The Price of Human Evolution: Cancer-Testis Antigens, the Decline in Male Fertility and the Increase in Cancer.

The increasing frequency of general and particularly male cancer coupled with the reduction in male fertility seen worldwide motivated us to seek a potential evolutionary link between these two phenomena, concerning the reproductive transcriptional modules observed in cancer and the expression of cancer-testis antigens (CTA). The phylostratigraphy analysis of the human genome allowed us to link the early evolutionary origin of cancer via the reproductive life cycles of the unicellulars and early multicellulars, potentially driving soma-germ transition, female meiosis, and the parthenogenesis of polyploid giant cancer cells (PGCCs), with the expansion of the CTA multi-families, very late during their evolution. CTA adaptation was aided by retrovirus domestication in the unstable genomes of mammals, for protecting male fertility in stress conditions, particularly that of humans, as compensation for the energy consumption of a large complex brain which also exploited retrotransposition. We found that the early and late evolutionary branches of human cancer are united by the immunity-proto-placental network, which evolved in the Cambrian and shares stress regulators with the finely-tuned sex determination system. We further propose that social stress and endocrine disruption caused by environmental pollution with organic materials, which alter sex determination in male foetuses and further spermatogenesis in adults, bias the development of PGCC-parthenogenetic cancer by default.

Evaluation of embryo aneuploidy (PGT-A) and endometrial receptivity (ERA) testing in patients with recurrent implantation failure in ICSI cycles.

OBJECTIVE: The objective of a study was to assess the ability of the pre-implantation genetic testing of embryos for aneuploidy (PGT-A) and Endometrial receptivity array (ERA)-alone or in combination to improve the clinical outcomes in intracytoplasmic sperm injection (ICSI) cycles in patients with repeated implantation failure (RIF). METHODS: This was a retrospective study of the 253 cycles with a history of the previous RIF. They were divided into four groups: Group I - frozen embryo transfers without any additional tests or procedures (pure FET), n = 72 cycles; Group II - FET with PGT-A, n = 87; Group III - FET with PGT-A and ERA, n = 72; Group IV - FET with ERA, n = 22. RESULTS: Median age of the entire study group for the females was 35 years. Only Group II (FET + PGT-A) showed statistically significant higher chance in achieving both biochemical (p = .01, OR = 5.5) and clinical pregnancy (p =.049, OR = 2.3), as compared to the Group I (FET with no additional tests). Both Group III and Group IV failed to demonstrate better clinical outcomes as compared to the Group I. CONCLUSIONS: Patients with RIF can benefit from testing for embryo aneuploidy using the PGT-A method, but the ability of the ERA test to improve the clinical outcome in ICSI cycles seems to be rather limited. Although the endometrium cycle is also weakened with age, the contribution of the embryo genetic quality is evidently more important for successful implantation, although in principle both factors reflect the reproductive health.

Prevalence of testosterone deficiency among aging men with and without morbidities.

In this cross-sectional study 1852 men aged 40-70 years attending primary health care were invited to fill out the aging male symptoms (AMS) scale. Out of these, 1222 men were found positive for the AMS and agreed to provide blood samples for the general blood test, lipid profile, glucose levels, and assessment of both total and free testosterone (T) levels. Men were screened for the following morbidities and syndromes: dyslipidemia, arterial hypertension, obesity, type II diabetes, metabolic syndrome, and chronic obstructive pulmonary disease (COPD). Testosterone deficiency was diagnosed if total T ≤ 3.46 ng/mL or free T ≤ 72 pg/mL. Among all 1222 men with positive AMS, decreased blood testosterone levels were detected in 669 men (55%). A total of 402 men were found healthy and 820 men were detected with different morbidities. Out of 669 men with testosterone deficiency, only 2.8% had no co-morbidities and 97.2% were men with co-morbidities. Testosterone levels were found significantly higher among healthy men (median 4.7 ng/mL) as compared to the men with morbidities (median 2.55 ng/mL, p<.001), adjusted for age. Testosterone deficiency was detected in significantly lower proportion of 402 men without co-morbidities as compared to the 820 men with co-morbidities: in 19 men (4.7) and in 650 men (79.3%, p<.05), respectively.

Semen quality of young men from the general population in Baltic countries.

STUDY QUESTION: What are the parameters of semen quality in Baltic men? SUMMARY ANSWER: Combined parameters of sperm concentration, motility and morphology revealed that 11-15% of men had low semen quality, 37-50% intermediate and 38-52% high semen quality. WHAT IS KNOWN ALREADY: Previous studies have revealed regional differences in semen parameters, and semen quality of Baltic men has been suggested to be better than that of other European men. STUDY DESIGN, SIZE, DURATION: This was a cross-sectional study of 1165 men aged 16-29 years from Estonia (N = 573), Latvia (N = 278) and Lithuania (N = 314) conducted in 2003-2004. PARTICIPANTS/MATERIALS SETTING METHODS: Men from the general population, median age 19.8 years, provided one semen sample each, had blood samples taken, had testis size determined, and provided information on lifestyle. Based on combined data of sperm concentration, sperm motility and morphology the cohort was classified into three categories: low, intermediate or high semen quality. Comparisons between groups (including subgroups of Estonian men of Russian versus Estonian ethnicity) were tested, adjusting for ejaculation abstinence and age. MAIN RESULTS AND THE ROLE OF CHANCE: The median sperm concentration of the Estonian, Latvian and Lithuanian populations of Baltic men was 63 mill/ml. Low semen quality was detected in 11-15% of the men, intermediate in 37-50% and high in 38-52%. No crucial differences between national subgroups were detected, except that a higher percentage (9.6%) of the subgroup of Russian Estonians reported having had cryptorchidism compared to the other men (2.5-3.6%, P < 0.001). Smoking had an adverse impact on both sperm concentration and total sperm counts (P < 0.001). LIMITATIONS REASONS FOR CAUTION: The semen quality data were collected >10 years ago. Thus, a recent change in semen quality cannot be excluded. Owing to the study design, it is assumed, but unproven, that the men were representative of the general populations. Some men were very young (16 years), however, this was also the case for other European studies of similar populations. Assessment of sperm motility is associated with inter-observer variation, and no quality control was undertaken for sperm motility assessment to account for that. Thus, estimates of sperm motility should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: Analysis of the semen variables separately did not identify that a considerable percentage of Baltic men had low semen quality. The combined analysis, however, showed that more than one out of nine men had semen quality at a level indicating reduced fertility chances. We suggest that future studies of semen quality should be carried out reporting both results of single semen parameters and estimates that combine the most frequently assessed variables. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the EU fifth framework project Number QLK4-1999-01422 'Envir.Repro.Health' extension to Baltic countries Number QLRT-2001-02911; Estonian Science Foundation, grant numbers 2991 and PUT181. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A New Baltic Population-Specific Human Genetic Marker in the PMCA4 Gene.

OBJECTIVES: The PMCA gene family consists of 4 genes and at least 21 splice variants; among these, the Ca2+ ATPase 4 (PMCA4) gene encodes a plasma membrane protein abundantly expressed in several tissues, including the kidney, heart, and sperm. Knockout of PMCA4 causes infertility due to immotile sperm in mouse models. We therefore investigated variants in this gene for potential association with infertility in groups of Estonian (n = 191) and Latvian (n = 92) men with reduced sperm motility. METHODS: All exons, exon-intron boundaries, 5' and 3' untranslated regions, and the promoter region of the PMCA4 gene were analysed by direct sequencing for a group of Estonian infertile men. Genotyping of guanine and adenine alleles of rs147729934 was performed, using a custom-designed TaqMan® probe for a group of Latvian infertile men as well as additional groups from Latvia and several groups of people with proven ethnicity from the Baltic region. RESULTS: Although we did not identify any significant associations between variants in the gene and infertility, our results indicated that in all studied Latvian and Estonian groups the adenine allele of the variant rs147729934 was present at a higher frequency than expected. Analysis of additional samples indicated that the adenine allele of rs147729934 likely originated once in the modern-day Baltic or western Russia area, as the frequency of the minor adenine allele observed in this region is remarkably higher than that in the general European population. CONCLUSIONS: Our results revealed no significant difference in frequencies of genetic variants in PMCA4 gene between men with normal and those with reduced sperm motility. The adenine allele of the variant rs147729934 is potentially an informative tool for future population studies concerning ancient Baltic and Finno-Ugric history.

Idiopathic Infertility as a Feature of Genome Instability.

Genome instability may play a role in severe cases of male infertility, with disrupted spermatogenesis being just one manifestation of decreased general health and increased morbidity. Here, we review the data on the association of male infertility with genetic, epigenetic, and environmental alterations, the causes and consequences, and the methods for assessment of genome instability. Male infertility research has provided evidence that spermatogenic defects are often not limited to testicular dysfunction. An increased incidence of urogenital disorders and several types of cancer, as well as overall reduced health (manifested by decreased life expectancy and increased morbidity) have been reported in infertile men. The pathophysiological link between decreased life expectancy and male infertility supports the notion of male infertility being a systemic rather than an isolated condition. It is driven by the accumulation of DNA strand breaks and premature cellular senescence. We have presented extensive data supporting the notion that genome instability can lead to severe male infertility termed "idiopathic oligo-astheno-teratozoospermia." We have detailed that genome instability in men with oligo-astheno-teratozoospermia (OAT) might depend on several genetic and epigenetic factors such as chromosomal heterogeneity, aneuploidy, micronucleation, dynamic mutations, RT, PIWI/piRNA regulatory pathway, pathogenic allelic variants in repair system genes, DNA methylation, environmental aspects, and lifestyle factors.

A novel EDA variant causing X-linked hypohidrotic ectodermal dysplasia: Case report.

Hereditary ectodermal dysplasias are a complex group of inherited disorders characterised by abnormalities in two or more ectodermal derivatives (skin, nails, sweat glands, etc.). There are two main types of these disorders - hidrotic and hypohidrotic/anhidrotic ectodermal dysplasias. Hypohidrotic ectodermal dysplasia (HED) or Christ-Siemens-Touraine syndrome (OMIM: 305100) occurs in 1 out of 5000-10,000 births [19] and has an X-linked recessive inheritance pattern (X-linked hypohydrotic ectodermal dysplasia - XLHED) [2]. The main cause of XLHED is a broad range of pathogenic variants in the EDA gene (HGNC:3157, Xq12-13) which encodes the transmembrane protein ectodysplasin-A [4]. We report here the case of a patient with a novel inherited allelic variant in the EDA gene - NM_001399.5:c.337C>T (p.Gln113*) - in the heterozygous state. Targeted family member screening was conducted and other carriers of this EDA gene pathogenic variant were identified and phenotypically characterised. The patient subsequently underwent in vitro fertilisation with preimplantation genetic testing for monogenic diseases (PGT-M).

When Three Isn't a Crowd: A Digyny Concept for Treatment-Resistant, Near-Triploid Human Cancers.

Near-triploid human tumors are frequently resistant to radio/chemotherapy through mechanisms that are unclear. We recently reported a tight association of male tumor triploidy with XXY karyotypes based on a meta-analysis of 15 tumor cohorts extracted from the Mitelman database. Here we provide a conceptual framework of the digyny-like origin of this karyotype based on the germline features of malignant tumors and adaptive capacity of digyny, which supports survival in adverse conditions. Studying how the recombinatorial reproduction via diploidy can be executed in primary cancer samples and HeLa cells after DNA damage, we report the first evidence that diploid and triploid cell sub-populations constitutively coexist and inter-change genomes via endoreduplicated polyploid cells generated through genotoxic challenge. We show that irradiated triploid HeLa cells can enter tripolar mitosis producing three diploid sub-subnuclei by segregation and pairwise fusions of whole genomes. Considering the upregulation of meiotic genes in tumors, we propose that the reconstructed diploid sub-cells can initiate pseudo-meiosis producing two "gametes" (diploid "maternal" and haploid "paternal") followed by digynic-like reconstitution of a triploid stemline that returns to mitotic cycling. This process ensures tumor survival and growth by (1) DNA repair and genetic variation, (2) protection against recessive lethal mutations using the third genome.

Case of Inherited Partial AZFa Deletion without Impact on Male Fertility.

Male factor infertility accounts for 40-50% of all infertility cases. Deletions of one or more AZF region parts in chromosome Y are one of the most common genetic causes of male infertility. Usually full or partial AZF deletions, including genes involved in spermatogenesis, are associated with spermatogenic failure. Here we report a case of a Caucasian man with partial AZFa region deletion from a couple with secondary infertility. Partial AZFa deletion, involving part of USP9Y gene appears to be benign, as we proved transmission from father to son. According to our results, it is recommended to revise guidelines on markers selected for testing of AZFa region deletion, to be more selective against DDX3Y gene and exclude probably benign microdeletions involving only USP9Y gene.

Sperm DNA damage in men from infertile couples.

AIM: To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. METHODS: A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay (SCSA) were carried out. RESULTS: Ninety-seven men (28% of the whole study group) had a DNA fragmentation index (DFI)> 20%, and 43 men (12%) had a DFI>30%. In the group of men with abnormal semen parameters (n = 224), 35% had a DFI>20%, and 16% had a DFI>30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters (n=126). Men with low sperm motility and abnormal morphology had significantly higher odds ratios (ORs) for having a DFI>20% (4.0 for motility and 1.9 for morphology) and DFI>30% (6.2 for motility and 2.8 for morphology) compared with men with normal sperm motility and morphology. CONCLUSION: In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique (ART).

Varicocele Is Associated with Impaired Semen Quality and Reproductive Hormone Levels: A Study of 7035 Healthy Young Men from Six European Countries.

BACKGROUND: Present knowledge on the impact of varicoceles on testicular function is largely based on studies of subfertile and infertile men, making it difficult to extrapolate the impact of varicocele on the general population. OBJECTIVE: To describe associations between varicocele and testicular function assessed by semen analysis and reproductive hormones in men from the general population. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional multicentre study of 7035 young men, median age 19 yr, from the general population in six European countries (Denmark, Finland, Germany, Estonia, Latvia, and Lithuania) were investigated from 1996 to 2010. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We analysed results from physical examination, conventional semen variables, and serum reproductive hormones using multivariable regression analyses. RESULTS AND LIMITATIONS: A total of 1102 (15.7%) had grade 1-3 varicocele. Increasing varicocele grade was associated with poorer semen quality, even in grade 1 varicocele. In grade 3 varicocele, sperm concentration was less than half of that in men with no varicocele. Presence of varicocele was also associated with higher serum levels of follicle-stimulating hormone, lower inhibin B, and higher levels of luteinising hormone; testosterone and free testosterone were not significantly different between men with and without varicocele. This study cannot draw a conclusion on the progressiveness of varicocele or the effect of treatment. CONCLUSIONS: We demonstrated an adverse effect of increasing grade of varicocele on testicular function in men not selected due to fertility status. PATIENT SUMMARY: The presence and increasing grade of varicocele is adversely associated with semen quality and reproductive hormone levels in young men from the general population.

Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples.

Controversy exists over levels of DNA integrity in the sperm of fertile and infertile men. In addition, the effect of leukocytospermia on sperm DNA in these 2 groups is unclear. We decided to address these questions by collecting semen samples from men known or presumed to be fertile and men from infertile couples. Samples were analyzed and assessed for sperm concentration, motility, and morphology. Samples failing to meet World Health Organization (WHO) standards in one or more of these parameters were judged abnormal. Samples were then arbitrarily assigned normalized scores in each of the above parameters, and scores were summed to give a normalized value for overall sperm quality. DNA abnormality was determined by an in situ DNA denaturation test with acridine orange and expressed as a percentage of cells with abnormal DNA integrity (ADI). Assessment of 187 samples revealed a moderate inverse correlation between ADI and sperm quality (r =.58), although a large degree of ADI dispersion was observed in abnormal semen samples. The average ADI for normal and abnormal semen samples was 18% +/- 2.8% and 36% +/- 5.8%, respectively, with the threshold of 95% probability set at 30%. When sorted for leukocytospermia, the difference in ADI between normal and abnormal semen groups without leukocytospermia was much smaller (17% +/- 2.2% and 22% +/- 4.6%; P =.023). Leukocytospermia had no significant effect on ADI in the normal semen group (P = .46); however, ADI was more than double the ADI in the abnormal semen group (18% +/- 2.4% and 50% +/- 11%; P < .001). The results of our analysis show that at least 3 factors affect net DNA integrity in leukocytospermic samples that fail to meet WHO standards: 1) primary DNA damage, which is moderately inverse to sperm quality, in particular to sperm concentration; 2) effect of leukocytes increasing primary or provoking potential DNA damage in a cascade-like manner, particularly in sperm with poor morphology and motility; and 3) a decreasing proportion of cells with damaged DNA in semen with the worst quality.

Reproductive physiology in young men is cumulatively affected by FSH-action modulating genetic variants: FSHR -29G/A and c.2039 A/G, FSHB -211G/T.

Follicle-Stimulating Hormone Receptor (FSHR) -29G/A polymorphism (rs1394205) was reported to modulate gene expression and reproductive parameters in women, but data in men is limited. We aimed to bring evidence to the effect of FSHR -29G/A variants in men. In Baltic young male cohort (n = 982; Estonians, Latvians, Lithuanians; aged 20.2 ± 2.0 years), the FSHR -29 A-allele was significantly associated with higher serum FSH (linear regression: effect 0.27 IU/L; P = 0.0019, resistant to Bonferroni correction for multiple testing) and showed a non-significant trend for association with higher LH (0.19 IU/L) and total testosterone (0.93 nmol/L), but reduced Inhibin B (-7.84 pg/mL) and total testes volume (effect -1.00 mL). Next, we extended the study and tested the effect of FSHR gene haplotypes determined by the allelic combination of FSHR -29G/A and a well-studied variant c.2039 A/G (Asn680Ser, exon 10). Among the FSHR -29A/2039G haplotype carriers (A-Ser; haplotype-based linear regression), this genetic effect was enhanced for FSH (effect 0.40 IU/L), Inhibin B (-16.57 pg/mL) and total testes volume (-2.34 mL). Finally, we estimated the total contribution of three known FSH-action modulating SNPs (FSHB -211G/T; FSHR -29G/A, c.2039 A/G) to phenotypic variance in reproductive parameters among young men. The major FSH-action modulating SNPs explained together 2.3%, 1.4%, 1.0 and 1.1% of the measured variance in serum FSH, Inhibin B, testosterone and total testes volume, respectively. In contrast to the young male cohort, neither FSHR -29G/A nor FSHR haplotypes appeared to systematically modulate the reproductive physiology of oligozoospermic idiopathic infertile patients (n = 641, Estonians; aged 31.5 ± 6.0 years). In summary, this is the first study showing the significant effect of FSHR -29G/A on male serum FSH level. To account for the genetic effect of known common polymorphisms modulating FSH-action, we suggest haplotype-based analysis of FSHR SNPs (FSHR -29G/A, c.2039 A/G) in combination with FSHB -211G/T testing.

Genetically determined dosage of follicle-stimulating hormone (FSH) affects male reproductive parameters.

CONTEXT: The detailed role of FSH in contributing to male testicular function and fertility has been debated. We have previously identified the association between the T-allele of the FSHB promoter polymorphism (rs10835638; G/T, -211 bp from the mRNA start) and significantly reduced male serum FSH. OBJECTIVE: In the current study, the T-allele carriers of the FSHB -211 G/T single nucleotide polymorphism represented a natural model for documenting downstream phenotypic consequences of insufficient FSH action. DESIGN AND SUBJECTS: We genotyped rs10835638 in the population-based Baltic cohort of young men (n = 1054; GG carriers, n = 796; GT carriers, n = 244; TT carriers, n = 14) recruited by Andrology Centres in Tartu, Estonia; Riga, Latvia; and Kaunas, Lithuania. Marker-trait association testing was performed using linear regression (additive, recessive models) adjusted by age, body mass index, smoking, and recruitment center. RESULTS: Serum hormones directly correlated with the T-allele dosage of rs10835638 included FSH (additive model, P = 1.11 × 10(-6); T-allele effect, -0.41 IU/liter), inhibin-B (P = 2.16 × 10(-3); T-allele effect, -14.67 pg/ml), and total testosterone (P = 9.30 × 10(-3); T-allele effect, -1.46 nmol/liter). Parameters altered only among TT homozygotes were reduced testicular volume (recessive model, P = 1.19 × 10(-4); TT genotype effect, -9.47 ml) and increased serum LH (P = 2.25 × 10(-2); TT genotype effect, 1.07 IU/liter). The carrier status of rs10835638 alternative genotypes did not affect sperm motility and morphology, calculated free testosterone, serum SHBG, and estradiol concentrations. CONCLUSION: We showed for the first time that genetically determined low FSH may have wider downstream effects on the male reproductive system, including impaired testes development, altered testicular hormone levels (inhibin-B, total testosterone, LH), and affected male reproductive potential.

Association of sperm apoptosis and DNA ploidy with sperm chromatin quality in human spermatozoa.

OBJECTIVE: To examine the relationship among sperm apoptosis, sperm chromatin status, and DNA ploidy in different sperm fractions. DESIGN: Prospective study. SETTING: Reproductive research center in a tertiary care hospital. INTERVENTION(S): Sperm prepared by density gradient were evaluated for sperm count, motility, apoptosis, and sperm chromatin assessment. MAIN OUTCOME MEASURE(S): Sperm count, sperm motility, toluidine blue (TB) results, DNA fragmentation index (%DFI), high DNA stainability, DNA cytometry, and early and late apoptosis. RESULT(S): Sperm motility was related to late apoptotic and subhaploid apoptotic sperm (r = -0.56 and -0.53, respectively). The sperm %DFI showed significant correlation with late apoptotic and subhaploid sperm (r = 0.62 and 0.68). TB-stained sperm were significantly correlated with late apoptotic sperm (r = 0.51). Significantly higher proportions of haploid sperm and light blue TB-stained sperm were seen in mature compared with immature fractions. CONCLUSION(S): Even in semen samples with low %DFI, semen processing results in a lower incidence of nuclear immaturity and subhaploidy, but the incidence of late apoptotic sperm remains unchanged. Therefore, simultaneous evaluation of apoptosis and sperm chromatin status is important for processing sperm in assisted reproductive procedures.

Sperm concentration in Latvian military conscripts as compared with other countries in the Nordic-Baltic area.

Recent studies of semen quality in men from the general population gave rise to the hypothesis of an East-West gradient in semen quality in the Nordic-Baltic area, with the highest sperm counts in Estonia, Lithuania and Finland, and the lowest in Denmark (30% difference in mean concentration). Genetic, lifestyle-related and environmental factors - alone or in combination - were suggested to be responsible for these differences. The aim of this study was to assess sperm concentration in men from the general population in Latvia and to investigate the impact of ethnic and lifestyle-related factors on this marker of male reproductive health. A total of 133 military conscripts from Latvia were investigated. We found that sperm counts among Latvian adolescents were at the same level (mean 74, median 63 x 10(6)/mL) as those previously reported from Estonia, Lithuania and Finland. Sperm concentration was somewhat higher than in Sweden without reaching the level of statistical significance (mean difference 3 x 10(6)/mL; 95% CI: -10, 16 x 10(6)/mL), and statistically significantly higher that in Denmark (mean difference: 17 x 10(6)/mL; 95% CI: 5, 2 x 10(6)/mL). The study also revealed an impact of ethnic factors on sperm numbers. Sperm concentration was significantly higher in men with both parents born in Latvia (77 +/- 60 x 10(6)/mL), compared with men with both parents born outside Latvia (55 +/- 45 x 10(6)/mL, p = 0.03).

Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm.

BACKGROUND: Sperm DNA integrity is of paramount importance in the prognosis of fertility. We applied image cytometry to a toluidine blue (TB) test we recently proposed. METHODS: Sperm samples from 33 men were assayed for standard sperm parameters and classified as normal or abnormal. Sperm smears were subjected to the TB test, DNA denaturation testing with acridine orange (AO), and terminal deoxyuridine triphosphate biotin nick end labeling (TUNEL). In CCD image analysis, TB-stained sperm cell heads were microscopically assigned to one of four color groups (dark, blue, light violet, and light blue). The optical densities of 6,600 cells in green and red CCD images were used to elaborate an algorithm for discrimination of these groups. RESULTS: The proportions of sperm in TB color groups, as estimated with the developed image cytometry algorithm, correlated with microscopic features. The number of TB dark cells correlated with the number of AO-red and TUNEL(+) cells. The proportion of TB dark cells in normal samples did not exceed 35%. Light-blue sperm cell heads prevailed in normal samples, whereas dark and blue sperm cell heads dominated in abnormal samples. CONCLUSIONS: The TB test was suitable for the assessment of sperm cell DNA integrity. The elaborated image cytometry algorithm can be used for this purpose and for finer determination of sperm nucleus status.