Publications by authors named "Paola Piomboni"

58 Publications

Sperm DNA Methylation at Metabolism-Related Genes in Vegan Subjects.

Front Endocrinol (Lausanne) 2021 9;12:633943. Epub 2021 Mar 9.

Unit of Andrology and Reproductive Medicine, Department of Medicine, University of Padova, Padova, Italy.

Objective: To investigate if epigenome of sperm cells could be dynamically affected by nutrition.

Design And Methods: We assessed 40 healthy volunteers with different dietary habits and collected their demographic characteristics, as well as clinical and anthropometric parameters. We compared methylation profiles in sperm quantified by bisulfite pyrosequencing, at promoter-associated CpG sites of genes involved in metabolism including fat mass and obesity-associated () and melanocortin-4 receptor () from six vegans and 34 omnivores. In addition, the rs9939609 (T>A) was genotyped.

Results: Higher DNA methylation levels were detected in the sperm of vegan at gene CpG1 (p=0.02), CpG2 (p=0.001), CpG3 (p=0.004), and CpG4 (p=0.003) sites and at -CpG2 site [p=0.016] as compared to sperm of omnivores. This association was not related to genotype.

Conclusions: Although limited by the small number of investigated cases, our data provide insight into the role of diet on sperm DNA methylation in genes involved in metabolism.
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http://dx.doi.org/10.3389/fendo.2021.633943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985526PMC
March 2021

Emerging role of embryo secretome in the paracrine communication at the implantation site: a proof of concept.

Fertil Steril 2021 Apr 23;115(4):1054-1062. Epub 2021 Jan 23.

Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy. Electronic address:

Objective: To assess the role of embryo secretome in modifying the molecular profile of glycodelin A (GdA) in endometrial organoids (ORG) mimicking the implantation window. To verify whether the use of embryo-conditioned culture medium at the time of the embryo transfer may increase in vitro fertilization outcome.

Design: Molecular study with human endometrial ORG and embryo-conditioned culture medium. Retrospective study using prospectively recorded data.

Setting: University hospital.

Patient(s): For isolation and culture of endometrial glandular ORG, endometrial biopsy specimens from five white women of proven fertility undergoing laparoscopy for tubal sterilization. A total of 75 women undergoing intracytoplasmic sperm injection for tubal and/or male infertility factor.

Interventions(s): In vitro fertilization.

Main Outcome Measure(s): Pinopodes presence in human endometrial ORG. Glycodelin A expression profile by means of two-dimensional electrophoresis. In vitro fertilization outcome.

Result(s): This in vitro study demonstrated that the treatment of endometrial ORG with the secretome of medium conditioned by the growing embryo increased the GdA relative abundance and induced a different glycoform pattern. Biochemical and clinical pregnancy rate significantly increased when the spent medium was loaded during the transfer (17.5% vs. 36.6% and 16.5% vs. 35.1%, respectively).

Conclusion(s): This study demonstrated that the secretome of implanting embryos is able to induce the expression as well as to determine the relative abundance and the glycosilation profile of endometrial GdA, a protein having a key role in the embryo-endometrial cross talk. Moreover, a significant increase in pregnancy rate was observed when the embryo transfer was performed by using the culture medium conditioned by the growing embryo.
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http://dx.doi.org/10.1016/j.fertnstert.2020.10.058DOI Listing
April 2021

Follicular microenvironment: Oxidative stress and adiponectin correlated with steroids hormones in women undergoing in vitro fertilization.

Mol Reprod Dev 2021 02 18;88(2):175-184. Epub 2020 Dec 18.

Department of Biomedical, Experimental, and Clinical Sciences "Mario Serio", University of Florence, Florence, Italy.

Research has been focused on determining the follicular microenviroment produced by the theca and granulosa cells since the molecular characterisation of this body fluid could lead to the understanding of several fertility problems. Oxidative stress may be one of the factors involved in female infertility since it plays a key role in the modulation of oocyte maturation and finally pregnancy. An increase in oxidative stress is correlated with inflammation and intense research was developed to understand the interaction between inflammation and adiponectin, based on the fact that many adipokines are inflammation related proteins linked to reactive oxygen species production. The aim of this study is to investigate the correlation between total adiponectin levels and oxidative stress amount in the serum and follicular fluid (FF) of women who undergone in vitro fertilization. Moreover we verified the expression of adiponectin in granulosa and cumulus cells. To clarify the predictive value of steroid hormones in human assisted reproduction, twelve steroid hormones in FF and serum, were quantified in a single run liquid chromatography/mass spectrometry, by using a multiple reaction monitoring mode and we related the serum and follicular fluids adiponectin levels with the concentration of the investigated steroid hormones.
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http://dx.doi.org/10.1002/mrd.23447DOI Listing
February 2021

Characterization of the Age-Dependent Changes in Antioxidant Defenses and Protein's Sulfhydryl/Carbonyl Stress in Human Follicular Fluid.

Antioxidants (Basel) 2020 Sep 28;9(10). Epub 2020 Sep 28.

Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy.

The oxidative stress, characterized by the imbalance between pro-oxidants and antioxidants molecules, seems to be involved in the pathogenesis of female subfertility. In particular, the presence of different markers of oxidative stress has been reported in human follicular fluid (FF) surrounding oocytes. Based on its distinctive composition and on the close proximity to the oocyte, FF creates a unique microenvironment having a direct impact on oocyte quality, implantation, and early embryo development. An imbalance in reactive oxygen species (ROS) production in ovarian follicular fluid may have a negative effect on these processes and, as a consequence, on female fertility. Therefore, the aim of this study was to evaluate the redox state of the FF through various methodological approaches. By means of 2D-electrophoresis we demonstrated that the main structural changes occurring in the proteins of the follicular fluid of normovulatory women were correlated to the age of the patients and to the antioxidant defenses present in the FF. Measurement of these parameters could have clinical relevance, since the assessment of the oxidative stress rate may be helpful in evaluating in vitro fertilization potential.
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http://dx.doi.org/10.3390/antiox9100927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599528PMC
September 2020

Relationship between the metabolic and lipid profile in follicular fluid of women undergoing in vitro fertilization.

Mol Reprod Dev 2020 09 3;87(9):986-997. Epub 2020 Sep 3.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

Among the follicular fluid (FF) components promoting the development of the oocyte are included glycoproteins, several fatty acids, and steroid hormones synthesized by the dominant follicle. For this, the analysis of the metabolites present in FF can determine the quality of the oocyte. FF composition is in part determined by local follicular metabolic processes and in part a plasma transudate. Since the causes of impaired fertility may be due to a metabolic imbalance, metabolomics is useful to identify low molecular weight metabolites. Oxidative stress is involved in human infertility and the use of metabolomics can be crucial to identify which other metabolites besides reactive oxygen species are involved in oxidative stress correlated to infertility. To obtain new information on the study of signaling molecules in FF, the knowledge of the lipid content will be important to improve information on the understanding of follicular development. The objective of this study is to identify (a) a metabolic profile and a lipid profile of FF in women undergoing in vitro fertilization and (b) to correlate the previous information obtained regarding adiponectin and oxidative stress with the metabolic and lipid profile obtained in the present study. As result, we found an increase in oxidative stress due to both an increase of androgens and an accumulation of lipids in the follicular environment and we suggest that this might be one of the causes of reduced fertility.
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http://dx.doi.org/10.1002/mrd.23415DOI Listing
September 2020

Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment.

J Clin Med 2020 May 28;9(6). Epub 2020 May 28.

Department of Molecular and Developmental Medicine, Siena University, 53100 Siena, Italy.

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.
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http://dx.doi.org/10.3390/jcm9061638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355669PMC
May 2020

Organoids of Human Endometrium: A Powerful In Vitro Model for the Endometrium-Embryo Cross-Talk at the Implantation Site.

Cells 2020 04 30;9(5). Epub 2020 Apr 30.

Department of Molecular and Developmental Medicine, University of Siena, Siena 53100, Italy.

Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.
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http://dx.doi.org/10.3390/cells9051121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291023PMC
April 2020

Expression of Matrix Metalloproteinases and Their Inhibitors in Endometrium: High Levels in Endometriotic Lesions.

Int J Mol Sci 2020 Apr 18;21(8). Epub 2020 Apr 18.

Department of Molecular and Developmental Medicine, Siena University, 53100 Siena, Italy.

Endometriosis is a condition defined as presence of endometrium outside of the uterine cavity. These endometrial cells are able to attach and invade the peritoneum or ovary, thus forming respectively the deep infiltrating endometriosis (DIE) and the ovarian endometrioma (OMA), the ectopic lesions feature of this pathology. Endometriotic cells display high invasiveness and share some features of malignancy with cancer cells. Indeed, the tissue remodeling underlining lesion formation is achieved by matrix metalloproteinases (MMPs) and their inhibitors. Therefore, these molecules are believed to play a key role in development and pathogenesis of endometriosis. This study investigated the molecular profile of metalloproteinases and their inhibitors in healthy ( = 15) and eutopic endometrium ( = 19) in OMA ( = 10) and DIE ( = 9); moreover, we firstly validated the most reliable housekeeping genes allowing accurate gene expression analysis in these tissues. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP3, and MMP10 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these tissues. In OMA lesions, all the investigated MMPs and their inhibitors were significantly increased, while DIE expressed high levels of MMP3. Finally, in vitro TNFα treatment induced a significant upregulation of , , and in both healthy and eutopic endometrial stromal cells. This study, shedding light on MMP and TIMP expression in endometriosis, confirms that these molecules are altered both in eutopic endometrium and endometriotic lesions. Although further studies are needed, these data may help in understanding the molecular mechanisms involved in the extracellular matrix remodeling, a crucial process for the endometrial physiology.
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http://dx.doi.org/10.3390/ijms21082840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215833PMC
April 2020

Expression of Taste Receptor 2 Subtypes in Human Testis and Sperm.

J Clin Med 2020 Jan 18;9(1). Epub 2020 Jan 18.

Department of Molecular and Developmental Medicine, Siena University, 53100 Siena, Italy.

Taste receptors (TASRs) are expressed not only in the oral cavity but also throughout the body, thus suggesting that they may play different roles in organ systems beyond the tongue. Recent studies showed the expression of several TASRs in mammalian testis and sperm, indicating an involvement of these receptors in male gametogenesis and fertility. This notion is supported by an impaired reproductive phenotype of mouse carrying targeted deletion of taste receptor genes, as well as by a significant correlation between human semen parameters and specific polymorphisms of taste receptor genes. To better understand the biological and thus clinical significance of these receptors for human reproduction, we analyzed the expression of several members of the TAS2Rs family of bitter receptors in human testis and in ejaculated sperm before and after in vitro selection and capacitation. Our results provide evidence for the expression of genes, with TAS2R14 being the most expressed bitter receptor subtype in both testis tissue and sperm cells, respectively. In addition, it was observed that in vitro capacitation significantly affects both the expression and the subcellular localization of these receptors in isolated spermatozoa. Interestingly, α-gustducin and α-transducin, two Gα subunits expressed in taste buds on the tongue, are also expressed in human spermatozoa; moreover, a subcellular redistribution of both G protein α-subunits to different sub-compartments of sperm was registered upon in vitro capacitation. Finally, we shed light on the possible downstream transduction pathway initiated upon taste receptor activation in the male reproductive system. Performing ultrasensitive droplets digital PCR assays to quantify RNA copy numbers of a distinct gene, we found a significant correlation between the expression of TAS2Rs and TRPM5 ( = 0.87), the cation channel involved in bitter but also sweet and umami taste transduction in taste buds on the tongue. Even if further studies are needed to clarify the precise functional role of taste receptors for successful reproduction, the presented findings significantly extend our knowledge of the biological role of TAS2Rs for human male fertility.
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http://dx.doi.org/10.3390/jcm9010264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019805PMC
January 2020

Proteostasis network alteration in lysosomal storage disorders: Insights from the mouse model of Krabbe disease.

J Neurosci Res 2020 04 3;98(4):718-733. Epub 2019 Dec 3.

Department of Life Sciences, University of Siena, Siena, Italy.

In Krabbe disease, a mutation in GALC gene causes widespread demyelination determining cell death by apoptosis, mainly in oligodendrocytes and Schwann cells. Less is known on the molecular mechanisms induced by this deficiency. Here, we report an impairment in protein synthesis and degradation and in proteasomal clearance with a potential accumulation of the misfolded proteins and induction of the endoplasmic reticulum stress in the brain of 6-day-old twitcher mice (TM) (model of Krabbe disease). In particular, an imbalance of the immunoproteasome function was highlighted, useful for shaping adaptive immune response by neurological cells. Moreover, our data show an involvement of cytoskeleton remodeling in Krabbe pathogenesis, with a lamin meshwork disaggregation in twitcher oligodendrocytes in 6-day-old TM. This study provides interesting protein targets and mechanistic insight on the early onset of Krabbe disease that may be promising options to be tested in combination with currently available therapies to rescue Krabbe phenotype.
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http://dx.doi.org/10.1002/jnr.24558DOI Listing
April 2020

Increased expression of neurogenic factors in uterine fibroids.

Hum Reprod 2019 11;34(11):2153-2162

Dept. of Molecular and Developmental Medicine, University of Siena, Siena, Italy.

Study Question: Are selective markers for the neuronal differentiation such as microtubule-associated protein 2 (MAP-2) and synaptophysin (SYP) as well as the nerve growth factor (NGF) expressed by fibroids, myometrium and eutopic endometrium?

Summary Answer: Neuronal markers NGF, MAP-2 and SYP are highly expressed in fibroids compared with matched myometrium, and this neurogenic pathway is upregulated by tumor necrosis factor (TNF) alpha in cultured smooth muscle cells (SMCs).

What Is Known Already: Uterine fibroids or leiomyomas are the most common benign tumors, accounting for approximately one-third of hysterectomies. The present trend is to improve the medical treatment avoiding surgery, also for fertility sparing; hence, the pathogenic mechanisms are investigated, aiming to develop new therapeutic strategy.

Study Design, Size, Duration: This laboratory-based case-control study is focused on fibroids and myometrial specimens obtained between 2015 and 2017 from 15 women of reproductive age at the proliferative phase of the menstrual cycle. Leiomyomas, matched myometrium and endometrium from each woman were analyzed. Control endometrium was obtained from women undergoing surgery for ovarian cyst (n = 15).

Participants/materials, Setting, Methods: qRT-PCR, western blotting and immunostaining were applied to evaluate the expression of neurogenic markers; the effects of TNF on NGF, MAP-2 and SYP expression in cultured SMCs from leiomyomas and matched myometrium were analyzed.

Main Results And The Role Of Chance: qRT-PCR analyses using tissues from clinical patients showed that the levels of NGF, MAP-2 and SYP mRNA were significantly higher in uterine leiomyomas compared with their matched myometrium (P < 0.05), whereas only NGF was significantly increased in eutopic endometrium compared with healthy endometrium. In primary SMCs, isolated from fibroids or from the adjacent myometrium, NGF, MAP-2 and SYP mRNA expression were significantly increased by TNF treatment (P < 0.05). Finally, human endometrial stromal cells prepared from the endometrium of patients affected by uterine fibroids display higher TNF expression (P < 0.001).

Limitations, Reasons For Caution: qRT-PCR analysis and immunofluorescence validation are robust methods demonstrating a clear upregulation of neurogenic factors in leiomyomas, even though additional studies are needed to establish a correlation between increased neuronal gene expression and degree of pain, as well as the involvement of inflammation mediators in the development of the neurogenic unhinge. Therefore, more in vivo studies are needed to confirm the results achieved from primary cultured SMCs.

Wider Implications Of The Findings: The increased expression of neurogenic factors in uterine fibroids and endometrium may contribute to explain the painful stimuli. Accordingly, these neurogenic pathways may represent potential therapeutic avenues to treat the fibroid-related disorders.

Study Funding/competing Interest(s): This study was supported by research grants from the University of Siena. The authors declare no conflict of interest.

Trial Registration Number: N/A.
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http://dx.doi.org/10.1093/humrep/dez182DOI Listing
November 2019

Clues to Non-Invasive Implantation Window Monitoring: Isolation and Characterisation of Endometrial Exosomes.

Cells 2019 08 1;8(8). Epub 2019 Aug 1.

Dept. of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy.

Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.
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http://dx.doi.org/10.3390/cells8080811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721457PMC
August 2019

Functional polymorphism within NUP210 encoding for nucleoporin GP210 is associated with the risk of endometriosis.

Fertil Steril 2019 08 27;112(2):343-352.e1. Epub 2019 Jun 27.

Department of Biology, University of Pisa, Pisa, Italy.

Objective: To investigate whether nucleoporin 210 (GP210, encoded by NUP210 gene) is involved in endometriosis.

Design: Immunohistofluorescence analysis for assessing whether GP210 is expressed in endometrial tissues from patients and controls; genotyping and case-control study for assessing the association between rs354476 within NUP210 and risk of endometriosis; in vitro luciferase assay for assessing the functional activity of rs354476.

Setting: University.

Patient(s): Histologically diagnosed cases (n = 175) of endometriosis: minimal or mild (stage I-II) in 48 cases (28%), moderate (stage III) in 69 cases (39%), and severe (stage IV) in 58 cases (33%). Controls (n = 557) were female blood donors collected at Meyer Hospital of Florence.

Intervention(s): None.

Main Outcome Measure(s): GP210 tissue expression; genotype distribution and risk of endometriosis; in vitro gene expression measurements.

Result(s): GP210 had positive nuclear immunohistofluorescence staining in endometrial glandular epithelium. Carriers of the variant allele were associated with increased risks: C/T, odds ratio (OR) 1.83, 95% confidence interval (CI) 1.04-3.21; T/T, OR 2.55, 95% CI 1.36-4.80. In vitro, luciferase assay showed that rs354476 is a bona fide target for hsa-miR-125b-5p.

Conclusion(s): Nucleoporin GP210 is involved in endometriosis. Rs354476 polymorphism affects the regulation of NUP210 gene expression by altering the binding with hsa-miR-125b-5p, a microRNA already known as playing an important role for endometriosis. This provides the rationale for the observed increased risk of endometriosis in carriers of the variant allele.
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http://dx.doi.org/10.1016/j.fertnstert.2019.04.011DOI Listing
August 2019

Taste Receptors: New Players in Sperm Biology.

Int J Mol Sci 2019 Feb 22;20(4). Epub 2019 Feb 22.

Department of Molecular and Developmental Medicine, Siena University, 53100 Siena, Italy.

Taste receptors were first described as sensory receptors located on the tongue, where they are expressed in small clusters of specialized epithelial cells. However, more studies were published in recent years pointing to an expression of these proteins not only in the oral cavity but throughout the body and thus to a physiological role beyond the tongue. The recent observation that taste receptors and components of the coupled taste transduction cascade are also expressed during the different phases of spermatogenesis as well as in mature spermatozoa from mouse to humans and the overlap between the ligand spectrum of taste receptors with compounds in the male and female reproductive organs makes it reasonable to assume that sperm "taste" these different cues in their natural microenvironments. This assumption is assisted by the recent observations of a reproductive phenotype of different mouse lines carrying a targeted deletion of a taste receptor gene as well as the finding of a significant correlation between human male infertility and some polymorphisms in taste receptors genes. In this review, we depict recent findings on the role of taste receptors in male fertility, especially focusing on their possible involvement in mechanisms underlying spermatogenesis and post testicular sperm maturation. We also highlight the impact of genetic deletions of taste receptors, as well as their polymorphisms on male reproduction.
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http://dx.doi.org/10.3390/ijms20040967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413048PMC
February 2019

Impact of asymptomatic genital tract infections on in vitro Fertilization (IVF) outcome.

PLoS One 2018 16;13(11):e0207684. Epub 2018 Nov 16.

Laboratory of Molecular Microbiology and Biotechnology (LA.M.M.B.), Department of Medical Biotechnologies, University of Siena, Siena, Italy.

Background: Infertility is estimated to affect approximately 9-30% of reproductive-aged couples. Several conditions involving one or both partners may contribute to infertility. The aim of this study is to evaluate the role of asymptomatic genital tract infections in the outcome of In Vitro Fertilization (IVF) in couples with infertility.

Methods: A total of 285 infertile couples were enrolled in the study. Vaginal/endocervical swabs and semen samples were collected and subjected to microbiological analysis. Spermiograms were carried out on semen specimens, and lactobacilli were quantified in vaginal swabs. Data were associated with IVF results and analysed by using non parametric tests and multivariate analysis.

Results: Microbiological analysis showed that 46.3% of couples presented with an asymptomatic genital tract infection. Spermiogram results showed a significantly diminished motility of sperm cells in samples positive to microbiological testing compared to negative specimens. Enterococcus faecalis was the most prevalent species (11.6%) in positive semen samples and was found to negatively affect both sperm morphology (p = 0.026) and motility (p = 0.003). Analysis of genital swabs from females showed that the presence of E. faecalis (p<0.0001), Escherichia coli (p = 0.0123), Streptococcus agalactiae (p<0.0001), and Gardnerella vaginalis (p = 0.0003) was significantly associated to reduced levels of vaginal lactobacilli. Association of microbiological data with IVF outcome showed that 85.7% of IVF+ couples was microbiologically negative, while IVF was successful in just 7.5% of couples infected with E. faecalis and/or U. urealyticum and/or M. hominis (p = 0.02).

Conclusions: The results show the negative impact of E. faecalis on sperm quality and the association of definite bacterial pathogens with reduced levels of vaginal lactobacilli. The presence of E. faecalis and/or U. urealyticum and/or M. hominis in genital samples of infertile couples is predictive for a negative outcome of IVF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207684PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239332PMC
April 2019

Recombinant FSH Improves Sperm DNA Damage in Male Infertility: A Phase II Clinical Trial.

Front Endocrinol (Lausanne) 2018 10;9:383. Epub 2018 Jul 10.

Laboratory of Seminology- Sperm Bank "Loredana Gandini", Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy.

Male infertility is a global health dilemma and Follicle-Stimulating Hormone (FSH) administration has shown promising results. Several studies showed that infertile men with normal semen parameters have low levels of DNA damage while infertile men with abnormal semen parameters have more damage at the DNA level. Sperm DNA damage may affect the reproductive outcome and has been associated with failure in the achievement of competent embryos and pregnancy fulfillment. The aim of this study was to evaluate whether the administration of recombinant FSH (Gonal-f® PEN 900 IU) could improve sperm DNA fragmentation in men with infertility. The secondary endpoints of this study were to evaluate the FSH effects on sperm parameters and hormonal assets. A longitudinal, prospective, multicenter, open-label clinical trial was carried out. Infertile couples were recruited from six Italian Reproductive Medical Centers and 115 infertile men were enrolled for this study. All participants were treated with subcutaneous injections of Gonal-f® 150 IU every other day, within a 3 month-time frame. The semen samples were examined in accordance to the 2010 World Health Organization criteria. Sperm DNA Fragmentation (DFI) was determined by fluorescence microscopy using terminal deoxynucleotidyl transferase-mediated d-UTP Nick-end Labeling (TUNEL) assay. Statistical analysis was performed using both the -test for paired samples and the Wilcoxon signed-rank test. FSH administration improved DFI in 67% of patients, with an average decrease of 35.4% compared to the baseline. This improvement is more evident in men with basal DFI lower than 17% and in those with FSH basal levels between 2.16 and 4.27 IU/L. In addition, FSH enhanced the gonadal function, increasing the hormones AMH and Inhibin B and semen parameters. Limitation of these results are represented by the absence of a placebo group and of FSHR genotype stratification sub-analysis. Recombinant FSH 150 IU is well tolerated and effective in eliciting a significant DFI reduction as well as in improving gonadal function.

Trial Registration: EUDRACT Number 2010-020196-23. Registred 14 April 2011.
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http://dx.doi.org/10.3389/fendo.2018.00383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048873PMC
July 2018

Matrix metalloproteinases and their inhibitors in human cumulus and granulosa cells as biomarkers for oocyte quality estimation.

Fertil Steril 2018 05;109(5):930-939.e3

Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; Center for Diagnosis and Treatment of Couple Sterility, S. Maria alle Scotte Hospital, Siena, Italy.

Objective: To study the molecular profile of metalloproteinases and their tissue inhibitors in granulosa and cumulus cells in a subset of fertile and infertile women.

Design: Molecular study with granulosa and cumulus cells.

Setting: University hospital.

Patient(s): Forty-four women undergoing assisted reproductive techniques for female infertility factor, with partners having a normal spermiogram and 15 normally fertile women with male partner affected by severe oligoasthenoteratozoospermia or nonobstructive azoospermia.

Intervention(s): In vitro fertilization.

Main Outcome Measurement(s): We investigated gene expression level of metalloproteinases (MMP2, MMP9, MMP11) and their tissue inhibitors (TIMP1, TIMP2) by means of quantitative reverse-transcription polymerase chain reaction, protein quantification by means of Western blot, and localization by means of immunofluorescence.

Result(s): We firstly validated HPRT1 as the most reliable housekeeping gene enabling correct gene expression analysis in both granulosa and cumulus cells. Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP9, and MMP11 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these cells. MMP9 is specifically expressed only in granulosa, whereas MMP2 is more expressed in cumulus and granulosa cells in cases of reduced ovarian response and decreased fertilization rate.

Conclusion(s): This study sheds light on MMP and TIMP expression in granulosa and cumulus cells, and it may help in understanding the fine regulation of oocyte maturation inside the follicle. Although further studies are needed to fully understand the molecular mechanisms involved in these processes, our findings may be useful in the identification of biomarkers of oocyte maturation, competence acquiring, and fertilization.
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http://dx.doi.org/10.1016/j.fertnstert.2018.01.030DOI Listing
May 2018

Soluble protein fraction of human seminal plasma.

J Proteomics 2018 03 27;174:85-100. Epub 2017 Dec 27.

Functional Proteomics Laboratory, Department of Life Sciences, Siena University, Via Aldo Moro 2, 53100 Siena, Italy.

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http://dx.doi.org/10.1016/j.jprot.2017.12.015DOI Listing
March 2018

Gene Expression and Apoptosis Levels in Cumulus Cells of Patients with Polymorphisms of FSHR and LHB Undergoing in Vitro Fertilization Program.

Cell Physiol Biochem 2017 27;43(6):2391-2404. Epub 2017 Oct 27.

Università degli Studi di Palermo, Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Palermo, Italy.

Background/aims: FSH receptor (FSHR) Ala307Thr and Asn680Ser and LHβ chain (LHB) Trp28Arg and Ile35Thr polymorphisms affect the response to pharmacological ovarian stimulation with r-FSH in women undergoing assisted reproductive treatment (ART). Here, we evaluated the expression level of selected genes involved in follicle maturation and the possible onset of apoptosis in cumulus cells of patients with single and double FSHR and LHB polymorphisms, as potential markers of oocyte competence.

Methods: Cumulus cells from 36 stimulated patients were collected and SNP genotyping performed by PCR. Gene expression was evaluated through real-time PCR, and apoptosis estimated via TUNEL assay, and cleaved caspase-3 and pAKT immunostaining.

Results: The cumulative data show significant correlations indicating that the genetic alteration of FSHR and/or LHB genes may lead to perturbations of the signaling network programmed to granulosa cell survival and follicle development. Notably, when double heterozygotes were compared to the rest of the patients, a higher level of apoptosis in terms of both DNA fragmentation index and amount of active caspase-3 was observed in cumulus cells.

Conclusions: These results may help to define personalized stimulation protocols in ART programs, to increase the success rate of ICSI procedures in accordance with the polymorphic condition of the individual patient.
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http://dx.doi.org/10.1159/000484392DOI Listing
January 2018

Dysregulation of GdA Expression in Endometrium of Women With Endometriosis: Implication for Endometrial Receptivity.

Reprod Sci 2018 Apr 7;25(4):579-586. Epub 2017 Jul 7.

2 Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy.

Glycodelin-A (GdA) has been proposed to represent a potential biomarker of endometrial function, but little is known about its expression during the different phases of the menstrual cycle and under pathological conditions. In the light of its potential importance also in embryo implantation, we aimed to evaluate the expression profile of GdA as well as the presence of different glycosylated glycoforms and the immunolocalization in endometrial tissue from women with endometriosis and in women with proven fertility, at different times during the menstrual cycle. Our results showed that GdA is synthesized by endometrial epithelial and stromal cells, both in healthy endometrium and eutopic endometrium from women with endometriosis, with a profile including several glycosylated glycoforms, differentially expressed in each phase of the menstrual cycle. During the secretory phase, a significant increase in GdA protein expression, with a different glycoforms profile, was observed in endometriotic eutopic endometrium. Protein localization in eutopic endometrial tissue resulted significantly different in comparison with endometrium from women with proven fertility. This study indicate that GdA is a complex glycoprotein including up to 6 different glycoforms specifically expressed during the different phase of the menstrual cycle; in pathologic conditions such as endometriosis, the expression profile is altered possibly related to the impaired endometrial receptivity.
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http://dx.doi.org/10.1177/1933719117718276DOI Listing
April 2018

Impaired spermatogenesis in the twitcher mouse: A morphological evaluation from the seminiferous tubules to epididymal transit.

Syst Biol Reprod Med 2017 Apr 19;63(2):77-85. Epub 2017 Jan 19.

a Department of Molecular and Developmental Medicine , University of Siena , Siena, Italy.

Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece-principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa.

Abbreviations: GALC: galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyde.
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http://dx.doi.org/10.1080/19396368.2016.1271918DOI Listing
April 2017

Single nucleotide polymorphisms of USP26 in azoospermic men.

Syst Biol Reprod Med 2016 Dec 11;62(6):372-378. Epub 2016 Oct 11.

a Department of Molecular and Developmental Medicine , University of Siena , Siena, Italy.

Some studies have focused on the association between male infertility and single nucleotide polymorphisms (SNPs) in the ubiquitin-specific protease 26 (USP26) gene, but the results are controversial. In this case-control study including both normozoospermic men and patients with nonobstructive azoospermia, we analyzed both the entire coding region and 5' and 3' untranslated regions of USP26 in order to identify genetic variants in this gene to investigate the role of USP26 on spermatogenesis. We reported variations in the USP26 gene sequence in 82% of azoospermic and in 50% normospermic men. The synonymous variation c.576G>A has a frequency significantly different in the azoospermic (60.2%) and normozoospermic (23.6%) groups, while the frequencies in the two groups of both c.1090C>T and c.1737G>A missense mutations did not reach statistical significance. A cluster mutation (c.371insACA, c.494T>C) was detected in 2 normozoospermic men (2.7%). In the 5'UTR we identified the -33C>T variation both in azoospermic (3.8%) and in normozoospermic (2.7%) men. In a normozoospermic man we detected the nonsense mutation c.882C>A, never reported to date. According to our results, we suggest that only the variation c.576G>A has a frequency significantly different in azoospermic compared to normozoospermic men. Moreover, the identification in a normozoospermic man of a nonsense mutation (c.882C>A) which causes the production of a truncated protein, suggests a marginal role of USP26 in male spermatogenesis. Additional studies may be useful as we cannot exclude that the other SNPs may represent risk factors for male fertility acting by an oligogenic/polygenic mechanism.
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http://dx.doi.org/10.1080/19396368.2016.1238116DOI Listing
December 2016

Suppression of galactocerebrosidase premature termination codon and rescue of galactocerebrosidase activity in twitcher cells.

J Neurosci Res 2016 11;94(11):1273-83

Department of Molecular and Developmental Medicine, Siena University, Siena, Italy.

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of β-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jnr.23790DOI Listing
November 2016

Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF.

Reprod Biol Endocrinol 2016 Sep 7;14(1):57. Epub 2016 Sep 7.

Department Molecular and Developmental Medicine, University of Siena, Policlinico Le Scotte Viale Bracci, Siena, 53100, Italy.

Background: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome.

Methods: This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated.

Results: Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased.

Conclusion: The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.
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http://dx.doi.org/10.1186/s12958-016-0184-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015196PMC
September 2016

Hormonal contraceptives: pharmacology tailored to women's health.

Hum Reprod Update 2016 09 15;22(5):634-46. Epub 2016 Jun 15.

Department of Molecular and Developmental Medicine, University of Siena, Policlinico Le Scotte, Viale Bracci 14, 53100 Siena, Italy.

Background: In recent years, several new oral contraceptives have become available. In some ways, they represent an evolution in terms of individualization and compliance on the part of women. The new formulations make it increasingly possible to prescribe a specific hormonal contraceptive on an individual basis.

Methods: A systematic literature search of PubMed was performed using the following combination of terms: 'oral contraceptives', 'estroprogestins' and 'combined oral contraceptive'. Only English-language papers published between January 2000 and July 2014 were included in our analysis. The present review analyzes all aspects of the choice of oral contraceptives in the different phases of a woman's life in detail.

Results: Regarding the estrogen component, lowering the dose of ethinylestradiol (EE) helped reduce associated side effects. Natural estradiol is now available and represents a valid alternative to EE. And regarding progestins, the dose has changed over time, as well as the endocrine and metabolic characteristics. These are the fruit of much research into improvement of old products (19-nor-progesterone-derived progestins) with androgenic effects and testing of new molecules with improved metabolic neutrality in terms of insulin sensitivity and lipid parameters. New progestins were a genuine turning point because they greatly reduced major side effects, such as water retention, and their anti-androgenic properties made them indicated for all forms of hyperandrogenism associated with acne and mild hirsutism. The associations of estradiol/dienogest and estradiol/nomegestrol acetate are the most suitable contraceptives for women with abundant menstrual bleeding and can increase the number of potential users of hormonal contraception.

Conclusion: Progress in the provision of new oral contraceptives has improved the risk/benefit ratio, by increasing benefits and reducing risks. The present challenge is to tailor contraceptives to individual needs in terms of efficacy and protection of reproductive health.
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http://dx.doi.org/10.1093/humupd/dmw016DOI Listing
September 2016

Protein pathways working in human follicular fluid: the future for tailored IVF?

Expert Rev Mol Med 2016 May 6;18:e9. Epub 2016 May 6.

Department of Molecular and Developmental Medicine,University of Siena,Viale Bracci 14,53100 Siena,Italy.

The human follicular fluid (HFF) contains molecules and proteins that may affect follicle growth, oocyte maturation and competence acquiring. Despite the numerous studies, an integrated broad overview on biomolecular and patho/physiological processes that are proved or supposed to take place in HFF during folliculogenesis and oocyte development is still missing. In this review we report, for the first time, all the proteins unambiguously detected in HFF and, applying DAVID (Database for Annotation, Visualization and Integrated Discovery) and MetaCore bioinformatic resources, we shed new lights on their functional correlation, delineating protein patterns and pathways with reasonable potentialities for oocyte quality estimation in in vitro fertilisation (IVF) programs. Performing a rigorous PubMed search, we redacted a list of 617 unique proteins unambiguously-annotated as HFF components. Their functional processing suggested the occurrence in HFF of a tight and highly dynamic functional-network, which is balanced by specific effectors, primarily involved in extracellular matrix degradation and remodelling, inflammation and coagulation. Metalloproteinases, thrombin and vitamin-D-receptor/retinoid-X-receptor-alpha resulted as the main key factors in the nets and their differential activity may be indicative of ovarian health and oocyte quality. Despite future accurate clinical investigations are absolutely needed, the present analysis may provide a starting point for more accurate oocyte quality estimation and for defining personalised therapies in reproductive medicine.
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http://dx.doi.org/10.1017/erm.2016.4DOI Listing
May 2016

Molecular Dissection Using Array Comparative Genomic Hybridization and Clinical Evaluation of An Infertile Male Carrier of An Unbalanced Y;21 Translocation: A Case Report and Review of The Literature.

Int J Fertil Steril 2016 Jan-Mar;9(4):581-5. Epub 2015 Dec 23.

Genitourinary Unit, University of Siena, Azienda Ospedaliera Universitaria Senese, Siena, Italy.

Chromosomal defects are relatively frequent in infertile men however, translocations between the Y chromosome and autosomes are rare and less than 40 cases of Y-autosome translocation have been reported. In particular, only three individuals has been described with a Y;21 translocation, up to now. We report on an additional case of an infertile man in whom a Y;21 translocation was associated with the deletion of a large part of the Y chromosome long arm. Applying various techniques, including conventional cytogenetic procedures, fluorescence in situ hybridisation (FISH) analysis and array comparative genomic hybridization (array-CGH) studies, we identified a derivative chromosome originating from a fragment of the short arm of the chromosome Y translocated on the short arm of the 21 chromosome. The Y chromosome structural rearrangement resulted in the intactness of the entire short arm, including the sex-determining region Y (SRY) and the short stature homeobox (SHOX) loci, although translocated on the 21 chromosome, and the loss of a large part of the long arm of the Y chromosome, including azoospermia factor-a (AZFa), AZFb, AZFc and Yq heterochromatin regions. This is the first case in which a (Yp;21p) translocation has been ascertained using an array-CGH approach, thus reporting details of such a rearrangement at higher resolution.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793181PMC
http://dx.doi.org/10.22074/ijfs.2015.4619DOI Listing
March 2016

Expression of microtubule associated protein 2 and synaptophysin in endometrium: high levels in deep infiltrating endometriosis lesions.

Fertil Steril 2016 Feb 18;105(2):435-43. Epub 2015 Nov 18.

Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy. Electronic address:

Objective: To assess whether healthy endometrium, eutopic endometrium, and endometriotic lesions express nerve growth factor (NGF), microtubule-associated protein 2 (MAP-2), and synaptophysin (SYP).

Design: Molecular study in tissue extracts.

Setting: University hospital.

Patient(s): A group of women (n = 70), divided as [1] healthy controls (n = 30) and [2] with endometriosis (n = 40), was included.

Intervention(s): From the healthy control group an endometrial specimen was collected by hysteroscopy (proliferative phase, n = 16; secretive phase, n = 14). Endometriotic and endometrial specimens were collected from women undergoing laparoscopic surgery for endometriosis, endometrioma (OMA) (n = 20), or deep infiltrating endometriosis (DIE) (n = 20).

Main Outcome Measure(s): To assess expression of NGF, MAP-2, and SYP messenger RNA (mRNA) levels in endometrium and in endometriosis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein localization by immunofluorescence. Cultures of human endometrial stromal cells were used to evaluate the effect of tumor necrosis factor (TNF)-α on NGF and SYP.

Result(s): Endometrial tissue from control expressed mRNA for NGF, MAP-2, and SYP, without any difference between proliferative and secretive phase. The DIE and OMA lesions showed the highest NGF mRNA expression, significantly higher than in eutopic endometrium and control. In DIE lesions SYP mRNA expression was higher than in OMA or in eutopic endometrium or controls. Immunofluorescence analysis of NGF, MAP-2, and SYP showed a slightly more intense positive signal in endometriotic lesions. Exposure to TNF-α increased NGF and SYP mRNA expression in endometrial culture cells.

Conclusion(s): The present study revealed the presence of two selected neuronal markers, MAP-2 and SYP mRNAs and protein expression, in eutopic endometrium and in endometriotic lesions.
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http://dx.doi.org/10.1016/j.fertnstert.2015.10.024DOI Listing
February 2016

The ICSI procedure from past to future: a systematic review of the more controversial aspects.

Hum Reprod Update 2016 Mar-Apr;22(2):194-227. Epub 2015 Nov 18.

Department of Molecular and Developmental Medicine, University of Siena, Policlinico Le Scotte, viale Bracci 14, 53100 Siena, Italy

Background: ICSI is currently the most commonly used assisted reproductive technology, accounting for 70-80% of the cycles performed. This extensive use, even excessive, is partly due to the high level of standardization reached by the procedure. There are, however, some aspects that deserve attention and can still be ameliorated. The aim of this systematic review was to evaluate the results of available publications dealing with the management of specific situations during ICSI in order to support embryologists in trying to offer the best laboratory individualized treatment.

Methods: This systematic review is based on material obtained by searching PUBMED between January 1996 and March 2015. We included peer-reviewed, English-language journal articles that have evaluated ICSI outcomes in the case of (i) immature oocytes, (ii) oocyte degeneration, (iii) timing of the various phases, (iv) polar body position during injection, (v) zona-free oocytes, (vi) fertilization deficiency, (vii) round-headed sperm, (viii) immotile sperm and (ix) semen samples with high DNA fragmentation.

Results: More than 1770 articles were obtained, from which only 90 were specifically related to the issues developed for female gametes and 55 for the issues developed for male gametes. The studies selected for this review were organized in order to provide a guide to overcome roadblocks. According to these studies, the injection of rescue metaphase I oocytes should be discouraged due to poor clinical outcomes and a high aneuploidy rates; laser-assisted ICSI represents an efficient method to solve the high oocyte degeneration rate; the optimal ICSI timing and the best polar body position during the injection have not been clarified; injected zona-free oocytes, if handled carefully, can develop up to blastocyst stage and implant; efficient options can be offered to patients who suffered fertilization failure in previous conventional ICSI cycles. Most controversial and inconclusive are data on the best method to select a viable spermatozoa when only immotile spermatozoa are available for ICSI and, to date, there is no reliable approach to completely filter out spermatozoa with fragmented DNA from an ejaculate. However, most of the studies do not report essential clinical outcomes, such as live birth, miscarriage and fetal abnormality rate, which are essential to establish the safety of a procedure.

Conclusions: This review provides the current knowledge on some controversial technical aspects of the ICSI procedures in order to improve its efficacy in specific contexts. Notwithstanding that embryologists might benefit from the approaches presented herein in order to improve ICSI outcomes, this area of expertise still demands a greater number of well-designed studies, especially in order to solve open issues about the safety of these procedures.
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http://dx.doi.org/10.1093/humupd/dmv050DOI Listing
October 2016

Morphological and molecular characterisation of Twitcher mouse spermatogenesis: an update.

Reprod Fertil Dev 2015 Feb 10. Epub 2015 Feb 10.

Spermatogenesis is a complex developmental program in which interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis. The Twitcher mouse is a model of Krabbe disease, characterised by the deficiency of galactosylceramidase, the enzyme that hydrolyses galactosylceramide and galactosylsphingosine. Galactosyl-alkyl-acyl-glycerol, a precursor of seminolipid, the most abundant glycolipid in spermatozoa, is also a substrate for galactosylceramidase. Altered sphingolipid metabolism has been suggested to be the cause of the morphological abnormalities reported previously in the spermatogenesis of Twitcher. However, given the frequency of infertility associated with neurological impairment, we hypothesised that an unbalanced hormonal profile could contribute to male infertility in this mutant. In order to clarify this issue, we investigated potential variations in the expression of hormones and hormone receptors involved in the regulation of spermatogenesis. Our data show that, in the brain of Twitcher mouse, gonadotrophin-releasing hormone (GnRH), LH and FSH gene expression is decreased, whereas expression of androgen receptor (AR) and inhibin ?A (INH?A) is increased. The changes in gene expression for the LH and FSH receptors and AR in the testes support the hypothesis that altered sphingolipid metabolism is not the only cause of Twitcher infertility.
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http://dx.doi.org/10.1071/RD14279DOI Listing
February 2015