Morphogenetic chromatin reorganization aspects at the preleptoten stage of human spermatogenesis

Male germ cells pool forms due to proliferation of germ cells during migration into the embryonic gonads and apoptosis. At the different stages of antenatal development a part of germ сells population in the seminiferous cords is represented by cells at the preleptotene stage of meiosis I. In newborns and infants a number of gametes at this stage of meiosis varies. Male germ cells enter meiotic development mainly in the puberty period. One of the theories of the unique chromatin condensation at the preleptotene stage (prochromosome) is a lack of special signal molecules responsible for the male gametes development. Another theory is that it is a modification that marks the germ сells capable of meiosis activation

Genetic and epigenetic mechanisms of regulation, chronology and dynamics of spermatogenesis of mammals

Genetic and epigenetic mechanisms of spermatogenesis – long process with many stages regulation are discussed. DNA code is the entirety of hereditary information, epigenetic mechanisms of gene regulation act without altering primary nucleotide sequences. Epigenetic regulation is a complex process, in which components of different groups of epigenetic modifications (non-coding RNAs, DNA methylation and histone modification) work together. Mistakes in any of the components of the process may cause impaired spermatogenesis and/or infertility, and may cause epigenetic diseases. Nowadays 90 imprinted genes and loci on 13 chromosomes are revealed. More then 10 human diseases involving genomic imprinting are known (Angelman syndrome, Prader–Willi syndrome, Russell–Silver syndrome, Beckwith–Wiedemann syndrome etc.). DNA methylation is essential for normal development and is associated with a number of key processes including animal growth and development, transcription, DNA replication and reparation, cell differentiation, genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and carcinogenesis. </p

Spermatological characteristics of mosaic and non-mosaic forms of Klinefelter syndrome

The study objective is to compare of ejaculate parameters in mosaic, non-mosaic Klinefelter syndrome (KS).Materials and methods. Eighty-five patients with KS were examined. The group 1 included 75 patients between the ages of 17 and 39 with non-mosaic KS (47,XXY), the group 2 included 10 males between the ages of 22 and 57 years with mosaic KS: 47,XXY/46,XY (n = 9), 48,XXY,der(X)/47,XXY/46,XY (n = 1).Results. KS patients semen volume was 1.9 ± 1.3 (0.1–5.5) ml in non-mosaic KS patients (47,XXY) and 1.5 ± 1.2 (0.05–4.00) ml in patients with a mosaic form of a KS, respectively, рН semen – 7.8 ± 0.5 (6.5–9.0) and 7.8 ± 0.2 (7.5–8.1), sperm count – 0.27 ± 1.42 (0.00–12.50) and 0.12 ± 0.28 (0.00–0.90) million/ml, respectively. The viscosity was increased (&gt;20 mm) at 41 % non-mosaic KS (group 1) and 22 % of mosaic KS (group 2) patients. The ejaculate sediment was investigated by quantitative karyological analysis of immature germ cells. The germ cells in 42 % samples of the ejaculate of the patients with a classical form of a KS and in 20 % samples of the ejaculate of the patients with a mosaic form was found. That indicates a partial preservation of spermatogenesis.Conclusion. The degree of spermatogenesis depletion in KS patients widely varied, ejaculate and germ cell parameters in the ejaculate sediment weren’t significantly different. Presence of few sperms (cryptozoospermia) and immature cells in the ejaculate sediment point to partial preservation of spermatogenesis

Quantitative karyological analysis of immature germ cells for the evaluation of spermatogenesis in patients with azoospermia or cryptozoospermia

The study objective is to evaluate spermatogenesis using the method of quantitative karyological analysis of immature germ cells from the ejaculate sediment (QKAIGC) in patients with azoospermia or cryptozoospermia with normal and low semen volume.Materials and methods. Seventy-two (72) semen samples from patients with suspected azoospermia were analyzed, 48 with normal semen volume (≥1,5 ml) and 24 with low semen volume (&lt;1,5 ml). A standard semen examination and QKA IGC were performed for all the samples.Results. By QKA IGC in 98.6 % of the samples spermatozoa were detected. In 16 (33 %) samples with normal ejaculate volume, incomplete spermatogenesis arrest at the pre-pachytene stages was revealed. It was accompanied by pachytene arrest (8 cases) and by diplotene arrest (3 cases). In samples with low ejaculate volume, 6 samples (25 %) showed signs of incomplete spermatogenesis arrest at the pre-pachytene stages; in 1 case it was accompanied by the meiotic arrest at diplotene of prophase I.Conclusion. Noninvasiveness, relative rapidity and possibility of multiple repeats of the test without risks for the patients’ health allow us to recommend QKA IGC for evaluation of spermatogenesis including dynamic evaluation. For suspected azoospermia, we recommend QKA IGC as an alternative for testis diagnostic biopsy

Antioxidant potential of seminal plasma in normozoospermia and asthenozoospermia

The study objective is to evaluate the antioxidant potential of seminal plasma in normozoospermia and asthenozoospermia.Materials and methods. Samples of ejaculate from 57 men of reproductive age were studied. Standard spermiologic examination and determination of the antioxidant potential of the seminal fluid using an original chemiluminescent method were performed. The method allowed to measure duration of the latent period from the moment of a decrease in luminescence of the solution containing reactive oxygen species (ROS) after addition of the seminal fluid until the most dramatic increase in luminescence corresponding to decreased antioxidant effect of the seminal fluid.Results. In the samples, the latent time varies from 4.6 to 17.5 minutes. Compared with normozoospermia, the antioxidant potential is significantly lower (p = 0.05) in men with asthenozoospermia (about 1.4 times), and even lower in patients with asthenoteratozoospermia (about 1.7 times) (p = 0.03). An inverse weak correlation was found between the activity of leukocytes and latent time in pathospermia (r = –0.23 and –0.18 for asthenozoospermia и asthenoteratozoospermia, respectively). A direct strong correlation was found between ROS-producing sperm activity and latent time in normozoospermia (p = 0.79), and inverse correlation in pathospermia (r = –0.26 and –0.62 for asthenozoospermia и asthenoteratozoospermia, respectively).Conclusion. Pathospermia is characterized by antioxidant seminal plasma deficiency, more pronounced for asthenoteratozoospermia. The antioxidant system of seminal plasma correlates with the ROS-producing ability of leukocytes and, to a greater extent, of the sperm. With normozoospermia, the oxidative balance is maintained, with pathospermia, a higher value of ROS production by sperm corresponds to a lower value of antioxidant capacity

Comprehensive semen examination in cystic fibrosis patients without seminal ducts obstruction

The study objective is to evaluate the semen parameters, quantitative and qualitative characteristics of spermatozoa, sperm ultrastructure and chromatin status in cystic fibrosis patients with preserved seminal ducts.Materials and methods. We examined 5 cystic fibrosis patients without bilateral obstruction of the vas deferens. Patients underwent standard semen analysis, quantitative karyological analysis of immature germ cells from the ejaculate sediment and transmission electronic microscopy of spermatozoa.Results. Of 5patients, 2 were asthenoteratozoospermic, 1 patient was oligoastenozoospermic, 2patients were asthenozoospermic and 1 was normozoospermic. Oligospermia was detected in 2 samples; increased viscosity of ejaculate was in 1 sample. The pH of the ejaculate was 7.0—7.8; the fructose of the ejaculate was low in one of 2 examined samples. However, oligospermia and low fructose concentration are only indirect signs of aplasy/hypoplasy to the seminal vesicles, since these patients did not perform instrumental visualization of the prostatove-sicular complex. Signs of partial meiotic arrest were revealed in all patients examined using quantitative karyological analysis of immature germ cells. An increased number of abnormal sperm heads, activated acrosomes, an increased content of "immature" chromatin in sperm nuclei were found by transmission electronic microscopy. Sperm DNA fragmentation was increased (32 %, normal range &lt;15 %) in 1 of 2 asthenoteratozoospermic patients.Conclusion. We found various sperm abnormalities and spermatology diagnoses (from normozoospermia to oligoasthenoteratozoospermia) in cystic fibrosis patients without semen ducts obstruction. No specific sperm morphology abnormalities were found. Oligospermia and a low ejaculate fructose indicate impaired seminal vesicles in some cystic fibrosis patients with no obstruction of the vas deferens

Semen parameters in polyzoospermic men

Polyzoospermia is extremely high (above 250 mln/ml) sperm concentration with normal volume of ejaculate. In the laboratory of genetic disorders of reproduction of Research Centre for Medical Genetics in 2007–2012 years we analyzed more than 14 000 semen samples from infertile men and men with reproductive disorders in order to study the distribution and characteristics of spermatogenesis in polyzoospermic men. Only polyzoospermic men semen analysis results were interpretered in present article. Polyzoospermia (sperm count over 250 mln/ml in volume over 1,5 ml) was detected in 191 semen samples (1.3 %). At the same time 15 % of the samples with polyzoospermia were normozoospermic. Among 85 % of the sperm pathology samples asthenozoospermia occured most frequently (77 %), astenoteratozoospermia was detected in 8 % of cases with polyzoospermia. The average proportion of vitality was 90.09 ± 10.02 %, normal morphology – 14.93 ± 8.51 %, and progressive motility – 17.72 ± 11.81 %. The average concentration of spermatozoa in the ejaculate in the examined samples was 313.29 ± 64.78 mln/ml. More than half of the tested samples with polyzoospermia had a concentration 250–300 mln/ml. Concentration of 450 mln/ml and more detected in 3 % of samples. The maximum total number of spermatozoa in the ejaculate in our study was more than 2 billion sperm cells and was observed in two polyzoospermic men. One of these patients had the maximum concentration (615 mln/ml) also. We found a high correlation (r = 0.89; p &lt; 0.01) between the volume of ejaculate and the total number of spermatozoa. Immature germ cells from ejaculate were estimated in 7 patients. There was a partial spermatogenesis arrest at meiosis I prophase in 5 out 7 of the examined semen samples

Comprehensive semen examination in patients with cystic fibrosis

Introduction. Cystic fibrosis (CF) is a common genetic disorder associated with male infertility. Almost all men with CF are infertile due to obstructive azoospermia.The objective is to evaluate semen parameters, excretion of seminal fluid, and spermatogenesis in CF patients.Materials and methods. We examined 44 male CF patients with pancreatic sufficiency (n = 20) and pancreatic insufficiency (n = 24). Standard semen analysis, quantitative karyological analysis of  immature germ cells from ejaculate sediment, biochemical semen  analysis (fructose, α-glucosidase, citric acid) were performed.Results. Semen analysis diagnosed azoospermia in 77.3 % patients, severe oligozoospermia (&lt; 1 million/ml) and other diagnoses (oligozoospermia, asthenozoospermia, normozoospermia) in 13.6 %  and 9.1 % patients, respectively. The signs of obstruction of the vas  deferens and aplasia of seminal vesicles were observed in 91 %  patients. Azoospermia, oligospermia, pH &lt;7.0 and low content of  fructose in the semen samples result from bilateral obstruction of the vas deferens and aplasia of the seminal vesicles.Conclusion. Cryptozoospermia and immature germ cells, detected in ejaculate sediment in all azoospermic patients with CF, indicated aplasia, but not congenital absence of the vas deferens. The majority of CF patients presented signs of seminal ducts obstruction and  impaired spermatogenesis and seminal fluid secretion. In contrast to pancreatic-sufficient CF male patients, pancreatic-insufficient CF  patients can be fertile and have no obstruction of seminal ducts and severe pathozoospermia

Comparative analysis of the results semen examination in patients with azoospermia caused by cystic fibrosis and congenital bilateral aplasia of vas deferens syndrome

The study objective is to compare the results of semen examination in patients with the congenital bilateral aplasia of vas deferens (CBAVD) syndrome and patients with cystic fibrosis.Materials and methods. Ninety-one patients with azoospermia were included in the study, 38 of them with CBAVD and 53 with cystic fibrosis. There were no significant differences in semen parameters (volume, viscosity and pH of the seminal fluid, the count of sperm and leukocytes) between the patients’ groups. In most samples of the ejaculate sediment, investigated by quantitative karyological analysis, spermatozoa and/or immature germ cells were found, indicating a partial preservation of patency of the vas deferens in most of CBAVD patients, also as azoospermic men with CF. Obtained data refute the generally accepted opinion about the absence of vas deferens in these patients, therefore, it is necessary to revise the terminology and further study the pathogenesis of abnormalities of vas deferens and seminal vesicles

Reproductive system status and the algorithm to solve fertility issues in men with cystic fibrosis

Rationale: Cystic fibrosis (CF) is a common hereditary disease related to the CFTR gene mutations and characterized by progression and multiple system involvement (primarily of the digestive tract and / or pulmonary system). Most men with CF are infertile. Due to new therapeutic options, the life expectancy of CF patients has increased, with reproductive issues becoming relevant.Aim: A multifaceted assessment of the reproductive system status and fertility in male patients with CF and improvement of the strategies to resolve their reproduction issues.Materials and methods: This cohort prospective study was performed 2006 to 2018 and included 81 unrelated Russian male patients with confirmed CF, aged from 15 to 69 years (mean age 25.6 ± 7.9 years). Forty two (42) patients had pancreatic sufficient and 39 pancreatic insufficient CF. The patients underwent clinical, andrological, laboratory and instrumental examination (scrotal ultrasonography, standard and biochemical semen examination and hormone levels).Results: Reproductive disorders and semen abnormalities found in CF patients varied from preserved fertility to infertility. The following andrological abnormalities were found: delayed puberty (48%), urological disorders (26%), uni- or bilateral testicular hypoplasia (42%), diffuse lesions and cysts of the epididymis (70%), diffuse lesions /calcifications of the prostate (50%), and decreased testosterone levels (24.2%). Azoospermia was diagnosed in 87.5% of the patients, “moderate” or “mild” pathozoospermia (oligo-/astheno-/teratozoospermia) in 11.1%, and normozoospermia in 1.4% of the patients. There were significant differences between the patients with pancreatic sufficient and pancreatic insufficient CF in the ejaculate volume (1.4 ± 1.5 ml vs. 0.6 ± 0.5 ml; р = 0.006), ejaculate pH (6.7 ± 0.7 vs. 6.1 ± 0.4; р &lt; 0.0001), and sperm concentration (19.6 ± 56.0 Mio/mL vs. 0.001 ± 0.008 Mio/ mL; p = 0.011). Normal ejaculate volume was more frequent (21.1% vs 14.7%; p &gt; 0.05) in patients under the age of 25. No bilateral obstruction of vas deferens was found in 71.4% patients with 3849+10kbC&gt;T mutation. There was a significant difference (p &lt; 0.00001) in the frequency of 3849+10kbC&gt;T mutation between the patients with vas deferens obstruction (9.5%) and without it (93.8%). We developed an algorithm to resolve infertility issues (including assisted reproductive technologies) in male CF patients depending on their fertility / presence and type of pathozoospermia and some other factors that may influence the conception and CF risk in the offspring. We also suggested practical recommendations for the andrological assessment, maintenance of reproductive health, and planning of childbirth in these patients.Conclusion: Male CF patients require a multifaceted assessment of their reproductive system. The prognosis of their reproductive functions, the strategy to maintain their reproductive health and making a decision on childbirth depends on the CF type, the CFTR genotype, the results of semen analysis, and the patient’s age. Pancreatic sufficient CF type, 3849+10kbС&gt;T mutation of the CFTR gene and younger age are favorable factors for potential maintenance of vas deferens patency and male fertility in CF patients