Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality.

BACKGROUND: We showed that in men with a constitutional chromosomal abnormality, DNA fragmentation was significantly higher in chromosomally unbalanced spermatozoa than in spermatozoa with a normal or balanced chromosomal content. These results could be explained by a phenomenon already described in infertile men: abortive apoptosis. OBJECTIVES: To determine whether magnetic-activated cell separation could select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men carrying a structural chromosomal abnormality. MATERIALS AND METHODS: The spermatozoa of ten males with a chromosomal rearrangement were separated into two populations using magnetic-activated cell separation (annexin V (-) and annexin V (+) fractions), in order to study meiotic segregation by fluorescence in situ hybridization, the percentage of spermatozoa with an externalization of phosphatidylserine by annexin V staining and DNA fragmentation by TdT-mediated dUTP nick-end labeling on the whole ejaculate and on selected spermatozoa in the same patient. RESULTS: For all patients, the percentage of spermatozoa with externalization of phosphatidylserine decreased in the annexin V (-) fraction and increased in the annexin V (+) fraction as compared to the frozen-thawed semen sample. The rates of DNA fragmentation were statistically much lower in the annexin V (-) fraction when compared to the rate before magnetic-activated cell separation for all but one patient. Conversely, we observed a statistically significantly higher rate of DNA fragmentation in the annexin V (+) fraction for six patients. After magnetic-activated cell separation, there was a significant increase of normal/balanced spermatozoa in the fraction of annexin V (-) for all patients. Conversely, we observed a significant decrease in the fraction of annexin V (+) for seven patients. DISCUSSION AND CONCLUSIONS: Magnetic-activated cell separation is a promising tool for increasing the selection of healthy spermatozoa, with a decrease in the number of spermatozoa with externalization of phosphatidylserine, DNA fragmentation, and chromosome unbalance, for use in assisted reproductive technologies such as intracytoplasmic sperm injection for males with a chromosomal structural abnormality.

Apoptotic sperm biomarkers and the correlation between conventional sperm parameters and clinical characteristics.

The principal aim of this retrospective study was to examine the relationship between sperm apoptotic biomarkers and the patient's biclinical characteristics, the conventional sperm parameters and the results of assisted reproductive technology. Sperm analysis, activated caspases, annexin V staining for phosphatidylserine (PS) externalisation and labelling assay for DNA fragmentation were assessed in 122 males of infertile couples. Fifty-seven couples were allocated to the natural conception group, and 65 couples underwent IVF or ICSI. Semen of IVF/ICSI patients showed a higher proportion of apoptotic spermatozoa in their spermatozoa when compared with a natural conception group (p < .05). Sperm apoptotic biomarkers correlated with age, FSH, and conventional sperm parameters. DNA fragmentation correlated positively with the percentage of semen having externalised PS (r = .78, p = 0) and activated caspases (r = .71, p = 0). Patients without clinical pregnancy had higher frequency of DNA fragmentation, externalised PS and activated caspases compared to patients with clinical pregnancy (p < .001). The best specificity and greater sensitivity were obtained with the test of the DNA fragmentation compared to the other biomarkers. Among the apoptotic biomarkers, only DNA fragmentation was found to predict natural or assisted pregnancy better than conventional sperm parameters.

[Does hyperandrogenic statute in the polycystic ovary syndrome constitute an obstacle to the success of in vitro fertilization?] / Le statut hyperandrogénique, dans le syndrome des ovaires polykystiques, est t-il une entrave à la réussite de la fécondation in vitro ?

OBJECTIVES: The aim of this study was to evaluate the impact of initial androgenic statute of women with polycystic ovary syndrome (SOPK) on the expected results of in vitro fertilization (FIV). MATERIALS AND METHODS: It is about an exploratory study carried out in 61 unfertile SOPK patients, having profited all from a cycle of FIV/ICSI (intracytoplasmic sperm injection). We have divided our population in two groups according to their hormonal androgenic statute: groups with hyperandrogenism and without hyperandrogenism. We proceeded to note the anthropometric and hormonal parameters as well as results of FIV for each of the two groups. RESULTS: SOPK patients with hyperandrogenism are more obese than their counterparts. Also, they have more oocytes collected after ovarian induction. In addition, we did not reveal any significant differences between our two study groups concerning parameters defining FIV response. CONCLUSION: Our findings suggest that hyperandrogenism statute in SOPK infertile women does not seem to compromise the results of FIV. On the contrary, it would make it possible to optimize the chances to puncture more significant number of oocytes.

Polymorphisms of transcription factor-7-like 2 (TCF7L2) gene in Tunisian women with polycystic ovary syndrome (PCOS).

BACKGROUND AND AIMS: Polycystic ovary syndrome (PCOS) is a common endocrine disorder that affects women in their child-bearing age, and is often associated with insulin resistance and type 2 diabetes (T2DM). Given the overlap between PCOS and T2DM, we investigated the association of transcription factor-7-like 2 (TCF7L2) variants rs4506565, rs7903146, rs12243326, and rs12255372 with the susceptibility to PCOS. SUBJECTS AND METHODS: Study subjects comprised 119 Tunisian women with PCOS (mean age 29.8±4.7years), and 150 control women (mean age 30.6±5.9years). TCF7L2 genotyping was done by the allelic discrimination/real-time PCR method. RESULTS: Minor allele frequencies (MAFs) of rs4506565 (P=0.61), rs7903146 (P=0.68), rs12243326 (P=0.56), and rs12255372 (P=0.60) were comparable between PCOS cases and control subjects. As the four tested TCF7L2 variants were in linkage disequilibrium, 4-locus (rs4506565, rs7903146, rs12243326, rs12255372) haplotype analysis demonstrated that haplotype 2111 was initially negatively associated with PCOS [P=0.035; OR (95% CI)=0.13 (0.02-0.85)], which was later lost upon correcting for multiple comparisons [Pc=0.248]. CONCLUSION: Our data suggest that there is weak or no contribution of TCF7L2 gene polymorphism to PCOS in Tunisian women. Further studies with larger samples are necessary to confirm this observation.

[Sperm immobilizing effect of leaves extracts of Cestrum parqui]. / Effet immobilisant des spermatozoïdes par les extraits des feuilles du Cestrum parqui.

OBJECTIVE: The leaves extracts of Cestrum parqui were reported to have spermicidal activity. The current investigation identified the spermicidal component of the extracts and evaluated its spermicidal potential in vitro, particularly the effects on sperm motility and vitality. METHODS: Sperms were prepared by discontinuous buoyant density gradient centrifugation and incubated with varying concentrations of extract from C. parqui (40-250 µg/ml) at 37°C and 5% CO(2). The mode of spermicidal action was evaluated by sperm motility and vitality at different intervals ranging from 5 to 240 minutes. Morphological changes in human spermatozoa after exposure to the extract were evaluated under transmission electron microscope. RESULTS: A dose- and time-dependent effect of this extract on sperm motility and viability was observed. The mean effective concentration of extracts that induced irreversible immobilization was 250 µg/ml. Transmission electron microscope revealed a significant damage to sperm membrane in head and acrosomal membranes, notable swelling and disruption. CONCLUSION: Our study indicates that this natural extract has potential spermicidal effect in vitro. It can adequately replace nonoxynol-9 in vaginal contraceptives to make them more vaginally safe and ecofriendly.

Obesity-induced impairment of endothelium-dependent vasodilation in Tunisian women.

OBJECTIVE: It is now well recognized that obesity is a major public health concern, and its prevalence has tremendously increased worldwide over the last decades, including Tunisia. As obesity is associated with cardiovascular diseases, the purpose of this study was to investigate the effect of obesity on forearm skin blood flow (FSBF) response to acetylcholine (Ach), an endothelium-dependent vasodilator, in Tunisian women over a wide range of body mass indices (BMIs). SUBJECTS: One hundred and eighty healthy women with an average age of 34+/-6 years, an average height of 162+/-7 cm and an average weight of 78+/-19 kg participated in this investigation. The mean BMIs of the 60 lean, 50 overweight and 70 obese subjects were 22.1+/-0.3, 27.7+/-0.2 and 38.4+/-0.7 kg m(-2), respectively. MEASUREMENTS: The FSBF was measured non-invasively using a laser Doppler flowmeter in response to local infusion of a cumulative dose of Ach. RESULTS: After adjusting for age, the mean response of FSBF to Ach was significantly greater in lean (1168%+/-78) than in overweight (643%+/-38) and obese subjects (323%+/-18) (P=0.002; P<0.0001, respectively), suggesting a reduction of the endothelium-dependent nitric oxide (NO) release by obesity. Our regression analysis also revealed that the maximum FSBF response to Ach (that is, its efficacy) was inversely correlated with BMI, waist and hip circumferences (r=-0.994, P=0.002; r=-0.2, P<0.0001, and r=-0.321, P=0.001, respectively). CONCLUSION: Our data demonstrate a reduction of skin vasodilatory reserve in obese patients and suggest a defect of both endothelial-dependent relaxation and wall compliance associated with obesity.

Detection of DNA fragmentation in human spermatozoa: correlation with semen parameters.

To determine the prevalence of high levels of sperm DNA damage among infertile men with normal and abnormal semen parameters, 90 patients were subdivided into the following three groups. Group A (n = 30): men with normal semen parameters who acted as the controls. Group B (n = 30): asthenozoospermic men and group C (n = 30): teratozoospermic men, suffering from male infertility. DNA damage was evaluated by the rate of DNA fragmentation index (DFI) as assessed by the terminal desoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. It was found that the difference was not significant between the percentage of DFI in patients with asthenozoospermia and the normospermic men (9.46% +/- 8.68 and 8.19 +/- 6.84 respectively, P-value not significant). The patients with teratozoospermia showed a significantly higher percentage of DNA fragmentation compared with the controls (respectively 21.37 +/- 17.26% and 8.19 +/- 6.84%, P < 0.001). There was a positive correlation between abnormal sperm morphology and the DFI (r = 0.44, P < 0.01) in group C. It is concluded that the impairments of sperm parameters were associated with an increase of DNA fragmentation; this association was strictly related to atypical forms.

Establishment of reference serum insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels of Tunisian children during childhood and adolescence.

BACKGROUND: The aim of the present study is to establish age- and sex-related reference ranges of serum IGF-I and IGF binding protein-3 (IGFBP-3) levels in a pattern of Tunisian children. SUBJECTS AND METHODS: Two hundred healthy Tunisian children (103 boys and 97 girls), aged between 6 and 16 yr, were considered in the study. RESULTS: Mean serum levels of IGF-I and IGFBP-3 are observed to be higher in girls compared to boys of the same age interval. However, these differences were statistically significant only in pubertal ages (11-14 yr) for IGF-I and in pre-pubertal ages (6-10 yr) for IGFBP- 3 (p<0.05). Steeper variations in IGF-I concentrations were obtained earlier in girls than in boys (11-12 vs 12-13 yr, Tanner stage 3-4). Peak of IGF-I levels are observed at almost the same age interval (12-14 yr). IGFBP-3 levels significantly increased at steeper variations of IGF-I for both sexes followed by steady values. CONCLUSIONS: Variations of IGF-I and IGFBP- 3 with the considered parameters (sex, age, and puberty stage), which concord with previous studies on various populations, emphasize the importance of locally established reference levels to construct a SD score prediction model. Establishment of reference serum IGF-I and IGFBP-3 ranges during childhood and adolescence in Tunisian subjects can help to enhance the diagnostic efficiency of IGF-I and IGFBP-3 in evaluating growth disorders in our population.

[DNA sperm methylation in assisted reproductive techniques]. / Méthylation du spermatozoïde en Assistance médicale à la procréation (AMP).

In the last few years, many tests were developed to study the fertilizing properties of the spermatozoa. However none of them was useful to obtain a prognostic factor. Indeed, the integrity of the spermatic DNA is also necessary to a successful fertilization for obtaining a pregnancy. DNA integrity could be evaluated by the measurement of the level of DNA methylation. Indeed, in the mammals, the methylation of the ADN is involved in diverse processes amongst them the regulation of the genome expression during the embryonic development. The objective of this study is to evaluate the impact of the level of methylation of the spermatic DNA in the success of in vitro fertilization (IVF), in terms of rate of fertilization, quality of the embryos and rate of pregnancy. The immunostaining of the 5-methylecytosine, then the quantification by image analysis or with flow cytometry, allowed an objective evaluation of the level of total methylation of spermatic DNA. Our data show that the level of DNA methylation influences neither the fertilization rate nor the embryos quality. On the other hand, the rate of pregnancy is decreased if the total level of DNA methylation is lower than a threshold value. The level of spermatic DNA methylation represents a new parameter of spermatic maturation.

[Ovarian hyperstimulation induced by a GnRH agonist. About one case]. / Hyperstimulation ovarienne induite par un agoniste de la GnRH. A propos d'un cas.

We report a case of ovarian hyperstimulation induced by a GnRH agonist (Decapeptyl in a patient aged of 23 years and having 3 years of primary infertility of male origin. Twelve days after agonist administration, several ovarian follicles, great-sized, and with a rate of elevated serum oestradiol have been noted. After triggering of the ovulation by 5000 IU of HCG, oocyte retrieval permitted the collection of 4 oocytes 3 of which were mature. Only one embryo with 4 cells has been transferred 48 hours after intracytoplasmic sperm injection fertilization (ICSI), but there was no pregnancy. Ovarian hyperstimulation induced by GnRH agonist is a rare event and only a few cases have been reported. The development of multiple follicles after the administration of an agonist is a paradoxal answer of the ovary to the pituitary desensitization without a clarified physiopathology. The hypothesis of a direct action of the agonist on the ovary is likeliest. Triggering of ovulation by human chorionic gonadotrophin (HCG) has been achieved by certain authors. Fertilization of oocytes and transfers of embryos have succeeded in certain cases, but only one pregnancy has been reported that led to a living birth.

[Factitious Cushing syndrome: two case reports]. / Le syndrome de Cushing factice: deux observations.

INTRODUCTION: Search of corticosteroid use is an important precaution before any exploration of hypercorticism. Nevertheless, this use may be factitious leading to serious misdiagnosis and pitfalls. EXEGESIS: We report two cases of 29 and 60 years old male patients, referred for hypercorticism with patent features including metabolic and trophical signs. Laboratories findings revealed unelevated urinary cortisol values (40 and 27 microg/ day) in both cases with normal ACTH and normal plasmatic cortisol response to synacthène (32,5 and 35 microg/dl). Corticosteroid use, initially denied, was finally approved by the patients (hydrocortisone and dexamethasone for the first patient and betametasone for the second). So, they were assigned to psychiatric therapy. CONCLUSION: Our cases emphasize that corticosteroid misuse must be evoked in patients with unexpected hormonal assessment of pituitary adrenal axis despite patent clinical features of hypercorticism. Several specific tests are actually available for detect factitious Cushing syndrome. However, differential diagnostic remains difficult mainly with pseudo Cushing and cyclical Cushing syndromes.

Parathyroid hormone concentrations during and after two periods of high intensity exercise with and without an intervening recovery period.

The purpose of this study was to examine the effect of a recovery period between two periods of exercise on bone metabolism and related hormones. Concentrations of serum parathyroid hormone ([PTH]), plasma ionized calcium ([Ca(2+)]) and total calcium were measured. A group of 12 healthy men aged 20-27 years participated in this study. They took part in two exercise protocols (P(1) and P(2)) on two separate weeks. The exercise in P(1) comprised two successive periods of 21 min each at 70% and 85% of maximal oxygen uptake; P(2) comprised two periods of exercise at the same intensities but separated by 40 min of recovery. Venous blood samples were collected 1 day before the sessions (control), before each protocol, during (7th and 21st min), at the end (42nd min in P(1) and 82nd min in P(2)) and after 24 h of recovery. The [PTH] was significantly elevated during the two protocols ( P<0.01), remained raised in P(1) after 24 h of recovery ( P<0.05) and was significantly lower ( P<0.01) at the end of P(2) when compared to P(1). The [Ca(2+)] decreased significantly during and at the end of the two protocols ( P<0.01) and had returned to control values after 24 h of recovery. Plasma lactate concentration increased during the two protocols ( P<0.01) and returned to control values after recovery. These results indicate firstly that [Ca(2+)] decreases during continuous exercise as [PTH] increases and remains raised after 24 h of recovery, secondly that a recovery period between two periods of exercise attenuates the variations in [Ca(2+)] and [PTH], and thirdly that recovery may have anabolic effects on bone. However, the small physiological changes observed prevent us from forming any firm conclusion about this.

[Post-partum autoimmune thyroiditis in a patient presenting with Sheehan's syndrome]. / Thyroïdite auto-immune du post-partum chez une patiente présentant un syndrome de Sheehan.

A 30-year-old women developed post-partum thyroiditis associated with post- partum pituitary infarction. Pituitary stimulation tests demonstrated global pituitary deficiency but there was no thyrotropin response to TRH stimulation and free T4 was normal. Associated goiter and high circulating levels of microsomal antibodies led to the diagnosis of post-partum thyroiditis associated with pituitary deficiency. Two cases of this unusual association have been reported. The underlying pathogenesis might involve post-partum immunological rebound and/or hypocorticism-induced immunological disorders.

Influence of head-down bed rest on the circadian rhythms of hormones and electrolytes involved in hydroelectrolytic regulation.

We investigated in six men the impact of a 17-day head-down bed rest (HDBR) on the circadian rhythms of the hormones and electrolytes involved in hydroelectrolytic regulation. This HDBR study was designed to mimic an actual spaceflight. Urine samples were collected at each voiding before, during and after HDBR. Urinary excretion of aldosterone, arginine vasopressin (AVP), cyclic guanosine monophosphate (cGMP), cortisol, electrolytes (Na+ and K+) and creatinine were determined. HDBR resulted in a significant reduction of body mass (P < 0.01) and of caloric intake [mean (SEM) 2,778 (37) kcal.24 h(-1) to 2,450 (36) kcal.24 h(-1), where 1 kcal.h(-1) = 1.163 J.s(-1); P< 0.01]. There was a significant increase in diastolic blood pressure [71.8 (0.7) mmHg vs 75.6 (0.91) mmHg], with no significant changes in either systolic blood pressure or heart rate. The nocturnal hormonal decrease of aldosterone was clearly evident only before and after HDBR, but the day/night difference did not appear during HDBR. The rhythm of K+ excretion was unchanged during HDBR, whereas for Na+ excretion, a large decrease was shown during the night as compared to the day. The circadian rhythm of cortisol persisted. These data suggest that exposure to a 17-day HDBR could induce an exaggeration of the amplitude of the Na+ rhythm and abolition of the aldosterone rhythm.

Effects of 17 days of head-down bed rest on hydro-electrolytic regulation in men.

Prolonged periods of head-down bed rest (HDBR) are commonly used to mimic the effects of microgravity. HDBR has been shown to produce, as in space, a cephalad redistribution of circulating blood volume with an increase in central blood volume which induces the early adaptations in blood volume regulating hormones. Changes in atrial natriuretic peptide (ANP), arginine vasopressin (AVP), renin activity and aldosterone have been observed. Many reports describe these endocrine adaptations but few investigations of rhythms are in the literature. We proposed to evaluate the circadian rhythms of the hormones and electrolytes involved in the hydro-electrolytic regulation during a HDBR study which was designed to simulate a 17-day spaceflight (Life and Microgravity Spacelab experiment, LMS, NASA).

Orthostatic tolerance and hormonal changes in women during 120 days of head-down bed rest.

BACKGROUND: Women will be included as mission specialists in the upcoming International Space Station program. This paper describes the changes in volume-regulating hormones and determines the degree of degradation in orthostatic tolerance in a group of women after 120 d of bed rest. The aim of this study was to test a countermeasure program to be used by women during long-duration spaceflights. METHODS: For 120 d of -6 degrees head-down bed rest (HDBR), eight healthy women were assigned either to a no-countermeasure (No-CM, n = 4), or to a countermeasure (CM, n = 4) group. In the countermeasure group, exercise began after 2 wk, pharmacological agents were given during the 1st and 3rd mo, and the "Centaur" suit was worn on the last day of bed rest and during the day time for several days after bed rest. Diet supplements were taken during the 1st and 4th mo of HDBR. Tilt tests were run before and after HDBR. RESULTS: After the HDBR, none of the CM subjects, had pre-syncopal or syncopal symptoms during tilt tests: BP was well maintained in the CM group, while heart rate and BP changed in the No-CM group. In plasma, atrial natriuretic peptide (ANP) increased in both groups and remained high throughout HDBR, while aldosterone increased and remained elevated in the No-CM group. Natriuresis was decreased during HDBR. CONCLUSION: The CM protocols used during this study were efficient and prevented orthostatic intolerance for the four CM subjects. It would be necessary to obtain more data regarding this set of CM protocols on female subjects to lead to statistical and formal conclusions.

Dietary docosahexaenoic acid-enriched phospholipids normalize urinary melatonin excretion in adult (n-3) polyunsaturated fatty acid-deficient rats.

Melatonin (MEL) plays an essential role in physiologic functions associated with darkness. We examined the effects of docosahexaenoic acid (DHA)-enriched phospholipids from pig brains (BPL) or hen eggs (EPL), as sources of DHA, on lipid FA composition of pineal membranes and daytime and nighttime concentrations of 6-sulfatoxymelatonin (aMT6) in adult male control and (n-3)-deficient rats fed BPL and EPL diets for 5 wk. In two experiments, at 3 wk of age, rats were divided into subgroups and fed semipurified diets containing either peanut oil [(n-3)-deficient group] or peanut plus rapeseed oil (control group) and two dietary formulas containing either 3.5 g/100 g diet of BPL (Experiment 1) or 5.0 g/100 g diet of EPL (Experiment 2). BPL and EPL diets provided approximately 200 mg of DHA/100 g diet. During the daytime, aMT6 concentrations were not significantly different among groups. Conversely, the (n-3)-deficient rats had significantly lower nighttime aMT6 concentrations than the control rats. BPL and EPL did not affect urinary nighttime aMT6 concentration in the control group, whereas (n-3)-deficient + BPL or EPL groups exhibited significantly higher nighttime aMT6 concentrations than the (n-3)-deficient group (76 and 110%, respectively). The level of DHA was significantly higher in the pineal glands of control rats than in (n-3)-deficient rats. In rats fed EPL and BPL, the level of DHA reached a plateau, between 10 and 11 mg/100 mg total fatty acids in control + BPL or EPL and (n-3)-deficient + BPL or EPL groups. These findings suggest that new DHA-enriched formulas may be used as an efficient alternative source of (n-3) polyunsaturated fatty acids to normalize MEL secretion.