 150 idiopathic non-obstructive azoospermic infertility patients (Group 2), 150 infertile severe oligozoospermic patients with left varicocele (Group 3), 150 idiopathic severe oligozoospermic infertility patients (Group 4), 150 infertile moderate oligozoospermic patients with left varicocele (Group 5), 150 idiopathic moderate oligozoospermic infertility patients (Group 6), 150 infertile mild oligozoospermic patients with left varicocele (Group 7), 150 idiopathic mild oligozoospermic infertility patients (Group 8), and 60 healthy unrelated men with proven fertility were recruited as control subjects (Group 9). We observed that our samples from Northeastern China had a higher frequency of microdeletions among the non-obstructive azoospermic individuals with varicocele, as compared with other Asian countries. Furthermore, the spermatogenic defect is due to the underlying Y chromosome microdeletion, and not the varicocele itself. Although varicocele is not the cause of male infertility, it may be associated with male infertility in the Northeastern Chinese population.

Male carriers of balanced reciprocal translocations in Northeast China: sperm count, reproductive performance, and genetic counseling.

Balanced chromosomal translocations in men can cause failure of spermatogenesis owing to meiotic impairment. Male carriers may exhibit normozoospermia, although clinical manifestations can include oligozoospermia or azoospermia, oligozoospermia or normozoospermia. Here, we reported the characteristics of balanced reciprocal translocations in men from northeastern China, and explored the relationship between sperm count and reproductive performance, to enable informed genetic counseling. The frequency of balanced reciprocal translocations was found to be 1.62%. Semen analysis showed that 5.9% of male carriers had azoospermia, 43.1% had oligozoospermia, and 51.0% had normozoospermia. Of the 25 men with a balanced reciprocal translocation and azoospermia or oligozoospermia, chromosome 1 was the most commonly often involved in the translocation. However, in the 26 normozoospermic men with a balanced reciprocal translocation and normozoospermia, chromosome 3 was most commonly implicated. Fifty percent of men with a balanced reciprocal translocation conceived a pregnancy that went to term. Our data suggest that of all chromosomes, chromosomes 1 and 3 are the most commonly involved chromosomes in balanced reciprocal such translocations in northeastern Chinese men. Karyotype analysis should be performed for men with azoospermia, oligozoospermia, and those in couples having suffered recurrent miscarriages. Natural conception should be discussed during genetic counseling for male carriers of balanced chromosomal translocations with normozoospermia.

Histological and hormonal testicular function in oligo/azoospermic infertile men.

We characterised and correlated the histological and hormonal aspects of a cohort of 261 azo/oligozoospermic men, applying a quantitative/qualitative evaluation of testicular tissue and serum and intratesticular hormonal measurements. One hundred and 93 azo/oligozoospermic patients were diagnosed as: complete sertoli cell only syndrome (cSCOS), n = 76; focal SCOS, n = 31; maturation arrest, n = 34; hypospermatogenesis, n = 17; mixed atrophy, n = 25; and severe atrophy, n = 10. Normal spermatogenesis was observed in 68 infertile men (controls). Patients with cSCOS, focal SCOS, mixed and severe atrophy had larger LC/clusters (11.5; 11.0; 10.7; 18.9 LC/cluster) than controls (6 LC/cluster; P < 0.001). cSCOS, focal SCOS, mixed and severe atrophy patients had higher FSH, LH and lower T/LH ratio serum levels than the other groups. Intratesticular testosterone concentrations were higher in tissues with complete or focal SCOS (45.6 ng mg(-1) protein) and mixed atrophy (79.0 ng mg(-1) protein) than normal tissues (20.3 ng mg(-1) protein; P = 0.03 and P = 0.007). Considering all subjects, significant correlations were found between T/LH ratio and Leydig cells/cluster (r = 0.510, P < 0.001), FSH levels (r = -0.692, P < 0.001) and with intratesticular testosterone (r = -0.354, P = 0.001); these correlations follow the pattern of severity of spermatogenic damage. By a thorough histological evaluation, we validate the concept that the severity of spermatogenic impairment is associated with major morphological and functional disturbance of the Leydig cell compartment.

Prognostic value of germ cells in the ejaculate: a case study.

The first approach to assessing male fertility is to study a spermiogram, where special attention is given to sperm count, motility and morphology, while less attention is given to other cells in the ejaculate. Normal spermatogenesis requires a balance between cell death and proliferation; therefore, the number of germ cells (GC) in the ejaculate is less than the number of sperm. We propose a new index for altered spermatogenesis, i.e., the rate GC/sperm. We investigated a patient with oligozoospermia and a GC/sperm ratio greater than one, which indicated that spermatogenesis had been damaged. Complementary cytological tests were employed to characterized GC status: Papanicolaou stain, transmission electronic microscopy (TEM), vitality test, AgNOR and TUNEL assay. We also correlated cell morphology with ultrastructure studies that showed apoptosis. Nuclear apoptosis is characterized by vacuolization, misshapen nuclei, and "half moon," dispersed, uncondensed, disrupted and smudged chromatin. Cytoplasmic apoptosis is characterized by vacuolization, cytoplasmic protrusions, lamellar bodies, and swollen endoplasmic reticulum and mitochondria. To date, only testicular biopsy has been used to diagnose complete or incomplete testicular arrest. Our investigation is the first to determine a cytological feature in semen samples that could be used as a biological marker for abnormal spermatogenesis and for predicting the transition from oligospermia to azoospermia.

Multinucleation of spermatozoa and spermatids in infertile men chronically exposed to carbofuran.

OBJECTIVE: To report the findings encountered in semen samples coming from two infertile men chronically exposed to carbofuran. METHODS: Semen samples were collected and analyzed as recommended by the World Health Organization. A morphological analysis was carried out by light microscopy. RESULTS: Seminal analysis revealed in the first case a total concentration of 42 million spermatozoa/mL with 17% motility and 20% normal shape. The second patient presented a total concentration of 5 million spermatozoa/mL with 6% motility and 2% normal shape. The patients presented a similar percentage of binucleated spermatozoa (28 and 26%) and of multinucleated spermatids (10 and 6%). CONCLUSION: To our knowledge, this is the first time that alterations in semen samples of men exposed to carbofuran are reported. More research in this area is needed to make conclusions on the subject.

Tratamiento con ácido fólico y zinc en hombres subfértiles: servicio de fertilidad Maternidad "Concepción Palacios" / Treatment with folic acid and zinc in subfertil men: service of fertility Maternidad “Concepcion Palacios”

Estudiar el efecto del tratamiento con ácido fólico y zinc, en pacientes masculinos subfértiles, con diagnóstico de astenospermia, oligospermia y/o teratospermia. Estudio prospectivo y descriptivo. Se administró tratamiento con ácido fólico (55 mg) y zinc (550 mg) diarios, por un período de 90 días, a pacientes con alteración de la motilidad, concentración y/o morfología espermática. Servicio de Fertilidad. Maternidad “Concepción Palacios ”. Caracas. Después de 90 días de tratamiento, la motilidad total aumento de 44,37 por ciento a 55,22 por ciento, (P=,0002). Las formas espermáticas normales pasaron de 54,11 por ciento a 55,46 por ciento (P=,0001) y las formas anormales disminuyeron e 44,29 por ciento a 4,25 (P=,0001). El tratamiento con ácido fólico y zinc, mejora significativamente la motilidad total y la morfología espermática, en el hombre sub-fértil.

The asynaptic chromatin in spermatocytes of translocation carriers contains the histone variant gamma-H2AX and associates with the XY body.

BACKGROUND: The close apposition of multivalents with the XY body has been repeatedly described in heterozygous carriers of chromosomal rearrangements. Because in many of these carriers spermatogenesis is deeply disturbed at the spermatocyte level, the association of autosomal chromatin with the XY body may impair the spermatocyte life. METHODS: Testicular biopsies from three men carriers of three different chromosomal rearrangements have been analysed by electron microscopy (EM) and immunolocalization of meiotic proteins. RESULTS: There is an ordered transition from isolated multivalents at early pachytene to XY body association in late pachytene, as shown in a carrier of a rob t(13;14) translocation by EM and in a reciprocal translocation t(9;14) carrier by immunofluorescence. The non-synapsed ends of the quadrivalent show BRCA1 located on the axes and the variant histone gamma-H2AX located on the chromatin. The area covered by gamma-H2AX increases with the association of the asynaptic ends with the XY body in the t(9;14) carrier, and the area covered with gamma-H2AX in the t(Y;15) carrier is larger than that of the XY body of controls. CONCLUSIONS: The affinity between the inactive XY body and asynaptic regions of multivalents is given a material basis, and transcriptional inactivation is probably shared by these two chromatin types.

Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia.

OBJECTIVE: To verify if the increase in sperm apoptotic DNA fragmentation during cryopreservation is greater in oligozoospermic patients than in normozoospermic controls. DESIGN: Controlled prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Forty-seven patients with oligozoospermia (concentration <10 x 10(6) sperm/mL) and 30 normozoospermic men. INTERVENTION(S): Sperm cryopreservation using a standard protocol with a test-yolk buffer, glycerol as the cryoprotectant. MAIN OUTCOME MEASURE(S): Rate of apoptotic sperm DNA fragmentation as assessed by the terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nickend labeling (TUNEL) assay, graded in apoptotic or nonapoptotic, before and after cryopreservation. RESULT(S): An increase in apoptotic DNA fragmentation was observed in all the groups, regardless of sperm concentration. Normozoospermic men presented a smaller rate of apoptotic DNA fragmentation than oligozoospermic patients, both in pre- and postcryopreservation samples. The increase in DNA fragmentation was similar in both groups. CONCLUSION(S): Cryopreservation induces apoptotic sperm DNA fragmentation in men, regardless of sperm concentration. Men with oligozoospermia present with higher precryopreservation and postcryopreservation apoptotic sperm DNA fragmentation. The increase in DNA fragmentation is similar in both groups.

Induction of spermatogenesis in azoospermic men after varicocelectomy repair: an update.

OBJECTIVE: To assess the treatment outcome after varicocele repair in azoospermic men and to correlate this outcome with the testicular histology patterns. DESIGN: Prospective study. SETTING: Academic medical centers. PATIENT(S): Medical records of 27 azoospermic men, who underwent testis biopsy and microsurgical repair of clinical varicocele between July 1999 and May 2004, were reviewed. INTERVENTION(S): Twenty-seven azoospermic men underwent testis biopsy and microsurgical repair of clinical varicocele. All patients had at least two semen analyses showing azoospermia taken before the surgery and two semen analyses postoperatively. MAIN OUTCOME MEASURE(S): Semen analysis after varicocelectomy. RESULT(S): Hypospermatogenesis was identified in 9, maturation arrest in 8, and germ cell aplasia in 10 men. Induction of spermatogenesis was achieved in nine men (33.3%). Of these, four had germ cell aplasia, three had maturation arrest, and two had hypospermatogenesis. The improvement in sperm concentration and motility ranged from 1.2 x 10(6)/mL to 8.9 x 10(6)/mL, and from 24% to 75.7%, respectively. Of these nine patients with improvement in semen quality, five relapsed into azoospermia 6 months after the recovery of spermatogenesis (four germ cell aplasia and one maturation arrest). One patient with maturation arrest established pregnancy. CONCLUSION(S): Azoospermic patients may have an improvement in semen quality after varicocelectomy. Semen samples may be cryopreserved after an initial improvement after varicocelectomy.

Recovery of spermatogenesis after microsurgical subinguinal varicocele repair in azoospermic men based on testicular histology

OBJECTIVE: Analyze whether testicular histologic patterns from a group of azoospermic men with varicocele is predictive of treatment outcome after subinguinal microsurgical varicocele repair. MATERIALS AND METHODS: Seventeen azoospermic men underwent bilateral open single testis biopsy and microsurgical subinguinal repair of clinical varicoceles. RESULTS: Histopathology of testicular biopsies revealed hypospermatogenesis (HYPO) in 6 men, maturation arrest (MA) in 5, and Sertoli cell-only (SCO) in 6. Overall, presence of spermatozoa in the ejaculates was achieved in 47 percent (8/17) of men after varicocele repair, but only 35 percent (6/17) of them had motile sperm in their ejaculates. Only men with testicular histology revealing HYPO (5/6) or maturation arrest (3/5) had improvement after surgery. Median (25 percent - 75 percent percentile) postoperative motile sperm count for both groups were 0.9 X 106/mL (0.1-1.8 X 106/mL) and 0.7 X 106/mL (0.1-1.1), respectively (p = 0.87). The mean time for appearance of spermatozoa in the ejaculates was 5 months (3 to 9 months). One (HYPO) of 8 (12.5 percent) men who improved after surgery contributed to an unassisted pregnancy. Postoperative testicular biopsies obtained from patients who had no improvement after surgery revealed that testicular histology diagnosis remained unchanged. Successful testicular sperm retrieval for intracytoplasmic sperm injection (ICSI) was achieved in 4 of 9 (44.4 percent) individuals who did not improve after surgery, including 1 man with testicular histology exhibiting SCO. CONCLUSION: Microsurgical varicocele repair in nonobstructive azoospermic men with clinical varicoceles can result in sperm appearance in the ejaculate when hypospermatogenesis or maturation arrest is found on testicular histology diagnosis.

Successful birth after injection of frozen human oocytes with frozen epididymal spermatozoa.

A couple (female 31, male 42 years old) with infertility due to obstructive azoospermy returned to the clinic in order to attempt pregnancy using their frozen oocytes and epididymal sperm cells, which had been cryopreserved at the time of a previous IVF attempt. Two days before the scheduled transfer, eight oocytes were thawed; 5/8 (63%) oocytes survived and 4/5 (80%) oocytes fertilized after intracytoplasmic sperm injection (ICSI) with the previously frozen epididymal spermatozoa. All four fertilized ova cleaved (100%). On day 2 after thawing, four embryos were transferred; three with two cells (grade II) and one with three cells (grade III). Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation, and the patient was delivered by Caesarean section at 40 weeks of gestation; the baby boy weighed 3025 g, and measured 51 cm, with Apgar of 10 in the 1st and 5th min. The cryopreservation and warming protocol used for this study yielded very favourable results, comparing well with reports in the literature. This case report demonstrates that it is possible to obtain high rates of oocyte survival following thawing and high rates of fertilization after ICSI, with viable development of the resulting embryos.

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients.

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis--Hypo, spermatogenic maturation arrest--MA, Sertoli cell only--SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 +/- 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8-20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33% of the procedures in patients with MA, 50% in patients with Hypo and 40% of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval.

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis-Hypo, spermatogenic maturation arrest-MA, Sertoli cell only-SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 n 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8 - 20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33 percent of the procedures in patients with MA, 50 percent in patients with Hypo and 40 percent of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval.