R. D’Agata

γ-Aminobutyric acid (GABA) a and b receptors mediate the stimulatory effects of GABA on the human sperm acrosome reaction: interaction with progesterone

OBJECTIVE To evaluate which gamma-aminobutyric acid (GABA) receptor mediates the stimulatory effects of this neurotransmitter on the human sperm acrosome reaction, and to examine the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA(A) receptor. DESIGN Prospective study. SETTING A university clinic of andrology. PATIENT(S) Men with normal sperm analysis parameters. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The acrosome reaction of motile spermatozoa. RESULT(S) The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA(A) receptor antagonist, and only partially by saclofen, a GABA(B) receptor antagonist. Accordingly, muscimol, a GABA(A) receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen, a GABA(B) receptor agonist, was less effective. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome-reacted spermatozoa compared with progesterone alone. However, this increase was less than a simple addition of effects, suggesting that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the ability of progesterone to induce acrosome reaction was tested in the presence of bicuculline, which suppressed the stimulatory effects of progesterone. Given that the GABA(A) receptor is linked to the chloride channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Picrotoxin completely suppressed the acrosome reaction induced by progesterone and only partially suppressed that caused by GABA. CONCLUSION(S) gamma-Aminobutyric acid stimulated the acrosome reaction in human spermatozoa, acting mainly through the GABA(A) receptor and to a lesser extent through the GABA(B) receptor. Progesterone interacted with the GABA(A) receptor to induce the acrosome reaction, and the functional integrity of the chloride channel was vital for this effect.

Spontaneous transmission from a father to his son of a Y chromosome microdeletion involving the deleted in azoospermia (DAZ) gene

Microdeletions of the so-called azoospermia factor (AZF) locus of the Y chromosome long arm (Yq) have been recognized as an etiological factor of severe oligozoospermia or azoospermia. Because of this, patients affected are generally infertile unless assisted reproductive techniques are used. We report the case of an oligozoospermic patient (proband) who inherited an extensive Yq microdeletion from his father through a spontaneous pregnancy. Leukocyte DNA was extracted using a commercially available kit. A total of 22 pairs of sequence-tagged site (STSs) based primers, spanning the entire AZF region, were used for the screening. Both the proband and his father carried a Yq microdeletion of the most distal AZF subregion (AZFc) where the deleted in azoospermia (DAZ) gene is located. The proband’s father was a sixty-nine-yr-old man who had 3 other children, 2 females and 1 male. This case adds further evidence that the deletion of the DAZ gene is associated with different phenotypic expressions, including normal fertility.

Glucocorticoids inhibit gonadotropin-releasing hormone by acting directly at the hypothalamic level

Glucocorticoids, the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, suppress gonadotropin release by acting at the level of the pituitary gland. However, experimental evidence suggests that they may also act at the hypothalamic level to suppress gonadotropin-releasing hormone (GnRH) release. The lack of a direct demonstration of this assumption, prompted us to evaluate the effects of glucocorticoids on hypothalamic GnRH release from individually-incubated hemi-hypothalami explanted from male rats. Since testosterone (T), dihydrotestosterone (DHT), and progesterone suppress GnRH release and androgens potentiate the effects of glucocorticoids on GnRH release, we studied also the interaction of these steroids with glucocorticoids on GnRH release. Corticosterone (B), the main glucocorticoid of the rodents with greater affinity for the type I glucocorticoid receptor, and dexamethasone (DEX), a synthetic type II glucocorticoid receptor agonist, were able to suppress basal GnRH release in a concentration-dependent fashion. DEX induced a more profound suppression of GnRH release. Neither T (0.1 nM) nor DHT (0.01 nM) modulated the suppressive effects of low (10 nM) or high (100 nM) concentrations of B on GnRH release. On the other hand, progesterone counteracted the suppressive effect of low concentrations of B (10 nM) on GnRH release, but had no effect on the suppression caused by a higher concentration of B (100 nM). The ability of glucocorticoids to inhibit directly GnRH release suggests that these stress-responsive hormones act also at the hypothalamic level to suppress the reproductive function. The suppressive effect of B was not modulated by androgens, but it was neutralized by progesterone, al least when B was used at low concentrations. We speculate that this steroid “protects” the GnRH-secreting neuron only during basal, but not stress-induced, HPA axis activity when the concentrations of glucocorticoids are more elevated.

Ultrasonographic evaluation of patients with male accessory gland infection.

MAGI is the inflammation of the accessory male glands that notoriously exerts a negative influence on male fertility. The diagnosis is integrated by clinical, laboratory and ultrasound evaluation. In particular, the ultrasound criteria were published in 1999. The aim of this study was to analyse the sensitivity and specificity of additional diagnostic ultrasound criteria as well as of conventional criteria in a selected category of infertile patients with MAGI. To accomplish this, 100 patients with MAGI were evaluated by scrotal and transrectal ultrasound by three different operators. The control group consisted of 100 age‐matched healthy men. Statistical analysis was performed to evaluate sensitivity, specificity, positive and negative predictive values, and ROC curve analysis. The results showed that additional ultrasound criteria had a diagnostic accuracy similar to traditional criteria. The threshold value of two criteria for each diagnostic category (traditional and additional criteria) obtained high values of sensitivity and specificity. In conclusion, this study confirms the validity of the ultrasound criteria of MAGI previously published; in addition, it suggests the clinical utility of other indicators in clinical practice with good diagnostic accuracy and finally it establishes a clear threshold ultrasonographic value for the diagnosis of MAGI.

High frequency of sexual dysfunction in patients with male accessory gland infections.

The aim of this study was to administer to two groups of patients with male accessory gland infection (MAGI), respectively, with positive or negative alterations in ultrasonography, a new diagnostic interview, arbitrarily named structured interview about MAGI (SI‐MAGI), to evaluate differences between these groups, especially about the prevalence of sexual dysfunction. After ultrasound examinations, patients with MAGI were divided into two age‐matched groups: positive and negative for ultrasound signs (US+ and US−). The SI‐MAGI was structured into four domains (urinary tract symptoms, ejaculatory pain or discomfort, sexual dysfunction and quality‐of‐life impact) for a total of 30 questions with four possible answers. Infertile patients of MAGI US+ group showed scores significantly higher than MAGI US− and healthy control group in all domains (anovaP < 0.005) in relation to scores of patients with MAGI US+ and US−: in domain 1 = 16.0 ± 0.5 vs 9.0 ± 0.5, domain 2 = 21.0 ± 1.0 vs 11.0 ± 1.0, domain 3 = 23.0 ± 0.5 vs 12.0 ± 1.0 and, finally, in domain 4 = 13.0 ± 2.0 vs 6.0 ± 1.0. In particular, a higher frequency of sexual dysfunction (52%) was detected in MAGI US+ group when compared with MAGI US− (28%). This study introduces a specific set of questions, which combined with the sperm analysis, microbiological and ultrasound investigations, that altogether better express the clinical presentation of MAGI. Finally, MAGI US+ group showed a high percentage of sexual dysfunction.

Environmental car exhaust pollution damages human sperm chromatin and DNA.

Objective: The adverse role of traffic pollutants on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. Methods: To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled. All of them were interviewed about their lifestyle. Hormone, semen samples, and environmental and biological markers of pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. Results: LH, FSH, and testosterone serum levels were within the normal range in tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were significantly lower in these men compared with controls. Motorway tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a late sign of apoptosis, compared with controls. A significant direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship. Conclusion: This study showed that car exhaust exposure has a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance of the men exposed, but also for the offspring health.

Semen alterations and flow-citometry evaluation in patients with male accessory gland infections.

Male accessory gland infections (MAGI) represent a major cause of male infertility mainly through the secretory dysfunction of the prostate, seminal vesicles, and epididymis. This study was undertaken to evaluate conventional and non-conventional sperm parameters in these patients, therefore 150 patients with MAGI were selected. Each of them underwent to two sperm analyses and evaluation of DNA fragmentation mitochondrial membrane potential (MMP), phosphatidylserine (PS) externalization, chromatin compactness, by flow cytometry. Results showed that patients with MAGI had a lower sperm progressive motility (11.4 ±5.0 vs 34.0 ±7.0%), and percentage of normal forms (9.0 ±3.7 vs 33.0 ±13.0%) compared to controls, instead, these patients showed higher number of seminal white blood cells (2.2 ±1.0 vs 0.4 ±0.6 106/ml). Patients with MAGI showed a higher number of spermatozoa with DNA fragmentation compared to controls (8.2 ±3.0 vs 1.0 ±1.0%). In addition, they have also a higher percentage of spermatozoa with low MMP (28.0 ±4.0 vs 2.0 ±2.0%). Patients with MAGI, showed a higher percentage of spermatozoa with PS externalization (8.0 ±4.0 vs 3.0 ±3.0%), an early sign of apoptosis, and lower percentage of viable spermatozoa (64.5 ±12.0 vs 88.0 ±10.0%). An increased percentage of spermatozoa with abnormal chromatin compactness (18.0 ±4.0 vs 5.0 ±3.0%) was found in patients with MAGI. In conclusion, patients with MAGI show alterations of conventional and biofunctional sperm parameters compared to controls. These results suggest to consider the flow cytometry evaluation among the diagnostic tools for male infertility.

High prevalence of thyroid dysfunction in pregnant women

Background/Aim: Maternal thyroid dysfunction during pregnancy has been associated with adverse obstetric and neonatal outcomes. This prospective study evaluates the prevalence of these disorders in pregnant women. Subjects and methods: Serum levels of TSH, free T4 (fT4), and thyroperoxidase antibodies (TPO-Ab) were measured in 951 women at different gestational ages of pregnancy. Trimester-specific reference ranges for TSH were used to classify pregnant women into five groups: 1) Overt hypothyroidism (OH); 2) Subclinical hypothyroidism (SCH); 3) Isolated hypothyroxinemia (IH); 4) Low TSH (isolated or associated with high fT4); and 5) Normal. A classification was made also according to the lower and upper ranges provided by the manufacturer for thyroid hormones. Pregnant women who were at a high risk of developing thyroid disease were identified. Results: Altogether, 117 women (12.3%) had hypothyroidism and 25 (2.6%) had low TSH. The prevalence of both OH and SCH was higher in the high-risk group than in the low-risk group, but 17.9% of women with hypothyroidism were classified at low-risk. A family history of thyroid disorders and TPO-Ab positivity increased the risk of SCH. Using non-pregnant reference range for TSH, 10.6% of women were misclassificated. Conclusions: The high prevalence of hypothyroidism observed in this study suggests that accurate thyroid screening with trimester specific reference ranges should be warranted, particularly in areas with mild to moderate iodine deficiencies.

Ultrasound characterization of the seminal vesicles in infertile patients with type 2 diabetes mellitus.

Male patients with type 2 diabetes mellitus (DM) may experience infertility because the disease affects negatively many aspects of reproduction, including seminal vesicle (SV) function. The aim of this study was to evaluate the ultrasound characteristics of the SVs of infertile patients with DM because no such data are available in these patients. To accomplish this, 25 infertile patients with type 2 DM and no other known causes of sperm parameter abnormalities were selected. Two different control groups were also enrolled: healthy men with idiopathic infertility (n=25) and infertile patients with male accessory gland infections (MAGI) (n=25), a well-studied clinical model of SV inflammation. Patients and controls underwent prostate-vesicular transrectal ultrasonography after 1 day of sexual abstinence before and 1h after ejaculation. The following SV ultrasound parameters were recorded: (1) body antero-posterior diameter (ADP); (2) fundus APD; (3) parietal thickness of the right and left SVs; (4) number of polycyclic areas within both SVs; (5) fundus/body ratio; (6) difference of the parietal thickness between the right and the left SV; and (7) pre- and post-ejaculatory APD difference. Patients with DM had a significantly (p<0.05) higher F/B ratio compared to controls and patients with MAGI. Only patients with MAGI had a significantly (p<0.05) higher number of polycyclic areas. Controls and MAGI patients have a similar pre- and post-ejaculatory difference of the body SV APD, whereas this difference was significantly (p<0.05) lower in patients with DM. In conclusion, this study showed that infertile patients with DM have peculiar SV ultrasound features suggestive of functional atony.

Effects of cancer and anti-neoplastic treatment on the human testicular function

Men with testicular neoplasia (TN) and Hodgkin’s disease (HD) are those who more frequently require treatment for fertility because these malignancies affect patients during the reproductive age and because the early diagnosis and the improved anti-neoplastic treatments cure most of these patients. Although some of them can father a child spontaneously, assisted reproductive techniques allow fatherhood to patients with severe spermatogenesis impairment and this possibility has raised concern about the long-term consequence of the testicular damage induced by chemo- and/or radiotherapy. This paper reviews the effects of cancer per se and of anti-neoplastic treatments on gonadal function, sperm aneuploidy rate and sperm DNA integrity. A debate is still open as to whether TN or HD per se may impair spermatogenesis. Many studies have shown that this is the case, albeit others have challenged this view. Chemo- and/or radiotherapy affects negatively gonadal function, rendering almost all patients azoospermics. However, spontaneous pregnancies and a high degree of spermatogenesis recovery occur with time. A large body of literature on sperm chromosome complement suggests an increased rate of structural and numerical chromosome abnormalities in patients with cancer during anti-neoplastic treatment. A minority of them has, however, shown that this effect disappears with time. An interesting and relatively new aspect is the study of sperm DNA integrity in patients with TN and HD particularly following chemo- and/or radiotherapy. The scanty information available seems to suggest that these patients have a permanent or at least a long-lasting DNA fragmentation in their spermatozoa.