The Role of Testosterone in Spermatogenesis: Lessons From Proteome Profiling of Human Spermatozoa in Testosterone Deficiency.

Testosterone is essential to maintain qualitative spermatogenesis. Nonetheless, no studies have been yet performed in humans to analyze the testosterone-mediated expression of sperm proteins and their importance in reproduction. Thus, this study aimed to identify sperm protein alterations in male hypogonadism using proteomic profiling. We have performed a comparative proteomic analysis comparing sperm from fertile controls (a pool of 5 normogonadic normozoospermic fertile men) versus sperm from patients with secondary hypogonadism (a pool of 5 oligozoospermic hypogonadic patients due to isolated LH deficiency). Sperm protein composition was analyzed, after peptide labelling with Isobaric Tags, via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) on an LTQ Velos-Orbitrap mass spectrometer. LC-MS/MS data were analyzed using Proteome Discoverer. Criteria used to accept protein identification included a false discovery rate (FDR) of 1% and at least 1 peptide match per protein. Up to 986 proteins were identified and, of those, 43 proteins were differentially expressed: 32 proteins were under-expressed and 11 were over-expressed in the pool of hypogonadic patients compared to the controls. Bioinformatic analyses were performed using UniProt Knowledgebase, and the Gene Ontology Consortium database based on PANTHER. Notably, 13 of these 43 differentially expressed proteins have been previously reported to be related to sperm function and spermatogenesis. Western blot analyses for A-Kinase Anchoring Protein 3 (AKAP3) and the Prolactin Inducible Protein (PIP) were used to confirm the proteomics data. In summary, a high-resolution mass spectrometry-based proteomic approach was used for the first time to describe alterations of the sperm proteome in secondary male hypogonadism. Some of the differential sperm proteins described in this study, which include Prosaposin, SMOC-1, SERPINA5, SPANXB1, GSG1, ELSPBP1, fibronectin, 5-oxoprolinase, AKAP3, AKAP4, HYDIN, ROPN1B, ß-Microseminoprotein and Protein S100-A8, could represent new targets for the design of infertility treatments due to androgen deficiency.

Perceived knowledge, coping and diagnostic-therapeutic pathways of infertile couples trying to conceive: an Italian survey.

BACKGROUND: The last decades' increasing infertility risk factors have brought to a growing number of infertile couples. Knowledge regarding infertility and possible treatments is-however- poor, leading to difficulty in coping and understanding. Knowing infertile couples' perceptions and needs may help clinicians develop services that better address these needs. METHODS: This study aimed at assessing perceived knowledge, coping mechanisms and diagnostic-therapeutic pathways concerning infertility and Assisted Reproductive Technology (ART) in a sample of Italian infertile couples trying to conceive. In this cross-sectional study, 199 subjects aged 25-55 years old who had difficulties conceiving were asked to fill a questionnaire regarding infertility and ART perceived knowledge, coping and diagnostic-therapeutic pathways they had been through. RESULTS: The perceived knowledge score was 42.5%. Couples had difficulties understanding ART's success rate and the potential risks for the woman and fetus. Subjects from North of Italy, who were previously engaged in an ART procedure, those who had signed a written informed consent and were offered a specific counselling session had significantly higher mean score of perceived knowledge. Couples were handling the situation in a satisfactory way, supported also by their family, but the procedures, clinical examinations, therapies, medications, were considered complex and stressful by 66.5% of the respondents. CONCLUSIONS: Healthcare providers should consider the couples' previous experience with infertility, the important role of informed consent and personalized counselling sessions. Counselling for infertile couples should have a broader, comprehensive approach, integrating psychological, social, ethical support, as well as health literacy, taking into consideration the infertile couples' preferences and needs.

Laparoscopic Ethanol Sclerotherapy for Ovarian Endometriomas: Preliminary Results.

STUDY OBJECTIVE: The purpose of this study was to evaluate the laparoscopic aspiration of endometriomas through 95% ethanol sclerotherapy. DESIGN: A single-center, retrospective study. SETTING: Endometriosis outpatient clinic of a tertiary university hospital, gynecology department of Agostino Gemelli University Foundation Polyclinic IRCCS, Rome, Italy. PATIENTS: Fifty-three women with 64 identified endometriomas measuring 4 to 10 cm with no suspected malignancy. INTERVENTIONS: Laparoscopic aspiration and 95% ethanol sclerotherapy of endometriomas were completed in the patients between September 2013 and September 2017. MEASUREMENTS AND MAIN RESULTS: Using standard laparoscopy, the ovarian endometriomas were aspirated and washed to remove all cystic material. A 95% ethanol solution was instilled in the cysts and left for 15 minutes, then removed. The patients were followed by ultrasound at 3, 6, 9, and 12 months, and then annually to identify recurrence. All patients were administered postoperative hormone therapy, suspended only in those desiring pregnancy or experiencing adverse effects. The mean age was 32 years (range 19-40 years), and the mean cyst diameter was 6 cm (range 4-10 cm). Thirteen of the 53 patients (25%) had had previous surgery for endometriomas. Forty-one patients (77%) had associated deep endometriosis, treated during the same laparoscopic procedure. No major ethanol-related complications were recorded. The mean length of follow-up was 31 months. Recurrence of endometriomas was observed in 5 patients (9%). Overall, pregnancy occurred in 16 of 28 patients (57%) desiring pregnancy. CONCLUSION: Laparoscopic aspiration and ethanol sclerotherapy as treatment for endometriomas, even in patients with bilateral endometriomas or with associated deep endometriosis, resulted in <10% recurrence and no major complications.

Testosterone and FSH modulate Sertoli cell extracellular secretion: Proteomic analysis.

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

Semen Proteomics Reveals the Impact of Enterococcus faecalis on male Fertility.

BACKGROUND: Infectious etiologies contribute to 15% of male factor infertility. Enterococcus faecalis (E. faecalis) is commonly identified in semen culture of infertile men and it is associated with significantly poorer semen quality. OBJECTIVE: Aim of this study was to identify new seminal biomarkers for the male tract infection by E. faecalis, using proteomic profiling, in order to understand the effect of E. faecalis on the physiopathology of male reproduction. METHODS: We included in the study ten patients seeking medical care for primary infertility with prostate-vesicular-epidydimitis and with microbiological analysis on semen and/or prostatic secretions positive for E. faecalis. Ten fertile men have been enrolled as a control group in the protocol. An aliquot of each seminal plasma was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLCnano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer. RESULTS: Eight proteins have not been identified in the group of controls and have been observed in a remarkable proportion of patients, mainly involved in immune system activity (CD177, Swiprosin-1 and 2-oxoglutarate dehydrogenase). Arylsulfatase has been identified in the group of controls and was absent in all patients with infection. Three proteins (TIMP-1, WFDC domain protein 2 and Carboxypeptidase E) have been observed significantly different in patients versus controls, mainly related with inflammation. CONCLUSIONS: This is the first application of MS-based proteomics aimed to reveal an array of proteins in the seminal plasma and reflecting the effect of the infection by E. faecalis on semen composition.

Seminal suPAR Levels as Marker of Abacterial Male Accessory Gland Inflammation in Hypogonadism.

BACKGROUND: Recent evidences suggest that hypogonadism is an important risk factor for lower urinary tract symptoms and benign prostatic hyperplasia. Several papers have discussed the role of chronic inflammation in the development of BPH, which may be modulated by the hypogonadal state. Soluble Urokinase-type Plasminogen Activator Receptor (suPAR), known protein marker of systemic inflammation, can be assayed in the seminal plasma and represents a reliable and sensitive marker of inflammation for the Male Accessory Gland Inflammation (MAGI). OBJECTIVE: The aim of this study has been to investigate if seminal suPAR is elevated in MAGI with hypogonadism and if suPAR represent a useful marker of abacterial inflammation in hypogonadism. METHODS: We included in the study twenty male patients aged between 25 and 55 year-old with secondary postsurgical hypogonadism. The same patients were also evaluated after a 3-month of Testosterone Replacement Therapy (TRT), to evaluate the effect of androgen replacement therapy on suPAR. Ten fertile men have been enrolled as a control group in the protocol. SuPAR concentrations were assayed on seminal plasma using an Enzyme-Linked Immunosorbent Assay (ELISA) kit. RESULTS: Hypogonadic patients presented significantly increased levels of seminal suPAR respect to controls (86.1±36.8 vs 55.2±20.0 ng/mL, p<0.05). TRT in hypogonadic patients has been associated with a significant reduction of suPAR levels as reported in the control group (50.9±22.91 vs 86.1±36.8 ng/ml p<0.05). CONCLUSIONS: These results confirm the role of suPAR as a protein marker of MAGI and support the hypothesis that hypogonadism induces a state of inflammation in male accessory glands which is involved in male infertility. Moreover demonstrated that testosterone treatment probably exerts a positive effect on MAGI and infertility as documented by reduction of suPAR levels in hypogonadic treated patients.

Synthetic PreImplantation Factor (PIF) prevents fetal loss by modulating LPS induced inflammatory response.

Maternal control of inflammation is essential during pregnancy and an exaggerated response is one of the underlying causes of fetal loss. Inflammatory response is mediated by multiple factors and Toll-like receptors (TLRs) are central. Activation of TLRs results in NALP-3 mediated assembly of apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 into the inflammasome and production of pro-inflammatory cytokines IL-1ß and IL-18. Given that preventing measures are lacking, we investigated PreImplantation Factor (PIF) as therapeutic option as PIF modulates Inflammation in pregnancy. Additionally, synthetic PIF (PIF analog) protects against multiple immune disorders. We used a LPS induced murine model of fetal loss and synthetic PIF reduced this fetal loss and increased the embryo weight significantly. We detected increased PIF expression in the placentae after LPS insult. The LPS induced serum and placenta cytokines were abolished by synthetic PIF treatment and importantly synthetic PIF modulated key members of inflammasome complex NALP-3, ASC, and caspase-1 as well. In conclusion our results indicate that synthetic PIF protects against LPS induced fetal loss, likely through modulation of inflammatory response especially the inflammasome complex. Given that synthetic PIF is currently tested in autoimmune diseases of non-pregnant subjects (clinicaltrials.gov, NCT02239562), therapeutic approach during pregnancy can be envisioned.

Untreated Psoriasis Impairs Male Fertility: A Case-Control Study.

BACKGROUND: Recent evidence indicates that a systemic state of inflammation may impair fertility, but data about psoriatic males are scarce. OBJECTIVES: The aim of this study was to assess gonadal function in psoriatic males implementing our knowledge about fertility in these subjects. METHODS: Male psoriatic patients, aged between 18 and 55 years, and a group of healthy subjects matched for age, BMI and geographic origin were enrolled. All subjects underwent a complete physical and andrological examination, standard semen analysis, complete microbiological analysis and ultrasound evaluation of sexual glands. Seminal levels of soluble urokinase-type plasminogen activator receptor (suPAR) and serum levels of testosterone, estradiol (E2), sex hormone-binding globulin (SHBG), luteinizing hormone and follicle-stimulating hormone were also assessed. RESULTS: Fifty patients and 50 controls fulfilled the inclusion criteria and were enrolled in our study. Testosterone and SHBG were found to be significantly decreased in patients with psoriasis compared with the control group. Higher levels of E2 were also reported in psoriatic patients. Total sperm count, sperm motility and percent of spermatozoa with normal morphology were significantly reduced in patients compared to controls. suPAR levels were significantly increased in patients compared to controls and found to be above the reference limits. Ultrasound signs of inflammation of the accessory glands were observed in 35/50 patients with psoriasis and in none of the controls. CONCLUSION: Our study suggests that untreated psoriasis may impair male fertility. We also found that this might be due to an impact of systemic inflammation on the hormonal profile and on sexual accessory gland inflammation.

Cervical mucus proteome in endometriosis.

BACKGROUND: Endometriosis is a chronic gynecological inflammatory disease characterized by the presence of functional endometrial glands and stroma outside of the uterine cavity. It affects 7-10% of women of reproductive age and up to 50% of women with infertility. The current gold standard for the diagnosis combines laparoscopic evaluation and biopsy of the visualized lesions. However, laparoscopy requires general anesthesia and developed surgical skills and it has a high procedural cost. In addition, it is associated with the risk, although rare, of potential intraoperative or postoperative complications. To date, several noninvasive biomarkers have been proposed; however, no definite diagnostic biomarker is yet available. The aim of this study was to characterize the CM proteome in patients with endometriosis using high resolution mass spectrometry-based proteomics, implemented by bioinformatic tools for quantitative analysis, in order to investigate the pathophysiological mechanisms of endometriosis. METHODS: Cervical mucus samples were collected from patients affected by endometriosis and fertile controls. An aliquot of the soluble acidic fraction of each cervical mucus sample, corresponding to 0.5 mg of total protein, was left to digest with sequencing grade modified porcine trypsin. The peptides were analyzed by LC-MS/MS on a high resolution Orbitrap Elite mass spectrometer and data were evaluated using bioinformatic tools. RESULTS: We aimed at the first total profiling of the cervical mucus proteome in endometriosis. From the list of identified proteins, we detected a number of differentially expressed proteins, including some functionally significant proteins. Six proteins were quantitatively increased in endometriosis, almost all being involved in the inflammatory pattern. Nine proteins were quantitatively reduced in endometriosis, including some proteins related with local innate immunity (CRISP-3 and Pglyrp1) and protection against oxidative stress (HSPB1). Fifteen proteins were not detected in endometriosis samples including certain proteins involved in antimicrobial activity (SLURP1 and KLK13) and related to seminal plasma liquefaction and male fertility (KLK13). CONCLUSIONS: This is the first application of high resolution mass spectrometry-based proteomics aimed in detecting an array of proteins in CM to be proposed for the noninvasive diagnosis of endometriosis. This chronic disease presents in CM an inflammatory protein pattern.

Olfactory Receptors in Semen and in the Male Tract: From Proteome to Proteins.

The estimated number of testicular olfactory receptors (ORs) in mammals range between 20 and 66. Previous data reported the role of hOR17-4 and mOR23 in sperm-oocyte chemiotaxis. Proteomic analysis was performed to understand which are the ORs expressed in seminal plasma. Seminal samples by four fertile men were analyzed by an Ultimate 3000Nano/Micro-HPLC apparatus coupled with an LTQ-Orbitrap XL hybrid mass spectrometer. Western blot analysis confirmed the expression of three identified ORs. The expression of ORs in sperm cells, testis, and epididymis was evaluated by confocal microscopy analysis. In seminal plasma eight different ORs were identified by proteomics and three ORs have been confirmed by western blot. Confocal microscopy analysis revealed that OR4S1, OR4C13, and OR1I1 are expressed on the surface of sperm cells. In testicular tissue, OR4S1 and OR1I1 are expressed in spermatocytes and spermatids and OR4C13 is expressed throughout all the tubules. In patients with spermatocyte maturation arrest OR4S1 and OR1I1 expression was reduced and a weak positivity for OR4C13 was detected in the spermatogonia. OR4S1, OR4C13, and OR1I1 had intense and diffuse staining in the epididymis. This study initiated a new methodology for screening OR repertoire in sperms, testis and epididymis. Our results open new insights into OR involvement in sperm maturation and migration.

Antibiotics Prophylaxis for Operative Hysteroscopy.

OBJECTIVE: To evaluate the incidence of infectious complications and effect of prophylactic antibiotic administration during operative hysteroscopic procedures. METHODS: A multicentric randomized controlled trial was conducted between January 2012 and December 2013. Women (n = 180) affected by endometrial hyperplasia, myomas, or endometrial polyps undergoing operative hysteroscopy were randomized to receive cefazolin 2 g intravenously 30 minutes prior to the procedure (n = 91) and no treatment (n = 89). RESULTS: No statistical difference in terms of postoperative fever (2.4% vs 2.3%, P = .99), endometritis (0% vs 0%), pain (6.0% vs 10.4%, P = .40), cervicitis-vaginitis (0% vs 0%), pelvic abscess (0% vs 0%), pelvic inflammatory disease (0% vs 0%), and bleeding (0% vs 0%) was noticed. No statistical difference in terms of side effects attributable to antibiotic prophylaxis such as allergy (0% vs 4.8%, P = .12), nausea (10.7% vs 17.4%, P = .27), vomiting (3.6% vs 4.6%, P = .99), diarrhea (4.8% vs 5.4%, P = .99), cephalea (9.5% vs 3.5%, P = .13), dizziness (4.8% vs 2.3%, P = .44), and meteorism (5.4% vs 3.4%, P = .99) was noticed. CONCLUSION: The results of the current study support the recommendation not to prescribe routine antibiotic prophylaxis prior to operative hysteroscopy.

Inflammosome in the human endometrium: further step in the evaluation of the "maternal side".

OBJECTIVE: To investigate the expression of inflammosome components (NALP-3, associated speck-like protein containing a CARD [ASC]) and their activation (caspase-1, interleukin [IL]-1ß, and IL-18 secretion) in the human endometrium from fertile and women with history of recurrent pregnancy loss (RPL). DESIGN: Experimental study. SETTING: University hospital. PATIENT(S): Ten fertile women (control group [CTR]) and 30 women with RPL. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Endometrial samples were collected by hysteroscopy during the putative window of implantation and evaluated for chronic endometrial inflammation by hystopathological analysis. Inflammosome expression was analysed by immunohystochemical staining (27 RPL and 10 CTR women). The expression of NALP-3 and ASC protein was quantified by Western blot (30 RPL and 10 CTR women). Caspase-1 activation and IL-1ß and IL-18 secretion was quantified by ELISA (30 RPL and 10 CTR women). RESULT(S): We observed a significantly increased expression of inflammasome NALP-3 and ASC protein, an increased activation of caspase-1, and increased levels of IL-1ß and IL-18 in RPL endometrium compared with CTR. CONCLUSION(S): Abnormal activation of endometrial innate immunity by means of inflammosome, stimulated by pathogen- or damage-associated molecular patterns, may represent an additional mechanism, currently not investigated, negatively interfering with endometrial receptivity. More studies are required [1] to identify the primary trigger of endometrial inflammosome activation and its clinical impact in the occurrence of RPL; and [2] to validate the inflammosome components as a novel family of endometrial biomarkers and promising therapeutic targets in RPL.

Comparison between the stripping technique and the combined excisional/ablative technique for the treatment of bilateral ovarian endometriomas: a multicentre RCT.

STUDY QUESTION: Is the combined excisional/ablative technique for the treatment of ovarian endometriomas better than the traditional stripping technique in terms of recurrence rate? SUMMARY ANSWER: There is no evidence that the combined excisional/ablative technique is better than the traditional stripping technique, as similar recurrence rates were observed for the two techniques. WHAT IS KNOWN ALREADY: The stripping technique is associated with better results compared with ablative, non-excisional techniques for the treatment of ovarian endometriomas. Excisional techniques, such as stripping, have, however, been associated with reduced ovarian reserve as evaluated with anti-Mullerian hormone, and surgical techniques that better preserve the ovarian reserve are needed. STUDY DESIGN, SIZE, DURATION: A prospective, multicentre, randomized blinded clinical trial was carried out on 51 patients with bilateral endometriomas larger than 3 cm. For each patient, serving as her own control, one ovary was randomized to the stripping technique and the contralateral to the combined excisional/ablative technique. Patients were enrolled between January 2013 and April 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients of reproductive age with pelvic pain and/or infertility affected by bilateral endometriomas larger than 3 cm were included (n = 51). The patients underwent laparoscopic removal of endometriomas with two different surgical techniques performed at either side after random assignment: complete removal by stripping on one side versus the combined technique, consisting of partial excisional cystectomy followed by completion with ablative surgery using bipolar coagulation, on the other side. Post-operative follow-up was performed at 1, 3 and 6 months after surgery for the evaluation of endometrioma recurrence (primary outcome) and of antral follicle count (AFC) and ovarian volumes (OVs) to assess ovarian reserve (secondary outcome). MAIN RESULTS AND THE ROLE OF CHANCE: Recurrence rates were 5.9% for the stripping technique versus 2.0% for the combined technique (odds ratio 3.00; 95% confidence interval: 0.24-157.5; P = 0.62). AFC in the ovaries treated with the stripping technique did not differ significantly from AFC in ovaries treated with the combined technique at all follow-up visits, whereas OV was significantly lower after the combined technique at the 6-month follow-up visit (P = 0.04). LIMITATIONS, REASONS FOR CAUTION: A major limitation of this study is the small sample size and particularly for ovarian reserve, the secondary outcome, for which no formal sample size calculation was performed. The lower-than-expected recurrence rates in the present series may be related to the shorter follow-up in our study compared with most studies in the literature. Further studies with larger sample sizes and longer follow-up are needed to confirm the findings of this study. The combined technique using CO2 laser energy instead of bipolar coagulation should also be evaluated. WIDER IMPLICATIONS OF THE FINDINGS: The traditional excisional technique, i.e. the stripping technique, should still be considered the gold standard approach for the surgical treatment of endometriomas. STUDY FUNDING/COMPETING INTERESTS: No commercial funding was received. The authors report no relevant conflict of interest. TRIAL REGISTRATION NUMBER: ANZCTR number ACTRN12614000653662. TRIAL REGISTRATION DATE: 23 June 2014. DATE OF FIRST PATIENT'S ENROLMENT: 1 January 2013.

Human IgG Antinuclear Antibodies Induce Pregnancy Loss in Mice by Increasing Immune Complex Deposition in Placental Tissue: In Vivo Study.

PROBLEM: A threefold higher prevalence of antinuclear antibodies (ANA) has been reported in patients with recurrent pregnancy loss (RPL). Nevertheless, the role of ANA in reproductive failure is still unclear. The aim of this study was to investigate the role of ANA during early pregnancy in vivo. METHOD OF STUDY: We used pregnant mice treated with immunoglobulin G (IgG) obtained from normal healthy subjects (NHS); ANA(+) sera of patients with RPL; and ANA(+) sera from women with uncomplicated pregnancies (HW). Placental immunohistochemical/immunofluorescence staining was performed to detect complement and immune complex deposition. ELISA was performed to evaluate complement levels. RESULTS: ANA(+) IgG from RPL women significantly increased embryo resorption rate, reduced C3, and increased C3a serum levels compared to NHS IgG or ANA(+) -HW IgG. Increased C3 deposition and increased immune complex staining in placental tissues from mice treated with ANA(+) -RPL IgG fraction compared to NHS- and ANA(+) -HW-IgG-treated mice were found. CONCLUSION: ANA(+) IgG injection in mice is able to induce fetal resorption and complement activation. The presence on placental tissues of immune complexes and complement fragments suggests the complement activation as a possible mechanism of placental damage.

Novel biomarkers of androgen deficiency from seminal plasma profiling using high-resolution mass spectrometry.

CONTEXT: The seminal plasma is made of secretions from the testis, the epididymis, and the male accessory glands, which are dependent on the presence of androgenic stimuli. OBJECTIVE: The objective of this study was to identify new seminal biomarkers for secundary male hypogonadism using proteomic profiling. DESIGN: Seminal plasma samples from patients affected by secundary hypogonadism and normogonadal controls were analyzed by an LTQ Orbitrap XL hybrid mass spectrometer and data were evaluated using bioinformatic tools. SETTING: The study was performed at a clinical research center. SUBJECTS: Twenty male patients, aged 25-55 years, affected by secundary hypogonadic were studied. Ten patients were reevaluated after 6 months of T replacement therapy (TRT). Ten normogonadic men were enrolled as a control group. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: The list of absent proteins in the samples of hypogonadic patients and identified after TRT was studied. Bioinformatic tools were used to functionally annotate the panel of androgen-dependent proteins. The interaction network of the differentially expressed proteins was built in silico, including the androgen receptor. RESULTS: A lower number of proteins was identified in hypogonadic patients compared with normogonadal men. Among the 61 proteins identified in normogonadal men, 33 proteins were absent in hypogonadic patients. Fourteen of 33 absent proteins were identified in seminal samples after 6 months of TRT. Functional annotation analysis revealed that binding and enzymatic activities are mainly deficient in male hypogonadism. Seven of 14 differentially expressed proteins can fall into one large protein-protein interaction network, which directly involves the androgen receptor. CONCLUSION: A high resolution mass spectrometry-based proteomic approach was first used to describe the alterations of seminal seminal proteome in secundary male hypogonadism. These proteins represent putative physiological in vivo targets for androgen deficiency.

Anatomical causes of female infertility and their management.

The main female anatomical causes of infertility include post-infectious tubal damage, endometriosis, and congenital/acquired uterine anomalies. Congenital (septate uterus) and acquired (myomas and synechiae) diseases of the uterus may lead to infertility, pregnancy loss, and other obstetric complications. Pelvic inflammatory disease represents the most common cause of tubal damage. Surgery still remains an important option for tubal factor infertility, with results in terms of reproductive outcome that compare favorably with those of in vitro fertilization. Endometriosis is a common gynecologic condition affecting women of reproductive age, which can cause pain and infertility. The cause of infertility associated with endometriosis remains elusive, suggesting a multifactorial mechanism involving immunologic, genetic, and environmental factors. Despite the high prevalence of endometriosis, the exact mechanisms of its pathogenesis are unknown. Specific combinations of medical, surgical, and psychological treatments can ameliorate the quality of life of women with endometriosis. In the majority of cases, surgical treatment of endometriosis has promoted significant increases in fertilization rates. There are obvious associations between endometriosis and the immune system, and future strategies to treat endometriosis might be based on immunologic concepts.

Potential new mechanisms of placental damage in celiac disease: anti-transglutaminase antibodies impair human endometrial angiogenesis.

Celiac disease (CD) is an autoimmune enteropathy triggered by gluten ingestion and characterized by circulating anti-transglutaminase type 2 (anti-TG2) autoantibodies. An epidemiological link between maternal CD and increased risk of pregnancy failure has been established; however, the mechanism underlying this association is still poorly understood. Because proper endometrial angiogenesis and decidualization are prerequisites for placental development, we investigated the effect of anti-TG2 antibodies on the process of endometrial angiogenesis. Binding of anti-TG2 antibodies to human endometrial endothelial cells (HEECs) was evaluated by ELISA. Angiogenesis was studied in vitro on HEECs and in vivo in a murine model. In particular, we investigated the effect of anti-TG2 antibodies on HEEC matrix metalloprotease-2 (MMP-2) activity by gelatin zymography, cytoskeletal organization and membrane properties by confocal microscopy, and activation of extracellular signal-regulated kinases (ERKs) and focal adhesion kinase (FAK) by Western blot analysis. Anti-TG2 antibodies bound to HEECs and decreased newly formed vessels both in vitro and in vivo. Anti-TG2 antibodies impaired angiogenesis by inhibiting the activation of MMP-2, disarranging cytoskeleton fibers, changing the physical and mechanical properties of cell membranes, and inhibiting the intracellular phosphorylation of FAK and ERK. Anti-TG2 antibodies inhibit endometrial angiogenesis affecting the TG2-dependent migration of HEECs and extracellular matrix degradation, which are necessary to form new vessels. Our results identify pathogenic mechanisms of placental damage in CD.

Antiphospholipid antibodies affect human endometrial angiogenesis: protective effect of a synthetic peptide (TIFI) mimicking the phospholipid binding site of ß(2) glycoprotein I.

PROBLEM: Aim of our study was to investigate whether TIFI, a syntetic peptide able to compete with anti-phospholipid antibodies (aPL) in the binding to endothelium, may restore aPL-inhibited endometrial angiogenesis. METHODS: The protective role of TIFI was evaluated on: i) aPL-inhibited of human endometrial endothelial cells (HEEC) angiogenesis in vitro; ii) aPL-inhibited vascular endothelial growth factor (VEGF) and metalloproteases (MMPs) expression; iii) aPL-inhibited Nuclear Factor-κB (NF-κB) and Extracellular signal-Regulated Kinase (ERK) activation and (iv) angiogenesis in vivo. RESULTS: TIFI restores in a dose-dependent manner: i) aPL-mediated inhibition of HEEC angiogenesis in vitro and in vivo (P < 0.05), ii) VEGF (P < 0.001) and MMP-2 (P < 0.05) expression and iii) NF-κB DNA binding and ERK-1/2 activation (P < 0.05) inhibited by aPL. CONCLUSION: Our results show for the first time the protective effects of TIFI, as represented by its ability to interfere with aPL mediated anti-angiogenic activity.

Positive end-expiratory pressure during laparoscopy: cardiac and respiratory effects.

STUDY OBJECTIVE: To determine the effect of positive end-expiratory pressure (PEEP) on the respiratory system and on cardiac function. DESIGN: Prospective randomized study. SETTING: Operating room. PATIENTS: 60 ASA physical status 1 women scheduled for pelvic laparoscopic surgery. INTERVENTIONS: Patients were ventilated normally during surgery; PEEP was modified depending on patient group allocation. Group A was the control group and did not receive PEEP. Group B received PEEP 5 cmH2O and Group C received PEEP 10 cmH2O. MEASUREMENTS: Respiratory parameters measured were partial pressure of arterial oxygen (PaO2), partial pressure of carbon dioxide (PaCO2), and end-tidal carbon dioxide tension (ETCO2). Cardiac parameters measured were left ventricular end-diastolic volume index (LVEDVI), ie, ratio of LVEDV/body surface area (BSA; [LVEDVI = end-diastolic volume [EDV]/BSA); left ventricular (LV) systolic function, tricuspid annular plane systolic excursion (TAPSE), right ventricular (RV) fractional area change (FAC), RV dimensions in the apical 4-chamber view, tracing basal and mid-cavity minor dimensions and longitudinal dimension, cardiac index, systolic pulmonary artery pressure (PASP), and systolic RV pressure (RVSP). Respiratory and cardiac measurements were recorded at T0 (baseline); T1 (after anesthesia induction, before pneumoperitoneum induction); at 10 (T2), 20 (T3), and 30 (T4) minutes after CO2 insufflation; and at the end of surgery (T5). MAIN RESULTS: Ventilation with PEEP at 10 cm H2O led to significant improvement in both respiratory and cardiac parameters. A reduction in pulmonary vascular resistance and enhanced washout of expiratory CO2 occurred. Ten and, to a lesser extent, 5 cm H2O of PEEP decreased LV stroke work. CONCLUSIONS: Ventilation with PEEP (up to 10 cm H2O) recruits the hypoventilated areas of the lungs and reduces cardiac afterload.