Comparative proteomics analysis of human FFPE testicular tissues reveals new candidate biomarkers for distinction among azoospermia types and subtypes.

Understanding molecular mechanisms that underpin azoospermia and discovery of biomarkers that could enable reliable, non-invasive diagnosis are highly needed. Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the FFPE testicular proteome of patients with obstructive (OA) and non-obstructive azoospermia (NOA) subtypes hypospermatogenesis (Hyp) and Sertoli cell-only syndrome (SCO). Out of 2044 proteins identified based on ≥2 peptides, 61 proteins had the power to quantitatively discriminate OA from NOA and 30 to quantitatively discriminate SCO from Hyp and OA. Among these, H1-6, RANBP1 and TKTL2 showed superior potential for quantitative discrimination among OA, Hyp and SCO. Integrin signaling pathway, adherens junction, planar cell polarity/convergent extension pathway and Dectin-1 mediated noncanonical NF-kB signaling were significantly associated with the proteins that could discriminate OA from NOA. Comparison with 2 transcriptome datasets revealed 278 and 55 co-differentially expressed proteins/genes with statistically significant positive correlation. Gene expression analysis by qPCR of 6 genes (H1-6, RANBP1, TKTL2, TKTL1, H2BC1, and ACTL7B) with the highest discriminatory power on protein level and the same regulation trend with transcriptomic datasets, confirmed the proteomics results. In summary, our results suggest some underlying pathways in azoospermia and broaden the range of potential novel candidates for diagnosis. SIGNIFICANCE: Using a comparative proteomics approach on testicular tissue we have identified several pathways associated with azoospermia and a number of testis-specific and germ cell-specific proteins that have the potential to pinpoint the type of spermatogenesis failure. Furthermore, comparison with transcriptomics datasets based on genome-wide gene expression analyses of human testis specimens from azoospermia patients identified proteins that could discriminate between obstructive and non-obstructive azoospermia subtypes on both protein and mRNA levels. Up to our knowledge, this is the first integrated comparative analysis of proteomics and transcriptomics data from testicular tissues. We believe that the data from our study contributes significantly to increase the knowledge of molecular mechanisms of azoospermia and pave the way for new investigations in regards to non-invasive diagnosis.

Y-chromosome haplogroup architecture confers susceptibility to azoospermia factor c microrearrangements: a retrospective study.

AIM: To assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment. METHODS: This retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology "Georgi D. Efremov," involved 486 men from different ethnic backgrounds referred for couple infertility from 2002-2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers. RESULTS: Our participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/gr duplications were significantly associated with haplogroup R1a (P<0.001 and P=0.003, respectively) and b2/b3 deletions with haplogroup E (P=0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P=0.031). CONCLUSION: Even though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements.

Molecular and histological characteristics of early triploid and partial molar pregnancies.

Molar pregnancy has the highest incidence of all gestational trophoblastic diseases. This is a heterogeneous group of diseases, composed of precancerous lesions and gestational trophoblastic tumours. The hydatidiform mole is characterised by varying degrees of proliferation of syncytiotrophoblastic and cytotrophoblastic cells and stromal oedema. Based on established morphological and cytogenetic criteria, molar pregnancy is divided into partial and complete. The risk of persistent trophoblastic disease is higher in complete moles compared with partial moles. The aim of this study was to assess the importance of additional molecular methods as a conjunction to the standard histopathological analysis to accurately determine the type and origin of triploidy and to detect partial molar pregnancy. This study examined a total of 24 cases of triploidy. Apart from the detailed histomorphological analysis, a molecular analysis of the placental tissue and maternal DNA was also performed. Digynic triploidy was found in 15 cases, whereas diandric triploidy was found in nine of the cases. The results showed that due to the histomorphological overlap between partial molar pregnancy and hydropic abortions, concomitant histopathological analysis of the placental tissue and molecular analysis of the placental and maternal DNA can lead to correct diagnosis.

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

BACKGROUND: Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. AIM: To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. MATERIAL AND METHODS: Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). RESULTS: AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. CONCLUSION: AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

Proteomics analysis of malignant and benign prostate tissue by 2D DIGE/MS reveals new insights into proteins involved in prostate cancer.

BACKGROUND: The key to a more effective diagnosis, prognosis, and therapeutic management of prostate cancer (PCa) could lie in the direct analysis of cancer tissue. In this study, by comparative proteomics analysis of PCa and benign prostate hyperplasia (BPH) tissues we attempted to elucidate the proteins and regulatory pathways involved in this disease. METHODS: The samples used in this study were fresh surgical tissues with clinically and histologically confirmed PCa (n = 19) and BPH (n = 33). We used two dimensional difference in gel electrophoresis (2D DIGE) coupled with mass spectrometry (MS) and bioinformatics analysis. RESULTS: Thirty-nine spots with statistically significant 1.8-fold variation or more in abundance, corresponding to 28 proteins were identified. The IPA analysis pointed out to 3 possible networks regulated within MAPK, ERK, TGFB1, and ubiquitin pathways. Thirteen of the identified proteins, namely, constituents of the intermediate filaments (KRT8, KRT18, DES), potential tumor suppressors (ARHGAP1, AZGP1, GSTM2, and MFAP4), transport and membrane organization proteins (FABP5, GC, and EHD2), chaperons (FKBP4 and HSPD1) and known cancer marker (NME1) have been associated with prostate and other cancers by numerous proteomics, genomics or functional studies. We evidenced for the first time the dysregulation of 9 proteins (CSNK1A1, ARID5B, LYPLA1, PSMB6, RABEP1, TALDO1, UBE2N, PPP1CB, and SERPINB1) that may have role in PCa. The UBE2N, PSMB6, and PPP1CB, involved in cell cycle regulation and progression were evaluated by Western blot analysis which confirmed significantly higher abundances of UBE2N and PSMB6 and significantly lower abundance of PPP1CB in PCa. CONCLUSION: In addition to the identification of substantial number of proteins with known association with PCa, the proteomic approach in this study revealed proteins not previously clearly related to PCa, providing a starting point for further elucidation of their function in disease initiation and progression.

SNaPshot assay for the detection of the most common CFTR mutations in infertile men.

Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1-2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF) and CFTR-related disorder (CFTR-RD) mutations are found in a high percentage (22.7%) of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.

Extreme gigantomastia in pregnancy: case report--my experience with two cases in last 5 years.

We present an extreme case of gigantomastia in pregnancy during the second gemelar pregnancy of a 30-year-old woman. Her first pregnancy was 8 years ago, was also gemelar and she delivered with caesarean section. From the beginning of her current pregnancy, the patient noted steady growth of both of her breasts that reached enormous dimensions at the end of the pregnancy. This kind of breast changes did not occur during her first pregnancy. The patient also suffered from myasthenia gravis that was in remission during this pregnancy, without any therapy. The patient was in the 38 weeks of gestation, and a delivery with caesarean section was performed in line with the reduction of her breasts. The main reasons that led me to perform these two interventions as one act were the fact that puerperal mastitis could develop on these enormous breasts, further the small regression of these huge breasts during the bromocriptine treatment, as well as the intention to avoid other operative traumas, considering possibility of exacerbation of myasthenia gravis. I had already performed bilateral reduction mammaplasty with free areola-nipple graft, when a tissue with total weight of 20 kg (2 × 10 kg) was removed. The patient had an excellent post-operation recovery course.

Granular cell tumor of the breast: a case report and review of literature.

A 22-year-old female patient presented with a breast mass lesion with a clinical suspicion of a fibroadenoma. Histological evaluation revealed a rare benign neoplasm - granular cell tumor.Granular cell tumor is rare neoplasm that may arise in virtually any body site, and in 5% it occurs in the breast. The histogenesis of this tumor is still rather controversial and currently the most acceptable theory is a Schwann cell origin. The main histological feature is granular cytoplasm of the tumor cells.From a clinical point of view there is a similarity between granular cell tumor and mammary carcinoma on mammography and ultrasound. Pathohistologically, sometimes, differential diagnostic difficulties exist concerning apocrine carcinoma, histiocytic lesions and metastatic neoplasms.

Penile strangulation: two case reports and review of the literature.

INTRODUCTION: Entrapment or strangulation of the penis is a rare emergency situation that can lead to a wide range of vascular and mechanical injuries. AIM: The aim of this article is to present our experience dealing with penile strangulation. A review of the literature is also summarized in this report. Current treatment options and outcomes are also evaluated. METHODS: We performed a computerized MEDLINE search followed by a manual bibliographic review of cross-references. These reports were analyzed and the important findings summarized. RESULTS: Penile strangulation has been first time reported in 1755. Since that time, sporadic reports have appeared in the literature describing a variety of foreign bodies on the penis that have in common only the property of circularity. We noted motives, types of objects, types of strangulation, symptomatology, trauma grades, diagnoses, including psychological involvement, as well as possible treatment options. Furthermore, two cases of penile strangulation from our clinical practice are presented involving different degrees of vascular insult leading to different pathogenesis, clinical presentation, and surgical approach. CONCLUSION: Penile strangulation is an unusual clinical condition and the consequences can be severe. Penile strangulation could lead to different degrees of vascular obstruction. Consequently, several clinical syndromes can occur: from mild nonsignificant vascular obstruction that resolves after decompression to severe gangrene of the penis accompanied with impaired renal function. The most common motive associated with foreign bodies on the penis is sexual or erotic in nature. The choice of method for removal depends upon type, size, incarceration time, trauma grade, and availability of the equipment. Prompt diagnosis and early treatment are essential to avoid the potential complications of ischemic necrosis and autoamputation.

Extreme gigantomastia in pregnancy: case report and review of literature.

We present an extreme case of Gigantomastia in pregnancy in a 24-year old woman, gravida 2, in a 28 weeks' of gestation, with a total breast weight of 33 kg, complicated by infection, ulcerations and subsequent hemorrhage. Thorough laboratory analyses did not reveal any hint as to the cause of this enormous breast enlargement. Gynecological examinations and ultrasound revealed a viable, progressive normal fetus. The severity of the problem is further emphasized by the patients' breathing problems and even big difficulty in standing and walking. We performed bilateral simple mastectomy as a life-saving procedure to prevent fatal complications. The procedure finished without any complications or large amount of blood loss. There are less than 100 cases of gravid gigantomastia reported, but never to such extreme breast weight. Etiology remains uncertain, and controversy exists in therapeutic modality. According to the literature the most reliable conservative treatment is bromocriptine therapy, but if the condition progresses surgical intervention, in the form of reduction mammoplasty or simple mastectomy, is the treatment of choice.

Chondrolipoma of the breast--case report and a review of literature.

Chondrolipoma of the breast is a rare mesenchymal tumor and only a few cases have been reported so far. We present a case of a patient with a breast mass that was surgically removed, diagnosed as a fibroadenoma, and pathohistologically verified as a chondrolipoma. Macroscopically, the tumor presented as a well-circumscribed, firm white nodule resembling a fibroadenoma, and was microscopically characterized as fat lobules with isles of hyaline cartilage. We also present a review of literature and differential diagnoses.