New insights into the role of the Brdt protein in the regulation of development and spermatogenesis in the mouse.

The bromodomain testis-specific (BRDT) protein belongs to the bromodomain extra-terminal (BET) family of proteins. It serves as a transcriptional regulator of gene expression during spermatogenesis, and is an essential factor for the normal spermatogenesis process. In this study, we characterized mice of several age groups who lacked the Brdt gene. The testes of Brdt mutant mice aged 8 weeks exhibited complete spermatocyte maturation arrest with a significantly increased number of apoptotic cells. The weights of the testes and accessory glands as well as the testosterone levels of the mutant mice were significantly lower compared to the normal mice. The mutant mice had delayed puberty, with normal levels of testosterone and accessory gland weights at the age of 14 and 28 weeks. The testes of the mutant mice at older ages also exhibited round spermatids. The presence of the BRDT protein was identified in the mice pituitary gland. Microarray analysis of mice pituitaries showed that 28 genes were down-regulated while 26 genes were up-regulated in the absence of the Brdt gene. Our results suggest that in addition to its critical role in the spermatogenesis process, the BRDT protein is also responsible for scheduling male puberty by regulation of the pituitary-gonad axis.

Thawed human sperm quality is influenced by the volume of the cryopreserved specimen.

OBJECTIVE: To test the effect of sperm specimen volume in the freezing-thawing process on specimen quality. DESIGN: Experimental prospective study. SETTING: Tertiary academic medical center. PATIENT(S): Fifty high-quality sperm donors donated ∼3 times each. Sperm samples were split into two aliquots and frozen in volumes of 0.25 mL and 0.5 mL. INTERVENTION(S): Semen analyses. MAIN OUTCOME MEASURE(S): Eight sperm quality parameters of thawed specimens. RESULT(S): Thawed 0.5-mL specimens had a higher percentage of motility and viability, progressive motility concentration, percentage of cells with high mitochondrial membrane potential, and intact chromatin compared with 0.25-mL specimens. Although there were fewer cells with intact acrosomes in the 0.5-mL thawed samples, they had a similar ability to respond to ionophore by acrosome reaction as the 0.25-mL specimens. Both groups had similar percentages of cells with oxidative stress and numbers of cells that bound to the zona pellucida. The remaining air volume in the straw and freezing medium composition had a minimal effect on tested parameters. CONCLUSION(S): Better quality thawed human sperm was achieved after cryopreservation of high volumes compared with low volumes of specimens. Air volume in the straw had no influence on specimen quality.

Preservation of sperm of cancer patients: extent of use and pregnancy outcome in a tertiary infertility center.

Sperm cryopreservation is the best modality to ensure future fertility for males diagnosed with cancer. The extent to which cryopreserved sperm is actually used for impregnation, the fertility treatment options that are available and the success rates of these treatments have not been investigated in depth. The medical records of 682 patients who cryopreserved sperm cells due to cancer treatment were analyzed. Seventy of these patients withdrew their frozen sperm for fertility treatments over a 20-year period (most within the first 4 years after cryopreservation). Sperm quality of different malignancies and outcomes of assisted reproduction treatment (ART) for pregnancy achievement in relation to the type of treatment and the type of malignancy were evaluated. The results showed that the rate of using cryo-thawed sperm from cancer patients for fertility treatments in our unit was 10.3%. Sperm quality indices differed between different types of malignancies, with the poorest quality measured in testicular cancer. Conception was achieved in 46 of the 184 ART cycles (25%), and resulted in 36 deliveries. The use of intracytoplasmic sperm injection (ICSI) methodology yielded a significantly higher pregnancy rate (37.4%) than intrauterine insemination (IUI; 11.5%) and was similar to other groups of infertile couples using these modalities. In vitro fertilization (IVF) failed to produce pregnancies. In conclusion, the rate of use of cryopresseved sperm in cancer patients is relatively low (10.3%). Achievement of pregnancies by ICSI presents the best option but when there are enough stored sperm samples and adequate quality, IUI can be employed. Cryopreservation is nevertheless the best option to preserve future fertility potential and hope for cancer patients.

Genetic and physiological study of morphologically abnormal human zona pellucida.

OBJECTIVE: To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance. STUDY DESIGN: The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed. RESULTS: No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3. CONCLUSIONS: The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.

Screening for partial AZFa microdeletions in the Y chromosome of infertile men: is it of clinical relevance?

OBJECTIVE: To evaluate the frequency of complete and partial AZFa Y-chromosome microdeletions among infertile Israeli men. To review the published frequencies and histologic findings of AZFa deletions. DESIGN: Retrospective study. SETTING: Academic medical center. PATIENT(S): A total of 1,260 infertile Israeli men. Literature review (2000-2010) of reports on men with AZFa deletions and their testicular findings. INTERVENTION(S): The DNA of 1,260 infertile men was evaluated for AZF microdeletions. The DNA of 657 of them with undetected microdeletions was analyzed for partial AZFa deletion in the USP9Y and DDX3Y genes using sequence-tagged sites beyond EAA/EMQN recommendations. MAIN OUTCOME MEASURE(S): The frequency of complete and partial AZFa microdeletions. Availability of sperm cells for intracytoplasmic sperm injection in men with complete/partial microdeletions. RESULT(S): Two men had complete AZFa deletion (a frequency of 0.28% among nonobstructive azoospermic men). None had partial AZFa deletions. CONCLUSION(S): The likelihood of finding sperm cells in men with complete AZFa deletions is negligible. Complete AZFa deletion is rare and usually associated with azoospermia and absence of sperm cells in testicular tissue. The low frequency of partial AZFa deletions and the inconsistent prospects for spermatogenesis reported in the literature question the need for routine assessment of microdeletions in genes, such as USP9Y or DDX3Y.

Freezability and semen parameters in candidates of sperm bank donors: 1992-2010.

There has been considerable concern worldwide about possible semen quality deterioration over the last 2 decades. The aim of this study was to evaluate freezability and semen quality of healthy young males during the years 1992-2010. A total of 1211 young (20-32 years old) candidates for sperm bank donation were recruited into the study with no exclusion criteria. They were instructed to observe 2 to 3 days of abstinence from sexual activity, and most of them supplied 2 specimens each. Average values of the various semen parameters, including freezing survival, were calculated for each participant. The change in different semen parameters over years, according to yearly and monthly average temperatures, was evaluated by SAS PROC SURVEYREG analysis. During that period, there were significant increases in motility and vitality percentages, as well as in the percentage of thawed sperm motility. The parameters of volume, concentration, normal morphology, total count, and total motile count showed a significant decrease with years (P < .01). The significant increase in average yearly temperature (P < .004) had limited, nonsignificant association with any of the semen variables. However, average monthly temperature contributed significantly to the trend of semen quality parameters (ie, specimen volume, concentration, percentage of normal morphology, and thawed motility). To the best of our knowledge, this is the first demonstration of the occurrence of an improvement in percent thawed motility over the years, and its significance lies in enabling a higher proportion of sperm bank candidates to be suitable for donation. It is suggested that the global warming phenomenon might have only partial contribution to semen variable changes over the years.

Expression of BET genes in testis of men with different spermatogenic impairments.

OBJECTIVE: To characterize the BET gene expression in human testis with spermatogenetic impairments; to examine BRDT protein expression in testis and semen. DESIGN: Prospective study. SETTING: Fertility clinic. PATIENT(S): Azoospermic men (n = 120) who underwent testicular sperm extraction and who were classified as either normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cells only according to their combined histologic and cytologic testicular findings and three normozoospermic men who donated sperm. INTERVENTION(S): Evaluation of testicular biopsies by qualitative and quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical staining, and analysis of spermatozoa by immunofluorescence. MAIN OUTCOME MEASURE(S): Expression of the four BET genes in testis and localization of BRDT protein in testicular tissue and ejaculated spermatozoa. RESULT(S): The BRDT gene was not expressed in testicular tissue from patients with Sertoli cells only, whereas the other three genes of the BET family retained expression in all the pathologies. The BRDT protein was localized in the nuclei of spermatocytes, spermatids, and ejaculated spermatozoa. Expression of BRDT protein was almost nil in testicular tissue specimens with spermatocyte maturation arrest despite normal transcript levels. CONCLUSION(S): Human BRDT expression pattern differs from mouse BRDT expression. In human, BRDT is the only BET gene expressed exclusively in testicular germ cells. Its expression in elongated spermatids and ejaculated spermatozoa raises the possibility that it is involved in unidentified additional functions.

CDY1 and BOULE transcripts assessed in the same biopsy as predictive markers for successful testicular sperm retrieval.

OBJECTIVE: To evaluate the molecular markers CDY1 and BOULE in the same testicular biopsy for predicting success of sperm retrieval in azoospermic men. DESIGN: Prospective study. SETTING: University-affiliated medical center. PATIENT(S): Azoospermic men (n = 92) who underwent testicular sperm extraction (TESE) and who were classified as normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cell only according to their combined histological and cytological testicular findings. INTERVENTION(S): Quantitative and qualitative evaluation of testicular biopsies by histological and qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) expression methodologies. MAIN OUTCOME MEASURE(S): CDY1 and BOULE expression and the presence of sperm cells in testicular tissue. RESULT(S): Both transcripts significantly predicted the presence of sperm cells by qualitative and quantitative methodologies. Although CDY1 had the best sensitivity by qualitative RT-PCR (98.3%), assessing both transcripts simultaneously had an additive efficacy compared with assessing CDY1 alone, improving the specificity from 84.4% to 96.3%. CONCLUSION(S): Assessing the expression of both CDY1 and BOULE by qualitative RT-PCR is a sensitive and feasible test for predicting the presence of sperm cells in testicular tissue and may serve as a predictive tool if repeated TESE is required.

The likelihood of finding mature sperm cells in men with AZFb or AZFb-c deletions: six new cases and a review of the literature (1994-2010).

OBJECTIVE: To reassess the predictive value of detecting sperm cells in men with AZFb or AZFb-c deletions. DESIGN: Retrospective analysis of previously reported men with AZFb or AZFb-c deletions and the addition of six new cases. SETTING: Fertility institution. PATIENT(S): Men with both sequence tagged site marker identification and testicular cytology/histology findings. INTERVENTION(S): Systematic review of reported men with microdeletions that included eligibility, data extraction and analysis. MAIN OUTCOME MEASURE(S): Availability of sperm cells for intracytoplasmic sperm injection (ICSI) in men with AZFb/AZFb-c microdeletions. RESULT(S): The average prevalences reported for AZFb, AZFb-c, partial AZFb, and partial AZFb-c in azoospermic men were 0.9%±0.07%, 2.7%±0.93%, 1.23%±0.9%, and 1%±0.6%, respectively. Sperm cells were identified in 7% and 3% of the 28 and 71 men with complete AZFb and AZFb-c and in 57% and 43% of the 14 and 7 men with partial AZFb and AZFb-c deletions, respectively. The likelihood of finding sperm cells in men with complete versus partial AZFb and AZFb-c deletions was significantly lower. As yet, no clinical or chemical pregnancy after ICSI in cases with complete AZFb/b-c microdeletions has been reported. CONCLUSION(S): Determining the extent of AZFb or AZFb-c deletions is critical considering the frequency and the reasonable prospect of finding sperm cells in partial AZFb/AZFb-c deletions. Referring men with complete AZFb/b-c microdeletions to testicular sperm extraction/ICSI programs should be revaluated.

Long-term cryostorage of sperm in a human sperm bank does not damage progressive motility concentration.

BACKGROUND: The use of quarantined cryopreserved semen is mandatory in donor insemination programs. Whether sperm cells can survive and retain their ability to fertilize after long-term storage remains a controversial issue. The objective of this study was to determine the effect of the duration of cryostorage in liquid nitrogen on the sperm cells' progressive motility concentration (PMC) in a large study group. METHODS: A total of 2525 thawed sperm specimens, packed in straws and donated by 72 sperm bank donors for intrauterine insemination (IUI), were evaluated in an assisted reproduction institute. PMC was recorded after 0.5-14.4 years of cryostorage. RESULTS: The mean (+/-SD) value of PMC of all study samples was 10.8 +/- 3.3 x 10(6)/ml after freezing/thawing and before cryostorage (T0), and 12.3 +/- 2.9 x 10(6)/ml after storage and before using the specimen for IUI (T1, P < 0.0001). Specimen storage for different lengths of time revealed that storage duration had no significant influence on the PMC of the specimens (r = -0.03, P = 0.08). The PMC of partially filled straws was lower than in full straws. Cryostorage duration made no difference in the PMC of raw and washed sperm specimens. CONCLUSION: Prolonged storage of donated sperm in liquid nitrogen had no influence on the PMC of the specimens and therefore should not alter the fertilization potency of donated sperm. The high post-storage values of the PMC compared with the pre-storage PMC values was probably an artifact of the small volume of the pre-storage sample.

Assessing the predictive value of hyaluronan binding ability for the freezability potential of human sperm.

OBJECTIVE: To evaluate the predictive value of a sperm maturation test using the hyaluronan-binding assay (HBA) for freezability potential; and to determine the effect of freezing-thawing on HBA results. DESIGN: Prospective study. SETTING: Andrology laboratory at a teaching hospital. PATIENT(S): Candidates for sperm bank donation (n = 113) and active sperm bank donors (n = 16). INTERVENTION(S): Semen analyses including HBA and sperm freezing-thawing. MAIN OUTCOME MEASURE(S): Percentage of sperm HBA results and other sperm parameters in relation to freezing-thawing results. RESULT(S): The predictive value of HBA for high freezability value (>or=40% postthaw motility) was significant. However, 1- and 4-hour percentage of motility had a higher predictive value for good freezability. A better prognostic value than that of HBA resutlts was also found for sperm concentration and percentage of normal morphology. Freezing-thawing had no significant influence on HBA results. CONCLUSION(S): To the best of our knowledge this is the first demonstration that sperm maturation, determined by the HBA test, has a low value for predicting freezing-thawing sperm survival.

[The involvement of the zona pellucida in unexplained infertile women].

Unexplained infertility (*UI) was a common problem before the IVF era. A couple was declared as UI only after they passed all the routine common tests and no reason was found for the infertility. The introduction of ICSI in the IVF clinics enabled the investigation of the quality of the couple's gametes. Thus, the definition of the term UI was refined and the number of couples encompassed by this term decreased dramatically. Zona Pellucida (ZP) incompetence was described as one of the causes of UI. We recently (2004) published a case study of an UI couple due to this cause. The oocytes of the woman collapsed during the preparation of the oocytes for sperm injection. These oocytes suffered from irregular ZP and abnormal appearance. Only after gentle treatment of the oocyte-cumulus complex and ICSI fertilization, embryo development and delivery of normal newborn was achieved. This woman and others who suffer from UI did not conceive in a natural way since the ZP of the ovulated eggs did not bind sperm cells and, thereby, were not fertilized. It only recently became possible to find the reason for the infertility of the couple and the solution, through the use of highly advanced technology in the IVF-ICSI process. One of the reasons for the deformatted ZP is the malfunction of the gene that encodes the 4 glycoproteins, which compose the ZP. This is now under investigation in our Institute.

Deoxyribonucleic acid-damaged sperm in cryopreserved-thawed specimens from cancer patients and healthy men.

A similarity was found between the percentage of thawed, DNA-damaged spermatozoa in cancer patients and that in candidates to become sperm bank donors who had low sperm cryofreezability. Both groups were significantly different from the sperm bank donor group. It is suggested that the higher rate of DNA fragmentation in sperm from cancer patients compared with sperm bank donors is apparently a result of selecting donors by the level of sperm cryofreezability (i.e., high), rather than a direct effect of an existing malignancy.

Effect of long-term storage on deoxyribonucleic acid damage and motility of sperm bank donor specimens.

The percentage of sperm DNA damage in samples from sperm bank donors was not significantly different (P=.17), whereas the percentage of motile cells was lower (P=.009) after long-term (9-13 years) compared with short-term (1-5 years) storage. Density gradient isolation reduced the difference in sperm motility between the two groups.

Histone H4 acetylation and AZFc involvement in germ cells of specimens of impaired spermatogenesis.

OBJECTIVE: To measure histone-H4 acetylation and involvement of the AZFc region in testicular mixed atrophy. DESIGN: Prospective study. SETTING: University-affiliated medical center. PATIENT(S): Azoospermic men (n = 23) who underwent testicular sperm extraction and preparation for intracytoplasmic sperm injection (ICSI) divided into obstructive azoospermia with complete spermatogenesis (group A), testicular mixed atrophy (group B), and testicular mixed atrophy associated with AZFc deletion (group C). INTERVENTION(S): Testicular biopsy evaluation by Western blotting and quantitative immunohistochemistry of histone-H4 hyperacetylation (Hypac-H4) and lysine-12 acetylation (Lys12ac-H4). MAIN OUTCOME MEASURE(S): Percentage of spermatogonia and spermatids stained by Hypac-H4 and Lys12ac-H4 antibodies in retrieved specimens. RESULT(S): The percentage of spermatogonia stained for Hypac-H4 and Lys12ac-H4 in groups B and C was statistically significantly reduced. The percentage of elongated spermatids showing positive staining to Hypac-H4 was statistically significantly lower in group B than group A. The percentage of Lys12ac-H4-labeled spermatids was similar for all groups. Hypac-H4 and Lys12ac-H4 processes were highly correlated in spermatogonia but not in spermatids. CONCLUSION(S): The reduced percentage of spermatogonia with Hypac-H4 and Lys12ac-H4 in groups B and C may contribute to lower sperm production in mixed atrophy. Spermatids Hypac-H4 impairment in mixed atrophy did not deteriorate further by AZFc region deletion.

Role of poly(ADP-ribosyl)ation during human spermatogenesis.

OBJECTIVE: Genomic stability of cells is known to be linked to their poly(ADP-ribosyl)ation capacity. We aimed to demonstrate, for the first time, the patterns of poly(ADP-ribosyl)ation during human spermatogenesis. DESIGN: Retrospective case-control study. SETTING: Teaching hospital. PATIENT(S): Azoospermic men who underwent testicular biopsy for sperm recovery. INTERVENTION(S): Testicular biopsy evaluation by immunohistochemistry for the expression of poly(ADP-ribose) polymerase-1 (PARP-1) enzyme and of poly(ADP-ribose) (PAR) (an indicator for PARP activity.) MAIN OUTCOME MEASURE(S): The subcellular localization of both markers in testes with full spermatogenesis (obstructive azoospermia), spermatocyte maturation arrest, or Sertoli cell-only syndrome. RESULT(S): Expression of both markers was localized in germ cell nuclei in full spermatogenesis: PAR expression, indicating PARP activity, was exhibited in round and elongating spermatids and in a subpopulation of primary spermatocytes. Strong immunoreactivity for PAR was identified in all of the spermatocytes in maturation arrest at the spermatocyte level. Sertoli cells lacked immunoreactivity for both markers, whereas other somatic testicular cells were rarely immunostained. CONCLUSION(S): The detection of PAR expression in germ-line cells and its subcellular localization in meiotic and postmeiotic prophases demonstrates chromatin modifications occurring during spermatogenesis and establishes a key role for poly(ADP-ribosyl)ation in germ cell differentiation, presumably to safeguard DNA integrity.

Use of sex chromosome bivalent pairing in spermatocytes of nonobstructive azoospermic men for the prediction of successful sperm retrieval.

OBJECTIVE: To find the most informative method of XY bivalent detection for spermatozoa presence in testicular tissue of nonobstructive azoospermic men. DESIGN: Prospective study. SETTING: Institute for the Study of Fertility, affiliated with a university medical faculty. PATIENT(S): Thirty-five men with azoospermia, divided into subgroups: complete maturation arrest (n = 10), mixed atrophy (n = 14), and obstructive azoospermia (n = 11). INTERVENTION(S): Testicular tissue biopsies for sperm extraction. MAIN OUTCOME MEASURE(S): Histopathologic and cytology analyses and the presence of XY bivalent formation by fluorescence in situ hybridization probes for centromere and subtelomere regions. Immunostaining of gamma-H2AX for sex body (SB) identification was also performed. RESULT(S): Percentage of spermatocytes with X-Y pairing, determined by the paired short arms pseudoautosomal region, was significantly higher than percentage of spermatocytes with long arm telomeres in proximity in all three groups. The parameter of q telomeres in proximity was the most sensitive index to distinguish one group from the other. Stained SB by gamma-H2AX was found to be the most informative for the prediction of successful sperm retrieval. CONCLUSION(S): Alignment of the X and Y axes that occurs in the late zygotene stage probably precedes the stage in which the SB is stained by gamma-H2AX. Consequently, because of the nonhomogeneity of the testis, when histology raises suspicion of complete maturation arrest percentage of spermatocytes with stained SB is the most informative parameter for sperm presence on sperm retrieval.

Comparison of efficacy of two techniques for testicular sperm retrieval in nonobstructive azoospermia: multifocal testicular sperm extraction versus multifocal testicular sperm aspiration.

To compare the efficacy of 2 sperm-retrieval procedures, testicular sperm extraction (TESE) and testicular sperm aspiration (TESA), during the same procedure using the same subjects as their own controls. The presence of mature testicular sperm cells and motility were evaluated in 87 men with nonobstructive azoospermia (NOA) by means of multifocal TESE and multifocal TESA, which were performed during the same procedure using the same subjects as their own controls. Sperm cells were recovered by TESE in 54 cases, but by TESA in only 36 cases. There were significantly more cases (n = 20) in which sperm cells were recovered by TESE only, compared with 2 cases in whom cells were recovered by TESA only (McNemar's test, P < .001). The mean number of locations in each testis in which sperm cells were detected was significantly higher in the TESE group. In significantly more cases (n = 27), motility was observed in TESE material only, compared with 3 cases in which motility was present in material extracted by TESA only (McNemar's test, P < .001). Mean number of locations in each testis with motile sperm cells was significantly higher in the TESE group. The TESE procedure yielded significantly more sperm cells, as was also reflected by the difference in number of straws with cryopreserved sperm. This comparative prospective clinical study revealed that multifocal TESE is more efficient than multifocal TESA for sperm detection and recovery in men with NOA and should be the procedure of choice for sperm retrieval for them.

Differentiating between primary and secondary Sertoli-cell-only syndrome by histologic and hormonal parameters.

The contribution of histologic differentiation between primary and secondary Sertoli-cell-only (SCO) syndrome in azoospermic men was evaluated. No correlation was found between the presence of sperm cells in the testis and the histologic findings or inhibin B or FSH levels, suggesting a low prognostic value for this differentiation.

Detection of calretinin expression in abnormal immature Sertoli cells in non-obstructive azoospermia.

The current study identified for the first time calretinin expression in abnormal Sertoli cells of azoospermic men who underwent testicular biopsy for sperm recovery and application of the retrieved sperm by in vitro fertilization techniques. Testicular biopsies with various spermatogenic impairments were evaluated immunohistochemically for the expression of the calretinin calcium-binding protein and the marker for immaturity of Sertoli cells, cytokeratin-18 (CK-18). Distribution of the markers was assessed in testes demonstrating a histological phenotype of mixed atrophy, Sertoli cell-only, or normal spermatogenesis (obstructive-azoospermia) and in men carrying a deletion in the azoospermia factor region located on the Y chromosome. Calretinin-immunopositive immature Sertoli cells revealed by co-localization of both markers, calretinin and CK-18, were identified in the mixed atrophy group in seminiferous tubules demonstrating spermatogenic failure. Sertoli cells expressing both markers were rarely detected in all other groups. Leydig cells in all the assessed biopsies expressed calretinin and served as a built-in control for immunoreactivity. This pattern of calretinin-selective expression in immature Sertoli cells suggests a functional relationship between calretinin expression and the degree of Sertoli cell differentiation. Disorders of Sertoli cell differentiation as indicated by calretinin and/or CK-18 expression contribute to the multifactorial mechanisms underlying spermatogenic failure.