Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) with single-color chromosome-specific probes was used to study the rates of disomy for chromosome 1, 16, X, and Y in sperm of fertile and infertile subjects. Diploidy rates were studied using a two-color cocktail of probes for chromosomes 17 and 18 in the same sperm samples. Two-color methodology was not available at the outset of the study. A total of 450,580 spermatozoa were studied from 21 subjects (9 fertile, 12 infertile). Significant differences were observed in the disomy rates between chromosomes with the highest frequency observed for chromosome 16 (0.17%) and the lowest for the Y chromosome (0.10%). No differences were observed between fertile and infertile subjects for either diploidy or disomy. Total disomy rates for chromosomes 1, 16, X and Y ranged from 0.34% to 0.84% among infertile subjects, and 0.32% to 0.61% among fertile subjects. Our data suggest that generalized aneuploidy in sperm is not a major contributor to unexplained infertility.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.     

Idiopathic infertility in married couples in the light of cytogenetic analysis and sperm penetration assay

We have selected 47 couples with unexplained infertility in order to analyse a possible link between sperm dysfunction studied in males in in vitro conditions and karyotype analysis of somatic cells. In order to identify so called "idiopathically infertile" couples we had to exclude any change in reproductive organs in both partners or in spermiogram which would qualify any of spouses into known category of infertility. We have revealed chromosome aberrations (translocations and marker chromosomes) in 19% of infertile males and in 6% of infertile females. Idiopathically infertile males had an overall decreased ability of sperm function (measured by proportion of penetrated hamster oocytes by human sperm) in comparison to fertile controls, however, still well placed within physiological range of values. Only sperm from a patient with identified translocation was clearly below the normal level of penetration (20% of penetrated oocytes), however, also the patients with revealed chromosome variant polymorphisms presented statistically lower values of penetration in comparison to fertile controls (39% vs 57%, p<0.05). On the contrary, patients with marker chromosomes did not exhibit affected sperm function. It can be speculated that only particular chromosome aberration in group of idiopathically infertile males may affect sperm functional capability (measured in vitro), however, the intragonadal genetic analysis has to be recommended in order to confirm such a causative link.     

Androgen receptor gene CAG repeats length in fertile and infertile Tunisian men

Several reports implicated a relation between the trinucleotide (CAG) repeat length in the androgen receptor (AR) gene and male infertility. But such result was not reproduced in others. To test this hypothesis, we investigated the number of (CAG) repeats in the AR gene among two groups of infertile (n = 129) and fertile Tunisian men (n = 98), using polymerase chain reaction (PCR) targeting the AR CAG repeat tract, followed by electrophoresis on polyacrylamide gel (6%). For statistical analysis we used Student, Kolmogorov-Smirnov (KS) and chi(2)-tests. Significance was reached when P < 0.05. No statistically significant difference in the mean length of the CAG repeat was found between infertile and control groups (P = 0.47). Moreover, using KS test, we have not found a difference in the distribution of allele frequencies between infertile and controls (D(obs) = 0.046 < D(crit) = 0.180). We also did not found a statistically significant relationship between the size of the CAG repeat and impaired sperm production in Tunisian population. Our results may be attributed to the high probability that infertile males may represent a heterogeneous group with respect to the causes of defective spermatogenesis.     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.     

High Levels of Cadmium and Lead in Seminal Fluid and Blood of Smoking Men are Associated with High Oxidative Stress and Damage in Infertile Subjects

We measured the levels of malondialdehyde (MDA), protein carbonyls, glutathione S-transferase (GST) and reducte glutathione (GSH) in seminal plasma and spermatozoa from 95 subjects including 50 infertile patients to evaluate the association between oxidative stress and damage and the components of the anti-oxidant defenses in seminal plasma and spermatozoa of infertile subjects and concentrations of cadmium (Cd) and lead (Pb) in the blood and seminal plasma because of tobacco smoke exposure. The reactive oxygen species (ROS) in spermatozoa were also evaluated by luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-enhanced chemiluminescence assay. The sperm count, motility, and morphology in the smokers infertile group were found to be lower than those in the fertile male group and nonsmokers infertile group (p < 0.001). Concentrations of Cd, Pb, MDA, protein carbonyls, and ROS levels in the smokers infertile group were significantly higher than those in the fertile male and nonsmokers infertile male groups (p < 0.001). However, GSH levels and GST activities were decreased in the smokers infertile male group than those in the fertile male and nonsmokers infertile male groups (p < 0.001). The results indicate that smoking could affect semen quality and oxidative lipid and protein damage in human spermatozoa. From Pearson correlation analysis, positive correlations were demonstrated between the seminal plasma Cd and seminal plasma protein carbonyls and between seminal plasma Pb and spermatozoa ROS levels in smokers of the subfertile group, while there was a significant positive correlation between blood Cd and ROS levels in smokers of the fertile group. There was also a significant negative correlation of the Cd level of the blood and GSH levels of the sperm and seminal plasma. These findings suggest that cigarette smoking enhances the levels of Cd and Pb in seminal plasma and blood and the extent of oxidative damage associated with a decrease in components of the anti-oxidant defenses in the sperm of infertile males.     

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.     

Association of the (TAAAA)n repeat and Asp327Asn polymorphisms in the sex hormone-binding globulin (SHBG) gene with idiopathic male infertility and relation to serum SHBG concentrations

Sex hormone binding globulin (SHBG) is involved in delivering sex hormones to target tissues. We investigated the association between the (TAAAA)n repeat polymorphism, and Asp327Asn polymorphism in the SHBG gene with semen quality and idiopathic male infertility. We studied 168 men with idiopathic infertility [oligoasthenoteratozoospermia (OAT)] and equal number of age-matched normal controls. The serum levels of SHBG, reproductive and thyroid hormones, and Inhibin B were measured. Semen parameters were also assessed. The genotype assays for the SHBG polymorphism were done using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Baseline SHBG levels tended to be lower in infertile men (21.1±7.2nmol/l) compared to normal fertile men (24.7±7.9nmol/l). SHBG levels tended to be higher among the subjects with the Asn/Asn (25.84±3.6nmol/l) and S/S (24.50±5.4nmol/l) genotypes compared to subjects with the Asp/Asn (24.38±3.2nmol/l) and L/L (18.44±4.2nmol/l) genotypes of the SHBG gene. The genotype frequencies of Asp/Asp were 80.9% in cases and 71.4% in controls (P=0.001). The variant Asp/Asn genotype was associated with a more than 50% reduced risk of infertility (OR: 0.46, 95% CI: 0.25-0.80, P=0.001). Genotype analysis demonstrated six SHBG (TAAAA)n alleles with 6-11 repeats. Long SHBG (TAAAA)n alleles (>8 repeats) were at greater frequency in infertile men than fertile subjects (P=0.001), whereas short SHBG (TAAAA)n alleles (≤8 repeats) tended to be more frequent in fertile men than cases (P=0.001). Men with the 9/X TAAAA repeat genotype displayed a 2.82-fold increased risk of infertility (95% CI: 1.27-4.79, P=0.01). There were strong and significant positive correlations between plasma SHBG and sperm count (r=0.672, P=0.01), sperm motility (r=0.721, P=0.01) and sperm morphology (r=0.574, P=0.02). We concluded that the SHBG Asp237Asn and (TAAAA)n polymorphisms may influence SHBG levels and as a result, male infertility. Multicenter large scale studies are warranted to better elucidate the role of SHBG gene polymorphism in male infertility.     

Evaluation of sperm genomic integrity of normozoospermic men: a prospective study

The objective of our study was to evaluate the incidence of spermatozoa with nuclear DNA strand breaks in patients with normal routine sperm parameters (26 subjects). Sperm DNA fragmentation was measured using TUNEL test assessed in flow cytometer. Variable percentages of sperm with damaged DNA (9.42 +/- 7.68%; range: 2-36) were found. Two categories of patients were distinguished: (1) patients (8 out of 26 subjects) with < or = 4% of TUNEL-positive sperm and (2) patients (18 out of 26 subjects) with > 4% of TUNEL-positive sperm. A significantly lower percentage of normal sperm forms was found in patients with > 4% of TUNEL-positive sperm than in patients with < or = 4% of TUNEL-positive sperm. Moreover, a significant negative correlation (r(s) = -0.50) was noted only between a proportion of normal sperm forms and a proportion of TUNEL-positive spermatozoa. In electron microscope, a large number of spermatozoa with immature chromatin was observed more frequently in subjects with > 4% of TUNEL-positive cells (11 out of 18 subjects). Our results suggest that in some patients with normal routine sperm parameters, DNA fragmentation may be associated with poor sperm morphology. The diminished sperm genomic integrity may result from molecular disturbances in nuclear remodeling process during spermiogenesis. TUNEL assay is a screening tool that may help to discriminate between fertile and infertile men and may help to predict successful in vitro fertilization.     

Antioxidant levels in blood and seminal plasma and their impact on sperm parameters in infertile men

Excess reactive oxygen species (ROS) beyond the scavenging capacity of antioxidants leads to DNA damage and oxidation of lipoprotein components at the cellular and subcellular level. The oxidative stress (OS) adversely affects sperm function by altering membrane fluidity, permeability and impairs sperm functional competence. In the present study, the OS status in seminal plasma and blood serum in infertile men and its relationship with spermatozoa parameters have been investigated. Four groups of infertile men viz., oligozoospermic (n = 15), asthenozoospermic (n = 17), teratozoospermic (n = 19), and oligoasthenoteratozoospermic (n = 9), and healthy fertile controls (n = 40) have been analyzed for superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) in seminal plasma and blood serum. Significant correlation between blood serum SOD and sperm count has been observed in patients (p = 0.018) and controls (p = 0.021). Similarly, significant correlation between blood serum GSH and sperm progressive motility in patients (p = 0.036) and controls (p = 0.029) is observed. The low seminal MDA is associated with increase in sperm progressive motility in patients (p = 0.039) and controls (p = 0.028). Positive correlation is found between increased seminal MDA levels and abnormal sperm morphology in both patients and controls (r = 0.523, p = 0.029; r = 0.612, p = 0.034 respectively). Correlations between blood SOD and sperm count and between blood GSH levels and progressive motility suggest that these can be important biochemical markers in assaying the sperm count and motility. A negative correlation of motility with seminal MDA indicates that sperm membrane lipid peroxidation affects the fluidity and thus mobility of sperm axoneme. This affects functional competence of the sperm and acts like a biological safeguard. The results of the present study suggest the prospects of using the blood serum and seminal plasma antioxidants as a valuable tool to evaluate the sperm reproductive capacity and functional competence.     

Semen Abnormalities,Sperm DNA Damage and Global Hypermethylation in Health Workers Occupationally Exposed to Ionizing Radiation

Background

Cytogenetic studies have demonstrated that low levels of chronic radiation exposure can potentially increase the frequency of chromosomal aberrations and aneuploidy in somatic cells. Epidemiological studies have shown that health workers occupationally exposed to ionizing radiation bear an increased risk of hematological malignancies.

Objectives

To find the influence of occupational radiation exposure on semen characteristics, including genetic and epigenetic integrity of spermatozoa in a chronically exposed population.

Methods

This cross sectional study included 134 male volunteers of which 83 were occupationally exposed to ionizing radiation and 51 were non-exposed control subjects. Semen characteristics, sperm DNA fragmentation, aneuploidy and incidence of global hypermethylation in the spermatozoa were determined and compared between the non-exposed and the exposed group.

Results

Direct comparison of the semen characteristics between the non-exposed and the exposed population revealed significant differences in motility characteristics, viability, and morphological abnormalities (P<0.05–0.0001). Although, the level of sperm DNA fragmentation was significantly higher in the exposed group as compared to the non-exposed group (P<0.05–0.0001), the incidence of sperm aneuploidy was not statistically different between the two groups. However, a significant number of hypermethylated spermatozoa were observed in the exposed group in comparison to non-exposed group (P<0.05).

Conclusions

We provide the first evidence on the detrimental effects of occupational radiation exposure on functional, genetic and epigenetic integrity of sperm in health workers. However, further studies are required to confirm the potential detrimental effects of ionizing radiation in these subjects.     

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.     

Cytogenetic alterations in field workers routinely exposed to pesticides in Bogota flowers farms

Frequency of cytogenetic alterations (micronuclei and chromosome aberrations), DNA repair deficiencies and acetylcholinesterase activity was determined for field workers in Bogotá, Colombia. These workers were regularly exposed to organophosphate and carbamate insecticides while employed on farms for flower growing. Interviews were conducted with 31 workers associated with occupational risk of pesticides exposure and 30 without exposure. A standard cytogenetic assay was used to determine chromosome aberrations and micronuclei frequencies. In addition, a challenge assay assessed response to gamma-rays as an indication of DNA repair deficiencies--cells were exposed to gamma-rays in vitro and the frequencies of chromosome aberrations in post-irradiation metaphase cells were quantified. The data were evaluated for percentage of aberrant cells, cells with chromosome aberrations and frequencies of chromatid breaks per 100 metaphase cells in each worker. The exposed group had a significantly higher frequency of cells with chromosome aberrations and micronuclei as compared with the non-exposed group (p = 0.02). However, the challenge assay did not indicate a significant difference (p > 0.1). These findings require confirmation by further analytical studies involving larger sample. Cytogenetic and toxicological studies, in conjunction with thorough clinical examination are recommended.     

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

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

Spectrum of chromosomal aberrations in peripheral lymphocytes of hospital workers occupationally exposed to low doses of ionizing radiation

Chromosome aberrations frequency was estimated in peripheral lymphocytes from hospital workers occupationally exposed to low levels of ionizing radiation and controls. Chromosome aberrations yield was analyzed by considering the effects of dose equivalent of ionizing radiation over time, and of confounding factors, such as age, gender and smoking status. Frequencies of aberrant cells and chromosome breaks were higher in exposed workers than in controls (P = 0.007, and P = 0.001, respectively). Seven dicentric aberrations were detected in the exposed group and only three in controls, but the mean frequencies were not significantly different. The dose equivalent to whole body of ionizing radiation (Hwb) did appear to influence the spectrum of chromosomal aberrations when the exposed workers were subdivided by a cut off at 50 mSv. The frequencies of chromosome breaks in both subgroups of workers were significantly higher than in controls (< or =50 mSv, P = 0.041; >50 mSv, P = 0.018). On the other hand, the frequency of chromatid breaks observed in workers with Hwb >50 mSv was significantly higher than in controls (P = 0.015) or workers with Hwb < or =50 mSv (P = 0.046). Regarding the influence of confounding factors on genetic damage, smoking status and female gender seem to influence the increase in chromosome aberration frequencies in the study population. Overall, these results suggested that chromosome breaks might provide a good marker for assessing genetic damage in populations exposed to low levels of ionizing radiation.     

Changes in human sperm motion during capacitation in vitro

Spermatozoa from 10 fertile donors and from 10 patients with infertile marriages were washed and centrifuged (time zero, T0), and incubated in vitro in capacitation media for 6 h (T6), or 24 h (T24). At each time individual spermatozoa were classified as being morphologically normal or abnormal, and their movement characteristics were determined using high-speed videomicrography. Zona-free hamster oocytes were added to the T24 sperm suspensions. At all times, morphologically normal spermatozoa from donors and patients swam faster and had greater rolling frequency, flagellar beat frequency and amplitude than did abnormally shaped cells. Morphologically normal spermatozoa from donors exhibited a significant change in their movement pattern at T6. This change, which resembles hyperactivation in other species, was characterized by higher values of amplitude of lateral head displacement, and lower values of linearity, beat frequency and flagellar curvature ratio. In contrast, normal spermatozoa from patients showed only a decrease in straight line velocity at T6, with no other significant changes in movement characteristics. No changes in sperm movement could be demonstrated for the abnormal cells in either group of subjects. In sperm suspensions from donors and patients examined at T24, sperm vigour declined regardless of the morphological type. Spermatozoa from all 10 donors were able to penetrate the zona-free hamster oocytes, but spermatozoa from 5 of the 10 patients failed to penetrate oocytes. Correlations between hamster oocyte penetration and indicators of sperm vigour were demonstrated only for spermatozoa of patients.     

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

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

不育症精子乳酸脱氢酶同功酶LDHx活性测定及其定位研究

采用聚丙烯酰胺凝胶电泳、酶联染色、光密度扫描、分光光度法以及电镜酶细胞化学等方法 ,对 12例生育男性(生育组 )和 14例不育男性 (不育组 )精子 L DHx进行了研究。结果显示 L DHx电泳区带位于 L DH3和 L DH4之间 ,生育组精子 L DHx绝对活性和相对活性均高于不育组 (P<0 .0 5 )。相关分析表明精子密度与 L DHx绝对活性和相对活性均具有相关性 ,在生育组呈正相关 (r=0 .8和 0 .75 ,P<0 .0 5 ) ,在不育组呈负相关 (r=- 0 .76和 - 0 .78,P<0 .0 5 )。 L DHx酶细胞化学定位分析显示 L DHx酶反应颗粒主要分布于精子型线粒体 (STM)和胞质内 ,少量分布于顶体及质膜表面。生育组精子各部位酶反应颗粒多于不育组 ,且不育组精子多有畸形并伴有超微结构改变。上述研究分析提示 ,精子 L DHx活性测定与定位分析可作为检查不育症精子质量的可靠指标 ,为男性不育症的临床诊断提供实验依据     

Decline in seminal quality in Indian men over the last 37 years

Background

Since the first report of a decline in semen quality in 1974, there have been several reports of similar declines across populations. Despite some scattered reports of declining semen quality in the Indian sub-continent, comprehensive studies analyzing semen quality over the last few decades have not been undertaken. We undertook the present study to investigate the temporal trend in semen parameters in Indian populations over a period of 37 years (1979–2016).

Methods

Publications providing semen analysis details for fertile and infertile men from the Indian sub-continent were collected by a thorough literature search. Semen quality data for 6466 normal fertile or presumptive normal men (from 119 studies/data sets) and 7020 infertile men (from 63 studies/data sets) published between 1979 and 2016 were retrieved. We undertook systematic review and quantitative analysis of mean sperm count, motility, normal morphology and other available parameters. Data were analyzed to estimate semen parameters reference values for Indian men and to assess temporal trends in infertile, fertile and all subjects.

Results

Seminal quality shows a decreasing temporal trend and the decrease is higher in infertile than fertile males. In pooled analysis for all individuals, significant (p?<?0.05 or?<?0.001) declines in sperm concentration and normal morphology are observed; however, isolated analysis for each group shows declines without statistical significance. The mean (± SD) semen volume, sperm concentration, total motility, rapid linear progressive motility, normal sperm morphology and sperm viability for Indian fertile men are 2.88?±?0.77 ml, 81.08?±?29.21 million/ml, 66.37?±?10.95%, 52.64?±?15.78%, 56.68?±?20.23% and 72.63?±?8.31%, respectively, whereas in infertile these are 3.07?±?1.27 ml, 37.94?±?26.41 million/ml, 40.22?±?13.76%, 26.79?±?15.47%, 36.41?±?21.66% and 55.25?±?11.99%, respectively. The mean seminal parameter values were significantly lower (p?<?0.001) in infertile as compared to fertile men, except semen volume.

Conclusions

Semen parameters in Indian men have declined with time and the deterioration is quantitatively higher in the infertile group. The study also provides reference values for semen parameters in Indian men.

     

Hyperprolactinemia and luteal insufficiency in infertile patients with mild and minimal endometriosis.

The objective of the present paper was to assess the presence of hormonal alterations in infertile women with stage I or II endometriosis (Group III, n = 20) compared to fertile women without endometriosis (Group I, n = 14) and to fertile women with endometriosis (Group II, n = 7). Serum levels of FSH, LH, estradiol, TSH, and PRL were measured between days 1 and 5 of the early follicular phase; in the luteal phase, three serum samples were collected for progesterone measurement, and endometrial biopsies were performed. Serum estradiol levels were lower (p = 0.035) in infertile patients with endometriosis than in fertile patients without endometriosis. Six infertile patients with endometriosis presented prolactin levels above 20 ng/ml. This was not observed in the other groups. Luteal insufficiency was more frequent in infertile patients with endometriosis (78.9%) than in fertile patients with (42.9%) or without endometriosis (0%). In a multiple logistic regression analysis, only the presence of endometriosis and infertility was significantly associated with luteal insufficiency. The serum levels of LH, FSH, and TSH were not significantly different among the groups. Luteal insufficiency and altered prolactin secretion were associated with endometriosis, and could be important mechanisms causing infertility in this group of patients.