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1.
Hum Reprod ; 33(5): 887-894, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29481647

ABSTRACT

STUDY QUESTION: Are cohesins SA1/SA2 and the NAD-dependent deacetylase SIRT1 involved in telomere homeostasis of cumulus cells and thus eligible as biomarkers of follicular physiology and ovarian aging? SUMMARY ANSWER: SA1/SA2 cohesins and SIRT1 are associated with telomere length in cumulus cells and may be eligible biomarkers of follicular physiology and ovarian aging. WHAT IS KNOWN ALREADY: In somatic cells, cohesins SA1/SA2 mediate sister chromatid cohesion at the telomere termini (for SA1) and along chromatid arms (for SA2). The NAD+-dependent protein deacetylase Sirtuin 1 (SIRT1), which preserves DNA integrity from oxidative stress, may also modulate genome stability and telomere length. STUDY DESIGN, SIZE, DURATION: Collectively 280 cumulus/oocyte complex samples were recovered from a total of 50 women undergoing in vitro fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cumulus cells were separated from the oocyte-cumulus complex. DNA and total mRNA were extracted from cumulus cells and assayed for telomere length and for SA1, SA2 and SIRT1 gene expression profiling. Telomere length was determined by quantitave PCR and analyzed relative to the single copy of the housekeeping gene (albumin) to generate a T/S ratio (Telomere/single copy gene). Gene expression levels of SA1, SA2 and SIRT1 mRNA were assayed by quantitative RT-PCR and confirmed by western blotting and immunofluorescent studies (SIRT1). SA1/SA2 and SIRT1 gene expression levels and telomere length analysis of patients/samples were ranked in relation to their clinical setting parameters (BMI, age) and to the number of oocyte retrieved. MAIN RESULTS AND THE ROLE OF CHANCE: SA1 and SA2 transcripts were both detected in all cumulus cells analyzed and the relative amount showed a clear decreasing trend according to the age of patients. A significant increase in SA1 and SA2 was disclosed in high responder women (>6 oocytes retrieved) compared to poor responders (<4 oocytes) (P < 0.05). Furthermore, statistically significant positive correlations were also recorded between the transcripts levels of the two cohesin molecules (r = 0.89; P < 0.05) and, to a lesser extent, between telomere length and SA1 (r = 0.42; P < 0.001) and SA2 (r = 0.36; P < 0.001) mRNA levels. SIRT1 expression was also significantly increased in high responders (>6 oocytes) compared to poor responders. Significant correlations were found between SIRT1 and SA1 (r = 0.69; P < 0.001), between SIRT1 and SA2 (r = 0.78; P < 0.001), and between SIRT1 and telomere length (r = 0.36; P < 0.001). However, in the older patient group (>38 years), SIRT1 mRNA levels were twice as high as the levels recorded in the younger patient cohort (<34 years). Western blot analysis and immunofluorescent studies confirmed the increments in SIRT1 protein levels in patients over 38 years old. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Cumulus/oocyte complexes were retrieved by patients undergoing ovarian stimulation protocol for IVF. We cannot exclude the possibility that different stimulation protocols affect the correlations highlighted in this study. Future investigations should shed light on cumulus cells molecular profile according to different stimulation protocols. WIDER IMPLICATIONS OF THE FINDINGS: The overall results of our study point to the involvement of cohesins SA1/SA2 and SIRT1 deacetylase in telomere homeostasis in cumulus cells and highlight their possible eligibility as biomarkers of follicular physiology and ovarian aging. STUDY FUNDING/COMPETING INTEREST(S): Merck Serono S.P.A Italy sponsored the study with financial support. There are no competing interests to declare.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cumulus Cells/metabolism , Ovary/metabolism , Sirtuin 1/metabolism , Telomere Homeostasis/physiology , Adult , Biomarkers/metabolism , Female , Humans , Oocyte Retrieval , Ovarian Follicle/metabolism , Ovulation Induction , Telomere/metabolism , Cohesins
2.
Hum Reprod ; 32(11): 2324-2331, 2017 11 01.
Article in English | MEDLINE | ID: mdl-29040583

ABSTRACT

STUDY QUESTION: Are polymorphisms of taste receptor genes associated with male infertility? SUMMARY ANSWER: This study has showed the associations between three single nucleotide polymorphisms (SNPs) in taste receptors genes (TASR) and male infertility. WHAT IS KNOWN ALREADY: Recent studies showed the expression of taste receptors in the testis and in spermatozoa, suggesting their possible role in infertility. The vast genetic variability in taste genes results in a large degree of diversity in various human phenotypes. STUDY DESIGN, SIZE, DURATION: In this study, we genotyped 19 SNPs in 12 taste related genes in a total of 494 Caucasian male patients undergoing semen evaluation at the Centre of Couple Sterility of the Siena University Hospital. Consecutive patients were enrolled during infertility investigations from October 2014 to February 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Median age of the patients was 36 years (18-58) and 141 were smokers. Genotyping was performed using the allele-specific PCR. The statistical analysis was carried out using generalized linear model (GLM) to explore the association between age, smoking, the genetic polymorphisms and sperm parameters. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that the homozygous carriers of the (G) allele of the TAS2R14-rs3741843 polymorphism showed a decreased sperm progressive motility compared to heterozygotes and (A) homozygotes (P = 0.003). Moreover, the homozygous carriers of the (T) allele of the TAS2R3-rs11763979 SNP showed fewer normal acrosome compared with the heterozygous and the homozygous carriers of the (G) allele (P = 0.002). Multiple comparisons correction was applied and the Bonferroni-corrected critical P-value was = 0.003. LIMITATIONS, REASONS FOR CAUTION: The analysis is restricted to SNPs within genes and to men of Caucasian ancestry. WIDER IMPLICATIONS OF THE FINDINGS: In silico analyses strongly point towards a functional effect of the two SNPs: TAS2R14-rs3741843 regulates TAS2R43 expression, a gene that is involved in cilia motility and therefore could influences sperm mobility; the (T) allele of TAS2R3-rs11763979 increases the expression of the WEE2 antisense RNA one gene (WEE2-AS1). According to Genotype-Tissue Expression (GTEx) project the WEE2 gene is expressed in the testes where presumably it has the role of down regulating meiotic cell division. It is plausible to hypothesize that the WEE2-AS1 increased expression may down regulate WEE2 which in turn can alter the natural timing of sperm maturation increasing the number of abnormal sperm cells. STUDY FUNDING/COMPETING INTEREST(S): None.


Subject(s)
Infertility, Male/genetics , Polymorphism, Single Nucleotide , Receptors, G-Protein-Coupled/genetics , Sperm Motility/genetics , Adolescent , Adult , Alleles , Genotype , Humans , Male , Middle Aged , Young Adult
3.
Andrology ; 3(2): 203-12, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25755131

ABSTRACT

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.


Subject(s)
Infertility, Male/complications , Spermatozoa/abnormalities , Humans , In Situ Hybridization, Fluorescence , Male , Microscopy, Electron, Transmission , Spermatozoa/ultrastructure
4.
Andrologia ; 47(9): 1012-9, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25382683

ABSTRACT

This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable.


Subject(s)
Alkanesulfonic Acids/blood , Aneuploidy , Asthenozoospermia/metabolism , Caprylates/blood , Chromosome Aberrations/statistics & numerical data , DNA Fragmentation , Fluorocarbons/blood , Oligospermia/metabolism , Spermatozoa , Adult , Alkanesulfonic Acids/metabolism , Asthenozoospermia/epidemiology , Asthenozoospermia/genetics , Caprylates/metabolism , Case-Control Studies , Chromatography, High Pressure Liquid , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , Diploidy , Environmental Exposure/statistics & numerical data , Flow Cytometry , Fluorocarbons/metabolism , Humans , In Situ Hybridization, Fluorescence , In Situ Nick-End Labeling , Italy/epidemiology , Male , Middle Aged , Oligospermia/epidemiology , Oligospermia/genetics , Semen/chemistry , Sperm Count , Sperm Motility/genetics , Spermatogenesis/genetics
5.
Reproduction ; 148(5): 453-67, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25118297

ABSTRACT

In studies carried out previously, we demonstrated that small ubiquitin-like modifier 1 (SUMO1) is associated with poor sperm motility when evaluated with a protocol that reveals mostly SUMO1-ylated live sperm. Recently, with another protocol, it has been demonstrated that SUMO is expressed in most sperm and is related to poor morphology and motility, suggesting that sumoylation may have multiple roles depending on its localisation and targets. We show herein, by confocal microscopy and co-immunoprecipitation, that dynamin-related protein 1 (DRP1), Ran GTPase-activating protein 1 (RanGAP1) and Topoisomerase IIα, SUMO1 targets in somatic and/or germ cells, are SUMO1-ylated in mature human spermatozoa. DRP1 co-localises with SUMO1 in the mid-piece, whereas RanGAP1 and Topoisomerase IIα in the post-acrosomal region of the head. Both SUMO1 expression and co-localisation with the three proteins were significantly higher in morphologically abnormal sperm, suggesting that sumoylation represents a marker of defective sperm. DRP1 sumoylation at the mid-piece level was higher in the sperm of asthenospermic men. As in somatic cells, DRP1 sumoylation is associated with mitochondrial alterations, this protein may represent the link between SUMO and poor motility. As SUMO pathways are involved in responses to DNA damage, another aim of our study was to investigate the relationship between sumoylation and sperm DNA fragmentation (SDF). By flow cytometry, we demonstrated that SUMO1-ylation and SDF are correlated (r=0.4, P<0.02, n=37) and most sumoylated sperm shows DNA damage in co-localisation analysis. When SDF was induced by stressful conditions (freezing and thawing and oxidative stress), SUMO1-ylation increased. Following freezing and thawing, SUMO1-Topoisomerase IIα co-localisation and co-immunoprecipitation increased, suggesting an involvement in the formation/repair of DNA breakage.


Subject(s)
Cell Shape , DNA Damage , SUMO-1 Protein/metabolism , Sperm Motility , Spermatozoa/metabolism , Antigens, Neoplasm/metabolism , Cold Temperature , Cryopreservation , DNA Fragmentation , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Dynamins , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins/metabolism , Humans , Infertility, Male/metabolism , Infertility, Male/pathology , Male , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/metabolism , Oxidative Stress , Signal Transduction , Sperm Head/metabolism , Sperm Head/pathology , Spermatozoa/pathology , Sumoylation
6.
J Assist Reprod Genet ; 31(10): 1269-76, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25113619

ABSTRACT

PURPOSE: The purpose of this study was to evaluate the oxidative stress status (OS) of follicular fluid (FF) and the oocyte quality in women with polycystic ovary syndrome (PCOS) undergoing different ovarian stimulation protocols. METHODS: FF samples were collected after gonadotropin administration in association or not with metformin or D-chiro-inositol (DCI). OS status was then evaluated by checking the follicular fluid protein oxidation profile after specific labeling of aminoacidic free-SH groups, and two-dimensional electrophoresis followed by qualitative and semiquantitative analysis. Oocyte quality was assessed by international morphological criteria. RESULTS: Our data indicated that both treatments, even if to different extent, recovered a significantly high level of free-SH groups in FF proteins of PCOS women clearly indicating a decrease of OS level with respect to that found in FF samples from gonadotropins alone treated women. A higher number of good quality MII oocytes was also observed in DCI (P < 0.05) or metformin (P < 0.05) study groups in comparison to untreated control group. CONCLUSION: A natural supplement and a drug both showed a statistically significant positive effect on follicular milieu by decreasing the oxidative damage on FF proteins, as well as in recovering good quality oocytes.


Subject(s)
Biomarkers/metabolism , Inositol/therapeutic use , Metformin/therapeutic use , Oxidative Stress/drug effects , Polycystic Ovary Syndrome/drug therapy , Protein Processing, Post-Translational/drug effects , Adult , Female , Fertilization in Vitro/methods , Follicular Fluid/drug effects , Follicular Fluid/metabolism , Gonadotropins/therapeutic use , Humans , Oocytes/drug effects , Oocytes/metabolism , Ovulation Induction/methods , Oxidative Stress/physiology , Polycystic Ovary Syndrome/metabolism , Protein Processing, Post-Translational/physiology
7.
J Proteomics ; 90: 61-76, 2013 Sep 02.
Article in English | MEDLINE | ID: mdl-23500131

ABSTRACT

Human follicular fluid (HFF) has been proven to contain biologically active molecules and proteins that may affect follicle growth and oocyte fertilization. Based on this concept, HFF proteomic characterization is having a significant impact in the delineation of a biomarkers' profile for oocyte quality estimation and, maybe, for in vitro fertilization (IVF) success improvement. Follicular fluid is characterized by a vast protein complexity and a broad dynamic range of protein abundances that hinder its analysis. In this study we determined a proper solubilization and resolution method of HFF in 2-DE, minimizing sample manipulation, protein loss, and experimental artifacts. According to our methodology some low-abundance proteins were detected and identified by MS. Identified proteins were then functionally cross-linked by a pathway analysis. The generated path highlighted the occurrence in HFF of a tight functional-network in which effectors and inhibitors control and balance a space- and time-dependent induction/inhibition of inflammation, coagulation, and ECM degradation/remodeling. Such fine modulation of enzymatic activities exerts a fundamental role in follicle development and in oocyte competence acquiring. Alpha-1-antitrypsin resulted in the core protein of the delineated net and we interestingly detected its differential incidence in FF and serum from two small cohorts of patients who underwent IVF. BIOLOGICAL SIGNIFICANCE: Human ovarian follicular fluid (HFF) is the in vivo microenvironment for oocyte during folliculogenesis. It contains biologically active molecules that may affect oocyte quality, fertilization, and embryo development. HFF is also one of the most abundant "waste product" in assisted reproduction. This makes HFF a readily accessible source of biomolecules for competence evaluation of collected oocytes. The methodological improvement we obtained in proteomics characterization of HFF lead to a wide overview on the functional correlation existing between several fluid components and on how their aberrant occurrence/activity may affect oocyte quality and ovulation.


Subject(s)
Follicular Fluid/metabolism , Proteome/metabolism , Proteomics/methods , Adult , Biomarkers/chemistry , Biomarkers/metabolism , Female , Follicular Fluid/chemistry , Humans , Proteome/chemistry
8.
Andrology ; 1(3): 401-7, 2013 May.
Article in English | MEDLINE | ID: mdl-23307477

ABSTRACT

We report the results of the first three trials of an external quality control (EQC) programme performed in 71 laboratories executing semen analysis in Tuscany Region (Italy). At the end of the second trial, participants were invited to attend a teaching course illustrating and inviting to adhere to procedures recommended by WHO (V edition). Results of the first three trials of the EQC documented a huge variability in the procedures and the results. The highest variability was found for morphology (CV above 80% for all the trials), followed by count (CV of about 60% for all the trials) and motility (CV below 30% for all the trials). When results of sperm count and morphology were divided according to the used method, mean CV values did not show significant differences. CV for morphology dropped significantly at the third trial for most methods, indicating the usefulness of the teaching course for morphology assessment. Conversely, no differences were observed after the course for motility and for most methods to evaluate count, although CV values were lower at the second and third trial for the laboratories using the Burker cytometer. When results were divided according to tertiles of activity, the lowest mean bias values (difference between each laboratory result and the median value of the results) for count and morphology were observed for laboratories in the third tertile (performing over 200 semen analysis/year). Of interest, mean bias values for concentration dropped significantly at the third trial for low activity laboratories. In conclusion, lack of agreement of results of semen analysis in Tuscany is mainly because of the activity and the experience of the laboratory. Our study points out the importance of participating in EQC programmes and periodical teaching courses as well as the use of WHO recommended standardized procedures to increase precision and to allow the use of WHO reference values.


Subject(s)
Andrology , Laboratories , Quality Control , Semen/chemistry , Humans , Italy , Male , Sperm Motility
9.
Minerva Ginecol ; 64(6): 531-8, 2012 Dec.
Article in Italian | MEDLINE | ID: mdl-23232537

ABSTRACT

AIM: Recent studies on the pathophysiology of infertility have shown that oxidative stress (OS) can be one of the causal factors. The OS is, by definition, an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense systems. It seems that oxidative stress plays an important role in almost all phases of human reproduction. In fact, ROS are involved in the modulation of a large spectrum of reproductive functions such as oocyte maturation, ovarian steroidogenesis, corpus luteum functions and are involved in the processes of fertilization, embryo development and pregnancy, but also in some diseases that cause infertility. Polycystic ovary syndrome (PCOS) has recently been associated with increased oxidative stress, often put in relation to the syndrome's typical metabolic disorder. Inositol is an intracellular mediator of insulin, currently much used as a therapeutic agent in PCOS. While its main action takes place via insulin sensitization, little is known about the possible effects of other disorders, such as oxidative stress, associated with PCOS. The purpose of this study was therefore to assess the effect of D-chiro-inositol on the state of oxidative stress in the follicular fluid of women with PCOS. METHODS: Follicular fluids were obtained from women who have turned to the Center for Diagnosis and Treatment of Sterility of Obstetrics and Gynecology of the University Hospital of Siena and Modena diagnosed with PCOS. The women were treated with D-chiro-inositol (500 mg x 2 per day) for 3 months before being subjected to cycles of in vitro fertilization (IVF). The state of oxidative stress was measured by marking of free thiol groups of proteins in the follicular fluid with 3-(N-Maleimidopropionyl)-biocytin. RESULTS: In our study we obtained a lesser presence of free thiol protein groups equal to 77.8% in the follicular fluid of women with PCOS not treated with D-chiro-inositolo, compared to patients who instead have carried out such treatment. CONCLUSION: These results suggest that in PCOS women there is an increase of the oxidation of thiol groups of proteins follicular, correlated to a progressive increase of the oxidative stress and that the administration of D-chiro-inositol in patients with this disease seems to reduce the oxidation of thiol groups.


Subject(s)
Antioxidants/therapeutic use , Follicular Fluid/chemistry , Inositol Phosphates/therapeutic use , Oxidative Stress/drug effects , Polycystic Ovary Syndrome/drug therapy , Polysaccharides/therapeutic use , Adult , Antioxidants/pharmacology , Blotting, Western , Female , Fertilization in Vitro , Humans , Inositol Phosphates/pharmacology , Isoelectric Focusing , Lysine/analogs & derivatives , Lysine/analysis , Maleimides/analysis , Oocyte Retrieval , Ovulation Induction , Oxidation-Reduction , Polycystic Ovary Syndrome/metabolism , Polysaccharides/pharmacology , Pregnancy , Proteins/analysis , Sulfhydryl Compounds/analysis
10.
Int J Androl ; 35(2): 109-24, 2012 Apr.
Article in English | MEDLINE | ID: mdl-21950496

ABSTRACT

Mitochondria of spermatozoa are different from the corresponding organelles of somatic cells, in both their morphology and biochemistry. The biochemical differences are essentially related to the existence of specific enzyme isoforms, which are characterized by peculiar kinetic and regulatory properties. As mitochondrial energy metabolism is a key factor supporting several sperm functions, these organelles host critical metabolic pathways during germ cell development and fertilization. Furthermore, spermatozoa can use different substrates, and therefore activate different metabolic pathways, depending on the available substrates and the physico-chemical conditions in which they operate. This versatility is critical to ensure fertilization success. However, the most valuable aspect of mitochondria function in all types of cells is the production of chemical energy in the form of ATP which can be used, in the case of spermatozoa, for sustaining sperm motility. The latter, on the other hand, represents one of the major determinants of male fertility. Accordingly, the presence of structural and functional alterations in mitochondria from asthenozoospermic subjects confirms the important role played by these organelles in energy maintenance of sperm motility. The present study gives an overview of the current knowledge on the energy-producing metabolic pathways operating inside human sperm mitochondria and critically analyse the differences with respect to somatic mitochondria. Such a comparison has also been carried out between the functional characteristics of human sperm mitochondria and those of other mammalian species. A deeper understanding of mitochondrial energy metabolism could open up new avenues of investigation in bioenergetics of human sperm mitochondria, both in physiological and pathological conditions.


Subject(s)
Energy Metabolism , Mitochondria/metabolism , Sperm Motility/physiology , Spermatozoa/metabolism , Adenosine Triphosphate/metabolism , Asthenozoospermia/metabolism , Asthenozoospermia/pathology , Calcium/metabolism , Fertilization , Humans , Male , Mitochondrial Membranes/metabolism , Reactive Oxygen Species/metabolism , Spermatozoa/chemistry , Spermatozoa/cytology
11.
Eur J Obstet Gynecol Reprod Biol ; 157(1): 63-6, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21530058

ABSTRACT

OBJECTIVES: To prospectively evaluate the safety of metformin administration during pregnancy in a group of PCOS patients by assessing its effect on the prevalence of gestational complications and neonatal outcome. STUDY DESIGN: Our prospective, single centre study included 98 pregnant women with PCOS treated with metformin throughout pregnancy and 110 normal pregnant controls. All PCOS patients were hyperinsulinemic and received metformin (1700-3000 mg/day) before conception and until 37 weeks' gestation. RESULTS: Metformin treatment in the pregnant PCOS patients resulted in significant decrease in miscarriage rate (9.1% vs 20%; p<0.05), gestational diabetes (0 vs 13%; p<0.005), and gestational hypertension (0 vs 11%; p<0.005) and a non-significant decrease in pre-eclampsia (0 vs 3%; p=.24), compared to the control group. Mean neonatal Apgar score, weight and length were comparable between the two groups. CONCLUSIONS: Continuing metformin therapy throughout pregnancy resulted in significant reduction in pregnancy complications with concomitant improved neonatal outcome, with no serious deleterious side effects.


Subject(s)
Hypoglycemic Agents/therapeutic use , Metformin/therapeutic use , Polycystic Ovary Syndrome/drug therapy , Pregnancy Complications/drug therapy , Pregnancy Complications/prevention & control , Abortion, Spontaneous/epidemiology , Abortion, Spontaneous/prevention & control , Adult , Blood Glucose/analysis , Diabetes, Gestational/epidemiology , Diabetes, Gestational/prevention & control , Female , Humans , Hyperinsulinism/prevention & control , Hypertension, Pregnancy-Induced/epidemiology , Hypertension, Pregnancy-Induced/prevention & control , Hypoglycemic Agents/adverse effects , Insulin/blood , Italy/epidemiology , Metformin/adverse effects , Polycystic Ovary Syndrome/blood , Pregnancy , Pregnancy Complications/blood , Pregnancy Complications/epidemiology , Pregnancy Outcome , Prevalence
12.
Int J Androl ; 34(3): 247-55, 2011 Jun.
Article in English | MEDLINE | ID: mdl-20546047

ABSTRACT

The role of mitochondria in sperm motility was the subject of several investigations. However, different views on this topic emerged among scientists. In particular, very little is known on the mechanisms of energy production occurring during human sperm capacitation and related processes. In this study, we have investigated the mitochondrial respiratory efficiency in human sperm samples from normozoospermic subjects before and after swim-up selection and incubation under capacitating condition. Sperm cells, selected by swim-up treatment, were incubated up to 24 h and then demembranated by hypotonic swelling at selected times. The oxygen uptake rate was measured in both basal and swim-up selected samples by a polarographic assay. Mitochondria of swim-up selected cells showed an impressive oxygen consumption rate, which was about 20 times higher than that measured in basal samples. The high mitochondrial respiratory efficiency remained stable up to 24 h after the swim-up treatment. The respiration control ratio, the substrate specificity and the inhibitor sensitivity in the swim-up selected samples were similar to those of basal samples thereby suggesting that the physiology of mitochondria was preserved after the swim-up treatment. Furthermore, the remarkably high mitochondrial respiration in swim-up selected samples allowed the oxygraphic analysis of just 200,000 sperm cells. Sperm selection and incubation under capacitating condition are therefore associated with a high activity of the mitochondrial respiratory chain. The sperm oxygen consumption rate could be useful to exclude mitochondria malfunctioning in male infertility.


Subject(s)
Mitochondria/metabolism , Oxygen Consumption , Sperm Capacitation/physiology , Spermatozoa/metabolism , Blotting, Western , Cell Respiration , Electrophoresis, Polyacrylamide Gel , Energy Metabolism , Humans , Male , Phosphorylation , Sperm Motility/physiology , Tyrosine/metabolism
13.
Int J Androl ; 34(6 Pt 1): 581-93, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21039605

ABSTRACT

Sumoylation is a post-translational modification involved in the regulation of several cell functions. Recent studies suggest its involvement in spermatogenesis, but occurrence and function of SUMO (small ubiquitin-like modifier) in mature spermatozoa remain unknown. We report the occurrence of several SUMO1-conjugated proteins, in a range of 20-85 kDa, in ejaculated spermatozoa. By cytofluorimetric analysis, we evaluated the percentage of SUMO1-positive spermatozoa in 58 subjects undergoing semen analysis in our laboratory and correlated the obtained values with semen parameters. We found that the percentage of SUMO1-positive spermatozoa was inversely correlated with total (r = -0.35, p < 0.01) and progressive motility (r = -0.29, p < 0.05). Such correlations become stricter when only asthenospermic subjects were included in the analysis (r = -0.58, p = 0.01 for progressive motility, n = 17) and were lost in non-asthenospermic subjects. By immunofluorescence and immunoconfocal fluorescence, we demonstrated that SUMO1 is mainly located in the nucleus and, occasionally, in the midpiece of spermatozoa. Immunoelectron microscopy as well as a long permeabilization protocol demonstrated a massive localization of SUMO-1 in the nucleus. By using a fluorescent probe to distinguish dead/live cells, we show that SUMO1 is mainly present in live spermatozoa. In conclusion, sumoylation of human spermatozoa may be involved in the regulation of motility.


Subject(s)
SUMO-1 Protein/metabolism , Semen/metabolism , Spermatozoa/metabolism , Blotting, Western , Fluorescent Antibody Technique , Humans , Male , Microscopy, Confocal , Microscopy, Immunoelectron
14.
Minerva Urol Nefrol ; 62(3): 213-8, 2010 Sep.
Article in Italian | MEDLINE | ID: mdl-20940690

ABSTRACT

AIM: Among the factors contributing to male infertility, asthenospermia constitutes both a health and a social problem frequently associated with alterations in sexual function. Studies have shown that acetyl carnitine and L-arginine improve sperm motility and that ginseng enhances libido and sexual performance. This study examined the effect of treatment with carnitine, acetyl carnitine, L-arginine and ginseng in men with idiopathic asthenospermia and altered sexual function. METHODS: The study population was 180 patients with asthenospermia randomly assigned to two groups: group A (90 men) received treatment and group B (90 men) did not. The sperm count was 16.6 ± 3.2 x 106/mL and the total sperm motility was 26.5 ± 3.4%. RESULTS AND CONCLUSION: Sexual satisfaction was measured using the sexual satisfaction index (SSI). At the end of therapy, a significant improvement was observed in progressive sperm motility on spermiogram evaluation and in SSI scores in the treatment group.


Subject(s)
Arginine/therapeutic use , Asthenozoospermia/drug therapy , Carnitine/therapeutic use , Panax , Phytotherapy , Sexuality/drug effects , Sperm Motility/drug effects , Acetylcarnitine/therapeutic use , Adult , Double-Blind Method , Humans , Male , Middle Aged , Semen Analysis , Vitamin B Complex/therapeutic use
15.
J Med Genet ; 46(1): 21-31, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18782837

ABSTRACT

BACKGROUND: Previous studies have compared sperm phenotypes between men with partial deletions within the AZFc region of the Y chromosome and non-carriers, with variable results. In this study, a separate question was investigated, the basis of the variation in sperm phenotype within gr/gr deletion carriers, which ranges from normozoospermia to azoospermia. Differences in the genes removed by independent gr/gr deletions, the occurrence of subsequent duplications or the presence of linked modifying variants elsewhere on the chromosome have been suggested as possible causal factors. This study set out to test these possibilities in a large sample of gr/gr deletion carriers with known phenotypes spanning the complete range. RESULTS: In total, 169 men diagnosed with gr/gr deletions from six centres in Europe and one in Australia were studied. The DAZ and CDY1 copies retained, the presence or absence of duplications and the Y-chromosomal haplogroup were characterised. Although the study had good power to detect factors that accounted for >or=5.5% of the variation in sperm concentration, no such factor was found. A negative effect of gr/gr deletions followed by b2/b4 duplication was found within the normospermic group, which remains to be further explored in a larger study population. Finally, significant geographical differences in the frequency of different subtypes of gr/gr deletions were found, which may have relevance for the interpretation of case control studies dealing with admixed populations. CONCLUSIONS: The phenotypic variation of gr/gr carriers in men of European origin is largely independent of the Y-chromosomal background.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Y/genetics , Genetic Variation , Phenotype , White People/genetics , Australia , Deleted in Azoospermia 1 Protein , Europe , Gene Dosage , Genetic Loci , Haplotypes , Heterozygote , Humans , Male , Models, Genetic , Nuclear Proteins/genetics , RNA-Binding Proteins/genetics , Semen/metabolism , Seminal Plasma Proteins/genetics
16.
Int J Androl ; 31(3): 337-45, 2008 Jun.
Article in English | MEDLINE | ID: mdl-17573845

ABSTRACT

In this work we report a relatively simple and fast method for analysing oxygen consumption and therefore mitochondrial functionality, in individual human ejaculates. This oxygraphic method requires a low number of cells, is highly reproducible and linearly correlates with sperm concentration. Our results have shown that oxygen uptake by mitochondria of demembranated sperm cells from normozoospermic subjects is significantly stimulated by a large set of respiratory substrates and ADP. The respiratory control ratio (RCR) values indicate a good coupling between respiration and phosphorylation by sperm mitochondria and thus a well preserved integrity of the mitochondria themselves. Interestingly, whereas the rates of oxygen uptake, as expected, changed with different sperm concentrations, the RCR values remained constant, thus demonstrating a linear response of the assay. In asthenozoospermic subjects, however, a significant decrease in the sperm respiratory efficiency was found. The results obtained suggest that this method, besides its potential clinical application, could be useful for a deeper understanding of the biochemical properties of sperm mitochondria and their role in ATP production in human spermatozoa.


Subject(s)
Biological Assay/methods , Energy Metabolism , Mitochondria/metabolism , Oxygen Consumption , Oxygen/metabolism , Spermatozoa/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Asthenozoospermia/metabolism , Cell Respiration , Humans , Hypotonic Solutions , Male , Mitochondria/ultrastructure , Osmotic Pressure , Oxidative Phosphorylation , Rats , Rats, Wistar , Reproducibility of Results , Spermatozoa/ultrastructure , Time Factors
17.
Minerva Ginecol ; 59(5): 473-9, 2007 Oct.
Article in Italian | MEDLINE | ID: mdl-17912173

ABSTRACT

AIM: Leukocytes are often present in human seminal plasma and more frequently in infertile men. Leukocytospermia is associated with sperm morphological and functional alterations. Immune cell activation leads to an increase of free radical production, without any antioxidant defence activation. Leukocyte presence during sperm maturation and migration through male genital tract and consequently exposure to reactive oxygen species led to sperm alteration: axonemal, acrosomal and nuclear structure damage, associated with necrosis. In order to evaluate the immune-modulating and antioxidative activity of beta-glucan, fermented papaya and lactoferrin associated with vitamins C and E, we analysed sperm characteristics of selected infertile male with astheno-teratospermia and abacterial leukocytosis. METHODS: We selected 20 patients referred to our Sterility Centre for semen analysis with leukocyte concentration higher than 1x106 cell/mL. Seminal quality evaluation was performed according to WHO guidelines (1999) using Papanicolau and eosin staining, before and after three months of treatment with beta-glucan, papaya, lactoferrin, vitamin C and E. RESULTS: After therapy, seminal analysis showed a significant reduction of leukocyte concentration and an increase of sperm motility and normal sperm morphology. CONCLUSION: Our results suggest that a combined immunomodulating and antioxidant treatment protect sperm cells during maturation and migration through the male genital tract, resulting in a functional rescue demonstrated by the improvement of semen quality.


Subject(s)
Adjuvants, Immunologic/therapeutic use , Antioxidants/therapeutic use , Infertility, Male/drug therapy , Leukocytosis/drug therapy , Spermatozoa/drug effects , Adult , Ascorbic Acid/therapeutic use , Carica , Case-Control Studies , Drug Therapy, Combination , Fruit , Humans , Lactoferrin/therapeutic use , Male , Middle Aged , Phytotherapy/methods , Treatment Outcome , Vitamin E/therapeutic use , beta-Glucans/therapeutic use
18.
J Assist Reprod Genet ; 24(5): 195-8, 2007 May.
Article in English | MEDLINE | ID: mdl-17342427

ABSTRACT

PURPOSE: Azoospermia may sometimes be related to the use of androgenic anabolic steroids. We report the case of an azoospermic man who had abused androgenic anabolic steroids and who recovered spermatogenesis six months after cessation of abuse and the administration of hormonal therapy. METHODS: An azoospermic 34-year-old man came to Regional Referral Center for Male Infertility. The recovery of spermatogenesis was observed after the cessation of abuse of steroids and the administration of hormonal therapy. Ultrastructural analysis of sperm was carried out by transmission electron microscopy, and the meiotic segregation of chromosomes 1, 9, 18, X, Y was investigated. RESULTS: Mathematically elaborated transmission electron microscopy data highlighted seminal features close to normal fertility. Fluorescence in situ hybridisation showed a high frequency of XY disomy in sperm. CONCLUSIONS: Our findings confirm the recovery of spermatogenesis but suggest a possible relationship between altered meiotic segregation and the abuse of androgenic anabolic steroids.


Subject(s)
Anabolic Agents/adverse effects , Androgens/adverse effects , Azoospermia/etiology , Infertility, Male/chemically induced , Spermatogenesis/physiology , Spermatozoa/cytology , Spermatozoa/drug effects , Adult , Hormones/blood , Humans , Male , Spermatogenesis/drug effects , Spermatozoa/ultrastructure
19.
Andrologia ; 38(4): 122-7, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16872463

ABSTRACT

Pericentric inversions involving the secondary constriction (qh) region of chromosome 9 are considered to be normal variants of human karyotype. A number of investigators have suggested that chromosomal anomalies can contribute to human infertility causing spermatogenetic derangement. The present study was aimed at verifying the influence of chromosome 9 inversion on human spermatogenesis. Semen samples of 18 male carriers of chromosome 9 inversion, analysed by light microscopy, revealed that five patients were azoospermic. PCR analysis demonstrated that two of them also had Y microdeletions. The other 13 showed generally normal sperm concentrations and reduced motility. The morphological characteristics of sperm were studied by TEM and the data were elaborated by a mathematical formula. Sperm pathologies resulted more frequently in the studied group compared to controls, particularly apoptosis. Partial sequences of the A-kinase anchoring protein (Akap) 4 and 3 genes were performed in all patients, as a previous study by our group highlighted Dysplasia of Fibrous Sheath (DFS) defect in two men with inv 9 investigations. The possible effect of chromosome 9 inversion on meiotic chromosome segregation was investigated by FISH, which showed an increased incidence of diploidy. We hypothesized that this inversion could have variable effects on spermatogenesis, from azoospermia to severely altered sperm morphology, motility and meiotic segregation.


Subject(s)
Chromosome Inversion , Chromosomes, Human, Pair 9 , Infertility, Male/genetics , Infertility, Male/pathology , Adolescent , Adult , Humans , In Situ Hybridization, Fluorescence , Lymphocytes/pathology , Lymphocytes/ultrastructure , Male , Microscopy, Electron , Polymerase Chain Reaction , Spermatozoa/pathology , Spermatozoa/ultrastructure
20.
Hum Reprod ; 21(9): 2252-6, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16785260

ABSTRACT

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most frequent cause of menstrual disorders in teenage girls. Little information is available about the effects of metformin in adolescent girls with PCOS and its dose and its efficacy in regulating menstrual cyclicity and hyperandrogenic symptoms. We evaluated the effects of metformin treatment on ovulatory function, hirsutism, acne, hormonal patterns and body weight in adolescent girls with PCOS. METHODS: Eighteen girls, ranging in age from 15 to 18 years, were enrolled in the study. Clinical diagnosis of PCOS was based on the consensus criteria for PCOS accepted in May 2003 at Rotterdam. All subjects received 1700 mg/day metformin as tablets continuously for 6 months. They were then followed up for 6 months. RESULTS: Two patients complained of side effects for >2 weeks and interrupted treatment; they were not evaluated. All the others showed an improvement in menstrual cyclicity. Menstrual periods were ovulatory, with progesterone levels up to 6 ng/ml in luteal phase and a significant reduction in testosterone, androstenedione and free testosterone. BMI was restored within normal limits in all girls between 21 and 24 kg/m(2). Six months after the end of metformin treatment, menstrual cycles continued to be regular and ovulatory with normal BMI. Side effects were slight. CONCLUSIONS: The present results confirm the positive effects of metformin on menstrual periods and show that the drug can be administered to young women to improve ovulation and hyperandrogenic symptoms such as hirsutism, acne and weight gain.


Subject(s)
Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Obesity/complications , Polycystic Ovary Syndrome/complications , Polycystic Ovary Syndrome/drug therapy , Adolescent , Estradiol/metabolism , Female , Follicle Stimulating Hormone/metabolism , Humans , Insulin Resistance , Luteinizing Hormone/metabolism , Menstrual Cycle , Time Factors , Treatment Outcome
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