Congenital absence of the vas deferens (CAVD) is a heterogeneous disorder, largely due to mutations in the cystic fibrosis (CFTR) gene. Patients with unilateral absence of the vas deferens (CUAVD) and patients with CAVD in association with renal agenesis appear to have a different aetiology to those with isolated CAVD. We have studied 134 Spanish CAVD patients [110 congenital bilateral absence of the vas deferens (CBAVD) and 24 CUAVD], 16 of whom (six CBAVD, 10 CUAVD) had additional renal anomalies. Forty-two different CFTR mutations were identified, seven of them being novel. Some 45% of the CFTR mutations were specific to CAVD, and were not found in patients with cystic fibrosis or in the general Spanish population. CFTR mutations were detected in 85% of CBAVD patients and in 38% of those with CUAVD. Among those patients with renal anomalies, 31% carried one CFTR mutation. Anomalies in seminal vesicles and ejaculatory ducts were common in patients with CAVD. The prevalence of cryptorchidism and inguinal hernia appeared to be increased in CAVD patients, as well as nasal pathology and frequent respiratory infections. This study confirms the molecular heterogeneity of CFTR mutations in CAVD, and emphasizes the importance of an extensive CFTR analysis in these patients. In contrast with previous studies, this report suggests that CFTR might have a role in urogenital anomalies.

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Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens

The presence of various cytokines, namely hepatocyte growth factor (HGF), interleukin-1 receptor antagonist (IL-1 RA), and interleukins (IL-1 alpha, IL-6, and IL-8), as well as the production of reactive oxygen species (ROS) was investigated in seminal plasma of fertile and infertile patients in order to evaluate the possible value of measuring these substances for the diagnosis of male accessory gland infection, and to assess the possible relationship between oxidative stress and cytokines during leucocytospermia and male accessory gland infection (MAGI). Our findings indicate that all of the measured cytokines seem to be produced locally as well as by white blood cells (WBC) and that, due to the presence of higher numbers of WBC, accessory gland infection may exert a deleterious effect on sperm quality through the production of ROS and/or of particular cytokines such as IL-1 alpha, IL-1 RA, and IL-8. The most specific marker for a sensitivity of 95% in discriminating between cases with or without MAGI is the measurement of IL-6 in seminal plasma. In the absence of WBC several cytokines are constitutively produced and correlate with sperm concentration (HGF, IL-8), alpha-glucosidase (IL-6), and gamma-glutamyltransferase activity (HGF). The measurement of these cytokines in semen may provide clinically useful information for the diagnosis of male accessory gland infection, as well as in the absence of WBC where it can provide information about certain mechanisms of male reproductive function and dysfunction.

The relation between reactive oxygen species and cytokines in andrological patients with or without male accessory gland infection.

Alpha-glucosidase, glycerophosphocholine, and L-carnitine were measured in sperm-free seminal plasma to determine whether these markers reflected the epididymal function of men attending an infertility clinic. The putative markers correlated well with each other (r = 0.66 to 0.70) and in 92% of 283 cases were accurate in categorizing semen as containing normal or subnormal amounts of markers. Glucosidase was considered the best index of epididymal function and was used for a further 306 samples. The ejaculate content of epididymal markers was correlated with testicular volume and serum testosterone below values of 30 ml and 30 nmol/l, respectively. Markers were also correlated with the concentration and motility of spermatozoa in semen. Seventy-one of 425 patients (17%) displayed subnormal epididymal secretions, mainly in association with hypogonadism (Klinefelter syndrome, Kallman syndrome, idiopathic hypogonadotropic hypogonadism) but also in cases of obstructed ducts, maldescended testicles, and local irradiation following hemicastration. Because azoospermic patients had reduced epididymal markers with both high and low FSH levels and a large proportion of men with reduced glucosidase and normal FSH suffered from testicular failure, it is suggested that other indices of testicular function are required for correct interpretation of reduced epididymal markers. Thirteen patients (3%) had low markers for which no cause was apparent; these may be cases of infertility due to isolated epididymal dysfunction.

Epididymal markers in human infertility.

Cryopreservation and freezing-thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post-thaw motility. Various studies have depicted the ultrastructural changes caused by freezing-thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing-thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen-DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing-thawing on sperm chromatin: the native DNA content decreased as did the Feulgen-DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an "overcondensation" state for sperm chromatin after freezing-thawing and a lower conception rate for human semen after cryostorage.

Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Feulgen-DNA cytophotometric studies.

Biochemical analysis was made of specific accessory gland products in the ejaculates of 362 men suffering from various acute inflammatory diseases of the reproductive tract and 33 normozoospermic patients acting as controls. The ejaculate content of the epididymal markers alpha-glucosidase and L-carnitine, but not glycerophosphocholine, was significantly reduced in ejaculates from men with epididymitis; citric acid was reduced in men suffering from prostatitis; both citric acid and alpha-glucosidase were reduced in men suffering from adnexitis. The ejaculate content of epididymal and prostatic markers in prostato-urethritis (adnexitis), where the exact localization of the inflammation was unclear, was not as low as in epididymitis or prostatitis. Seminal vesicle function, as judged from semen volumes and seminal fructose, was not different in these groups of patients. The results, although strongly related to the clinical diagnosis, were unrelated to the microbiological flora of the semen and indicate that both the epididymis and the prostate glands are involved in some forms of adnexitis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2605.1990.tb01040.x

The influence of inflammation of the human male genital tract on secretion of the seminal markers alpha-glucosidase, glycerophosphocholine, carnitine, fructose and citric acid.

Azoospermic semen was obtained from 39 vasectomized men and 93 patients consulting for infertility. The latter underwent bioclinical investigations including measurement of plasma FSH and testicular biopsies. Carnitine content and alpha-glucosidase activity in semen samples were measured. The activity of alpha-glucosidase was determined systematically by a semiquantitative microtechnique and was verified by a spectrophotometric assay. A positive correlation was observed between carnitine and alpha-glucosidase activity. Both parameters were severely diminished in semen from the vasectomized men and the patients suffering from a complete obstruction of the genital tract. Enzyme activity was the most discriminant parameter in terms of sensitivity and specificity. The measurement of alpha-glucosidase in semen is a simple and sensitive method for determining the origin of azoospermia when used in conjunction with assays for plasma FSH.

α-Glucosidase as a Specific Epididymal Enzyme Marker Its Validity for the Etiologic Diagnosis of Azoospermia

The preservation of human semen in liquid nitrogen is now a common practice which has permitted a considerable extension in the application of artificial insemination, and whose advantages are now well proven. However, there is one major problem with the cryo- preservation of human semen: The incontestable drop in fertilizing capacity. Several authors have correlated this with the reduction in motility of a sample after thawing which is, indeed, more marked for human semen than for the majority of domesticated species(especially cattle). It would, however, seem clear that post-thaw motility is not a sufficient criterion of fertilizing power of a frozen semen sample. According to Behrman “The post thaw motility of a frozen preserved semen is not a determinant or index of its subsequent fertility” .3 In addition, certain frozen semen samples appear to loose all their fertility capacity even though residual motility is good.

Biochemical Modifications of Frozen Semen

A review of the recent results of the authors in the field of morphometric studies of Feulgen-stained cell nuclei is given. The data acquisition with Quantimet 720 and data processing with computer were used in the study. The discrimination between male and female cell nuclei from buccal smears on the basis of morphometric data is described. The morphometry-based classification of cells from vaginal smears according to the computer procedure is presented. The results are discussed in the context of the general strategy of morphometric work.

Morphometric studies of cell nuclei by means of image analysis and computer data processing.

Summary Feulgen-stained cell nuclei from a hamster vaginal smear were subjected to measurements by an image analyzing computer, Quantimet 720. Twenty features were measured for each nuclear image. The data were then processed by computer using the TAXI classification procedure. This resulted in the partition of the sample of nuclear images into five classes.

Jean-Claude Czyba

Papers

α-Glucosidase as a Specific Epididymal Enzyme Marker Its Validity for the Etiologic Diagnosis of Azoospermia

Biochemical Modifications of Frozen Semen

Morphometric studies of cell nuclei by means of image analysis and computer data processing.

Computer classification of cells from hamster vaginal smears based on morphometric measurements of Feulgen stained cell nuclei.

Effect of tenotomy on self-reinnervated and randomly reinnervated soleus muscle of rat.