Late Maturation Arrest Research Articles

Cytological Findings in Testicular FNAC in Azoospermia Patient: A Study of 395 Cases

Background: In the age of assisted reproduction, search for the cause of male infertility is increasing in numbers. As finding of obstructive azoospermia give the male a hope there are increased requisition to the cytopathologist to evaluate the spermatogenesis status of a male. The aim of the study was to see the cytomorphological pattern in azoospermia patients and to determine the procedure's safety.
 Materials and methods: From January 2015 to June 2022, in Chattogram, Bangladesh, 395 patients with azoospermia were taken in this descriptive study. After all aseptic precautions and local anesthetization aspiration was done from both testes. Cytomorphological analysis was done in the following classes i) Positive for spermatogenesis ii) Hypo-spermatogenesis iii) Early maturation arrest iv) Late maturation arrest v) Sertoli cell only vi) Atrophic.
 Results: Cytological diagnosis yielded 161 (40.76%) positive for spermatogenesis, indicating obstructive azoospermia, seconded by 'Sertoli cell only' in 115 (29.11%) patients. Both conditions were highest in the age group of 31-40, 93(57.76%) and 72 (62.6%). Hematoma was seen in 04(1.01%) cases and extended period of pain was experienced by 05 (1.26%) patients.
 Conclusion: FNA of the testis is a much simpler and easily accessible procedure to assess the spermatogenesis status of an azoospermia patient.
 Chatt Maa Shi Hosp Med Coll J; Vol.22 (1); January 2023; Page 53-56

Touch print smear of testicular tissue with thionine stain for intraoperative diagnosis in azoospermic men.

To compare the diagnostic value of testicular tissue touch print smear (TPS) conducted on azoospermic patients with results obtained from histopathology and in vitro fertility (IVF) lab findings. Microdissection testicular sperm extraction was performed on a group of 148 azoospermic patients and testicular samples obtained intraoperatively. Using TPS, the samples were smeared onto a sterile slide, followed with staining using thionine. The testis tissue bulk samples were also transferred to the IVF lab, and determinations of sperm presence or absence obtained from IVF lab tests were compared with the TPS sample results. Needle testis biopsy was separately performed on a group of 360 azoospermic patients, and results of pathohistology review on the biopsies were further compared with determinations of spermatogenesis stage obtained from TPS for those patients. When compared with IVF lab results, TPS was found to have 100% (126/126) positive predictive value and 95.5% (25/26) negative predictive value for predicting sperm presence or absence, respectively. Furthermore, TPS was further found to have a 93.6% correlation (337 of 360 biopsies) with results of histological diagnoses performed by needle biopsy. Results from histology and TPS for the detection of sperm presence were concordant in 96.1% (346/360) of biopsies. Diagnosis of SCO by TPS shows the highest correlation with histopathology (98.6%), followed by complete spermatogenesis (97.5%), early maturation arrest (78.9%), and late maturation arrest (27.3%). The results support the continued use of TPS in testicular tissue analysis for more rapid assessment of spermatogenesis and for detection of spermatozoa in azoospermic subjects.

FSH levels and testicular volumes are associated with the severity of testicular histopathology in men with non-obstructive azoospermia

PurposeThe purpose of this study is to assess a potential association between FSH levels and testicular volumes with the severity of testicular histopathology on testicular biopsy in men with non-obstructive azoospermia (NOA) undergoing microdissection testicular sperm extraction (microTESE).MethodsA retrospective chart review was performed from the electronic health records of men who underwent microTESE with NOA.ResultsEighty-six men with NOA underwent microTESE with concomitant testicular biopsy for permanent section to assess the testicular cellular architecture. The histopathological patterns were categorized by severity indicating the odds of sperm retrieval into 2 categories. The unfavorable category included Sertoli cell only pattern and early maturation arrest (n = 50) and the favorable category included late maturation arrest and hypospermatogenesis patterns (n = 36). In the men with unfavorable histopathologic patterns, the mean FSH level was 22.9 ± 16.6 IU/L, and the mean testicular volume was 10.4 ± 6.0 cc. This was in comparison to men with favorable histopathologic patterns revealing a mean FSH level of FSH 13.3 ± 12.0 with a mean testicular volume of 13.3 ± 5.9 cc. There was a statistically significant higher FSH level in men with unfavorable histopathology than favorable (p = 0.004) as well as a significant smaller mean testicular volume in men with unfavorable histopathology (p = 0.029).ConclusionsHigher serum FSH levels and smaller testicular volumes are associated with more severe testicular histopathological patterns in men with NOA.

P–008 The diameter of standard seminiferous tubules varies with the different histological patterns in patients with non-obstructive azoosperm

Abstract Study question Does the standard, not-dilated seminiferous tubules (STs) diameter vary according with different testis histology patterns in patients with non-obstructive azoospermia (NOA)? Summary answer The standard STs diameter differed significantly among cases with different testis histology: hypospermatogenesis (HYPO) had the highest STs diameter compared to the other histological subgroups. What is known already During microdissection testicular sperm extraction (mTESE), the identification of dilated STs, as subjectively evaluated at high magnification by comparing their apparent caliber with that of the surroundings, is crucial to identify residual foci of intact spermatogenesis and to retrieve sperm. Previous studies have demonstrated that dilated STs contain sperm in most cases, but it is not clear why in some cases an apparent normal tubular caliber does not correspond to spermatogenesis integrity. Aim of the present study was to assess whether different histology patterns could affect the STs diameter. Study design, size, duration We retrospectively evaluated 168 patients with NOA undergoing unilateral (N = 91) or bilateral (N = 77) mTESE from 2018 through 2019. One or more biopsy samples representative of the overall appearance of the testicular parenchyma were taken from one (for unilateral mTESE) or both testes (for bilateral mTESE), was fixed in Bouin’s solution and sent to the pathologist. Histological analysis was conducted by the same experienced pathologist, who examined at least 100 different tubule sections per biopsy sample. Participants/materials, setting, methods Each tubule section (N = 100 per sample) was evaluated at 10X magnification with a micrometer to measure the tubule diameter, then the mean ST diameter was computed. The basal membrane (BM) thickening was evaluated in every section, and a score was assigned by multiplying the degree of BM fibrosis (mild=1, moderate=2, severe=3) for the number of sections (e.g. BM score for moderate fibrosis in 50 sections=2x50=100). Leydig cells hyperplasia (LCH), if present, was also annotated. Main results and the role of chance The median + interquartile range STs diameter was 140; 110–185 mm, while the median BM score was 100; 10–150. Sertoli cell only syndrome (SCO) was found in 51.1% of cases, focal SCO (FSCO) in 4,7%, early (EMA) and late (LMA) maturation arrest in 10.2 and 2.73% respectively, HYPO in 26.17% and hyalinosis (HL) in 5% of cases. LCH was found in 46.88% of samples. STs diameter, BM score and LCH differed significantly among the different histological patterns: STs diameter was 125; 100–148 in SCO, 162; 102–187 in FSCO, 130; 100–175 in EMA, 145; 130–195 in LMA, 205; 170–240 in HYPO and 57.5; 42.5–100 in HL. HYPO samples also had the lowest BM score (20;1–100; p < 0.0001) and LCH prevalence (23.8%, p < 0.0001) compared to the other histological subgroups (HS). A multinomial logistic regression for prediction of different histological subgroups was run with STs diameter, BM score and LCH as candidate predictors: the model explained the 29% of variability of the outcome and correctly classified 69% of cases. STs diameter significantly predicted FSCO (RR 1.02, 95% CI 1.0–1.04), LMA (RR 1.02, 95% CI1–1.04) and HYPO (1.03, 95%CI 1.02–1.04), while BM score significantly predicted HL (RR 1.07, 95%CI 1.02–1.13). Limitations, reasons for caution The STs were carefully cut before extraction in order to preserve their structural integrity, however the accuracy of such a method of estimating the STs diameter needs to be assessed by further studies. Wider implications of the findings: The identification of dilated STs remains the best strategy to retrieve sperm by mTESE, since larger STs diameters are mostly associated with the more favorable histological patterns. However, dilated STs may be also found in cases with LMA, which explains why in some cases dilated STs do not contain sper. Trial registration number Not applicable

Sperm impairing microbial factor: potential candidate for male contraception

BackgroundDespite significant advances in contraceptive options for women, vasectomy and condoms are the only options available for male contraception. Due to this limitation, the burden of contraception resides on the shoulders of females only. Therefore, there is an urgent need to develop a safe, effective and reversible method of contraception for men. Amongst the alternative approaches, microbial derived products are gaining attention of the scientific world to combat unintended pregnancies. Earlier in our laboratory, sperm impairing microbial factor (Sperm immobilization factor) isolated from Staphylococcus aureus has shown excellent contraceptive efficacy in female mice. Keeping this in mind, the present study was carried out to exploit the sperm immobilization factor (SIF) as potential male contraceptive using vas deferens for administration in mouse model.MethodsSIF (10, 50, 100 or 200 μg) was inoculated in the lumen of right vas deferens whereas the left vas deferens served as control. The mice were sacrificed at Day 3, 7, 14, 21, 30, 45, 60 and 90 after inoculation and the results in terms of change in body weight, seminal parameters, Tissue somatic indices (TSI), haematological parameters, serum level of testosterone, lipid peroxidation and histology were studied. In order to ratify the SIF induced azoospermia SIF (200 μg) was administered with different doses viz. 100, 200, 300, 400 or 500 μg of SIF binding receptor extracted from mouse spermatozoa.ResultsThe weight profile studies of all the experimental groups showed no significant change in the initial and final body weight. In case of seminal parameters, the results revealed that right vas deferens treated with SIF showed azoospermia and with 200 μg of SIF it persisted up to 90 days. TSI of reproductive organs and non-reproductive organs showed no significant change in all the experimental groups. The haematological indices were found to be unaltered throughout the course of investigation however significant decrease in testosterone level was observed in the treated mice. The treatment also affected the oxidative status of the testis. Further, histological studies revealed hypospermatogenesis and late maturation arrest on treated side whereas the left side which served as control showed normal tissue histology. SIF induced azoospermia was ameliorated when administered with 400 μg of SIF binding receptor from mouse spermatozoa.ConclusionSIF, when administered via intra vas deferens route, could lead to complete azoospermia. Therefore, it could be considered as a potential male contraceptive.

SEMINAL TEX-101 MAY PREDICT RESTORATION OF SPREMATOGENESIS AFTER VARICOCELECTOMY IN AZOOSPERMIC MEN WITH PALPABLE VARICOCELE

around 40% of men with non-obstructive azoospermia (NOA) and palpable varicocele may benefit from varicocelectomy with appearance of sperms in ejaculate. Testicular histopathology predicts the outcome of varicocelectomy and men with hypospermatogenesis or late maturation arrest have better prognosis compared to men with early maturation arrest or Sertoli cell only (SCO) syndrome.

Undescended Testis Does Not Affect the Outcome of Microdissection Testicular Sperm Extraction

ObjectiveTo determine the sperm retrieval rates (SRRs) and predictive factors of patients with a history of undescended testis after testicular sperm extraction (TESE). MethodsA total of 311 patients were diagnosed with nonobstructive azoospermia (NOA) and underwent TESE were included in this study.These patients were divided into 2 groups: an undescended group consisting of 62 patients who had a history of undescended testes and an idiopathic group consisting of 249 patients. Of the 62 patients with a history of undescended testes, 26 had a history of bilateral orchidopexy, 15 had a history of unilateral orchidopexy, and 21 had no history of surgery. ResultsThe testicular spermatozoa were found in 134 (53.8%) and 31 (50%) patients in the idiopathic NOA and undescended testes groups, respectively. Similar to patients with idiopathic NOA, the overall SRRs were strongly associated with histopathology for patients with a history of undescended testes. These SRRs were 34.2%, 33.3%, 71.4%, 100%, and 100% for Sertoli Cell Only, late maturation arrest, early maturation arrest, hypospermatogenesis, and normal spermatogenesis, respectively (P <.001). In the undescended group, the SRRs of patients who underwent orchidopexy were not different than patients without a history of orchidopexy. However, patients who underwent unilateral orchidopexy had a higher SRR than those who underwent bilateral orchidopexy (P = .031). ConclusionTESE is a successful treatment modality for men with NOA associated with or without a history of undescended testis. The testicular histopathology and unilateral undescended testis were identified as independent predictors of SRRs for men with a history of undescended testis.

MP38-08 SELECTIVE LYSINE SPECIFIC DEMETHYLASE 1 INHIBITOR, NCL1, COULD CAUSE TESTICULAR TOXICITY VIA THE REGULATION OF APOPTOSIS

INTRODUCTION AND OBJECTIVE: Recent studies show that epigenetic alterations, such as lysine-specific demethylase 1 (LSD1), lead to oncogenic activation, suggesting such alterations as therapeutic target molecules. However, studies evaluating the effect of LSD1 inhibitors on male fertility are lacking. Here, we first analyzed the potential toxicity of new selective LSD1 inhibitor, NCL1, in the testis. METHODS: Immunohistochemistry of LSD1 using human testicular samples was undertaken. Six-week-old male C57BL/6J mice were injected intraperitoneally with dimethyl sulfoxide vehicle, 1.0 mg/kg, or 3.0 mg/kg NCL1 twice a week, and 20 mg/kg busulfan once. After five weeks, testicular toxicity and protein expression were analyzed in testicular and blood samples. Cell viability, flow cytometry, and western blot analyses of GC-1, TM3, and TM4 cell lines treated with NCL1 were also undertaken. RESULTS: In human samples, LSD1 was mainly expressed in Sertoli and germ cells, with intensity levels significantly decreasing in a progressively meiosis-dependent manner. Expression patterns among germ cells were similar in normal spermatogenesis, late maturation arrest, and early maturation arrest. Histological examination revealed significantly increased abnormal seminiferous tubules in busulfan and 3.0 mg/kg NCL1- treated mice compared to control; cellular detachment, sloughing, vacuolization, eosinophilic changes and TUNEL-positive cells were significantly greater. NCL1 also reduced serum total testosterone levels. Western blot of mouse testicular samples revealed NCL1 induced an elevation in cleaved caspases 3, 7, and 8, and expression of connexin 43 proteins. NCL1 significantly reduced the cell viability of GC-1, by apoptosis, but not TM3 and TM4 cell lines in a dose-dependent manner. CONCLUSIONS: High-dose NCL1 targeting LSD1 caused dysfunctional spermatogenesis and induced caspase-dependent apoptosis. These results add to our knowledge of the effect of LSD1 inhibitors on male fertility.Source of Funding: none

Fine-Needle Aspiration Cytology of the Testes for the Classification of Azoospermia and Its Value in the Assessment of Male Infertility

Background: Infertility is an ever-increasing problem in today’s world. It can be due to male or female causes. Azoospermia seen in 5–10% of infertile men is due to obstructive or non-obstructive causes. Traditionally, testicular biopsy is the gold standard for evaluation. Fine-needle aspiration (FNA), however, is minimally invasive, provides qualitative and quantitative information about spermatogenesis, and can aid in assisted reproductive techniques making it a novel technique for the evaluation of male infertility. Objective: We aimed to classify different causes of azoospermia into different patterns based upon FNA, and assess the utility of cell indices in classifying cases into different patterns. Method: We conducted a prospective and a retrospective study of 42 azoospermic males, confirmed on semen analysis, over a period of 5 years. Patients were subjected to FNA of the testes. Smears were prepared, air-dried, wet-fixed, and then stained with May-Grünwald Giemsa and Papanicolaou stains, respectively. Cells were identified using predetermined morphologic criteria, and various indices were calculated followed by statistical analysis of the observations. Results: The mean age of 40 patients who satisfied the adequacy criteria was 32.75 years (range 22–48 years). Thirty-four patients had primary infertility and 6 had secondary infertility. Of these, 12 had normal spermatogenesis, 8 had hypo-spermatogenesis, 3 had early and 7 had late maturation arrest, 6 had Sertoli cell-only syndrome (SCOS), and there were different results in each testicle in 4 cases. The Sperm Index (SI) was significantly higher in all cases of normal spermatogenesis than in any of the hypo-spermatogenesis cases (p = 0.009). The Sertoli Index (SEI) in cases of hypo-spermatogenesis and maturation arrest was significantly higher than in cases of normal spermatogenesis (p < 0.001). The Sperm-Sertoli Index (SSI) also showed significant differences between cases of hypo-spermatogenesis and normal spermatogenesis (p < 0.001). These indices were useful in categorising patients with azoospermia. Conclusion: FNA helps to easily and accurately identify all types of testicular cells without biopsy. SI, SEI, and SSI are powerful cell indices for assessing the extent of spermatogenesis and classifying various causes of azoospermia. Bilateral sampling and multiple aspirations give a better mapping of spermatogenesis within the testes. Testicular FNA can thus play a very important role in the evaluation of male infertility.

Relationship between testicular histopathology and the success of testicular sperm extraction in patients with non-mosaic Klinefelter syndrome

ObjectiveTo investigate the effect of testicular histopathology on the success of microscopic testicular sperm extraction (mTESE) and the factors that could predict the success of mTESE in patients with non-mosaic Klinefelter syndrome (KS). Material and methodsForty-one KS patients diagnosed with non-obstructive azoospermia (NOA) who had undergone mTESE at our clinic were included in the study. The patients were divided into 5 groups according to the histopathology results: hyalinisation of tubules (HT), sertoli cell only (SCO), early maturation arrest (EMA), late maturation arrest (LMA), and hypospermatogenesis (HS). The groups were compared with regard to age, duration of infertility, hormone profile, testicular volume, and sperm retriveal rate. The clinical features of the patients with mTESE from whom sperm could or could not be obtained were also compared with the aim of investigating the predictive value of testicular histopathology and the other variables for prediction of the success of mTESE. ResultsSperm could be obtained through mTESE in 13 out of 41 patients (31.7%). A statistically significant difference was determined between the groups with regard to the rate of sperm collection. No significant difference was determined between the histopathology groups with regard to the other variables. A statistically significant difference was determined between the groups from whic sperm could be collected or not with regard to age, Johnsen criteria, SCO, EMA and LMA variables. Multi-variate analysis revealed that age and Johnsen score were the independent variables predictive for success of mTESE. ConclusionThe present study has revealed that impairment in testicular histopathology negatively affects the success of mTESE and that it is a predictive factor for the success of mTESE in patients with KS. Increased patient age was also determined to negatively affect the success of mTESE and the operation was demonstrated to be more successful before 34 years of age.

A Novel Dual Immunostain to Characterize Sloughed Cells in Testicular Biopsies for Infertility.

Evaluation of testicular biopsies from azoospermic men requires recognition of phases of germ cell maturation as organized architecturally within the seminiferous tubule, as well as distinguishing the inability to generate mature spermatozoa (germ cell aplasia or maturation arrest) from normal spermatogenesis, which may be associated with a reversible obstruction. While traditional fixatives (eg, Bouin solution) provide exquisite nuclear detail and preserve the architectural integrity of the seminiferous tubule, formalin fixation yields biopsies with relatively poor nuclear detail and frequent luminal sloughing of cells, making it difficult to assess sperm maturation. One clone of the anti-DOG1 antibody was recently found to be expressed in late (postspermatogonial) germ cells. We developed a dual stain including DOG1 and SF-1 to mark late germ cells and Sertoli cells, respectively, in both sloughed and intact cells. Consecutive testicular biopsies (N=28) from men with azoospermia were classified by hematoxylin and eosin morphology and stained with a dual SF-1 (Perseus)/DOG1 (Cell Marque) immunohistochemical stain. Histologic patterns included normal spermatogenesis (5 cases), hypospermatogenesis (5 cases), late maturation arrest (2 cases), Sertoli cell only pattern (15 cases), and extensive tubular hyalinization (1 case). Architectural disruption of seminiferous tubules with sloughing of cells into the lumens was noted in all biopsies, at least focally. SF-1 (nuclear) was expressed in sloughed Sertoli cells; DOG1 (cytoplasmic) in sloughed postspermatogonial germ cells (spermatocytes and spermatids). This resulted in two distinct immunophenotypes: SF-1(+)/DOG1(-) sloughed cells in cases with the Sertoli cell only pattern and SF-1(+)/DOG1(+) sloughed cells in all other histologic patterns (normal spermatogenesis, hypospermatogenesis, and maturation arrest). Because the rate of sperm retrieval is lower in men with the Sertoli cell only pattern, this immunohistochemical stain may assist pathologists in the proper interpretation of testicular biopsies, allowing better-informed decision making by patients and clinicians regarding the subsequent use of assisted reproductive technologies.

Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders.

Histone H3.5 (H3.5) is a newly identified histone variant highly expressed in the human testis. We have reported the crystal structure, instability of the H3.5 nucleosome and accumulation around transcription start sites, mainly in primary spermatocytes, but its role in human spermatogenesis remains poorly understood. Testicular biopsy specimens from 30 men (mean age: 35years) with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction and 23 men with obstructive azoospermia (OA) were included. An H3.5-specific mouse monoclonal antibody recognizing an H3.5-specific synthetic peptide was generated, and immunohistological staining for H3.5 and proliferating cell nuclear antigen (PCNA) was performed on Bouin's solution-fixed sections. Expression and localization of H3.5 were compared with patient background, germinal stage, and PCNA expression. In testes of patients with normal spermatogenesis, differentially expressed H3.5 was specifically localized in either spermatogonia or preleptotene/leptotene-stage primary spermatocytes, especially during germinal stages VI-X. In NOA testes, mRNA expression of H3.5 (H3F3C) was significantly reduced compared with other H3 histone family members, and expression of H3.5 was significantly lower than that in OA. Additionally, the number of H3.5-positive germ cells was higher in hypospermatogenesis or late maturation arrest than in early maturation arrest in NOA testes (p<0.01). A significant positive correlation was observed between H3.5 and PCNA expression (p<0.05) but not TUNEL-positive cells, and expression of H3.5 was enhanced after hCG-based salvage hormonal therapy. Different from other testis-specific histones, which are often expressed during the histone-to-protamine transition during meiosis, H3.5 was expressed mainly in immature germ cells. H3.5 may play roles in DNA synthesis, but not apoptosis, and its expression is regulated by gonadotropins, indicating that such epigenetic regulations are important in normal spermatogenesis and spermatogenic disorders.

The predictive value of parameters of clinical presentations for sperm yield in patients with nonobstructive azoospermia receiving microdissection testicular sperm extraction

ObjectiveWe analyzed a cohort of nonobstructive azoospermia (NOA) patients receiving microdissection testicular sperm extraction (mTESE) to examine the relationship of sperm yield and the parameters of clinical presentations. We aim to identify the parameters that might positively predict a positive sperm yield after mTESE. Materials and methodsA total of 200 patients with NOA who had undergone mTESE were enrolled. Among them, 112 (56%) had received a prior testicular needle biopsy. Clinical data including physical findings, underlying genetic abnormalities, pathologic findings in needle biopsy, and sperm retrieval rate (SRR) during mTESE were reviewed and analyzed. ResultsThe pathological findings of prior needle biopsy demonstrate a predictive value of sperm yield during mTESE. Hypospermatogenesis had SRR of 93.3% during mTESE, early maturation arrest had SRR of 13.3%, late maturation arrest (LMA) had SRR of 66.7%, and Sertoli cell-only syndrome had SRR of 18.1%. Regarding parameters of clinical presentation, we found that SRR during mTESE was 85.7% for patients with hypogonadotropic hypogonadism, 60.0% for men with undescended testes (UDT) history, 50.0% for patients who had been exposed to chemotherapeutics due to malignancy of other organs, 100% for prior mumps infection, 50.0% for AZFc deletion, 50.0% for Klinefelter syndrome, and 33.3% for other sex chromosome-related abnormalities. No sperm was found in patients with AZFa or AZFb microdeletion. The consistency of histopathological findings between initial testis biopsy and mTESE was 77.7%. As much as 17.4% of cases had upgraded on spermatogenesis at later mTESE. ConclusionClinical presentations or phenotypes can be used as predictive factors for successful sperm retrieval during mTESE in patients with NOA. Hypogonadotropic hypogonadism and cases with UDT history have a higher chance of sperm retrieval. Initial testicular needle biopsy, if available, can provide valuable information about chances of sperm retrieval. Hypospermatogenesis predicts high sperm yield rate, and LMA can have best upgrade results of sperm yield after mTESE.

MP07-02 POLYA TAG LIBRARY PREPARATION FOR NEW GENERATION SEQUENCING (NGS) IN HUMAN TESTIS FAILS TO DETECT NON-CODING AND TRANSLATED RNAS IMPORTANT IN TESTICULAR FUNCTION AS COMPARED TO RIBOSOMAL RNA DEPLETION METHOD.

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology I1 Apr 2017MP07-02 POLYA TAG LIBRARY PREPARATION FOR NEW GENERATION SEQUENCING (NGS) IN HUMAN TESTIS FAILS TO DETECT NON-CODING AND TRANSLATED RNAS IMPORTANT IN TESTICULAR FUNCTION AS COMPARED TO RIBOSOMAL RNA DEPLETION METHOD. Ryan Flannigan, Anna Mielnik, Alex Bolyakov, Phil V. Bach, Peter Schlegel, and Darius Paduch Ryan FlanniganRyan Flannigan More articles by this author , Anna MielnikAnna Mielnik More articles by this author , Alex BolyakovAlex Bolyakov More articles by this author , Phil V. BachPhil V. Bach More articles by this author , Peter SchlegelPeter Schlegel More articles by this author , and Darius PaduchDarius Paduch More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.268AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Non-coding RNAs (ncRNAs) are emerging as important but poorly understood regulators of mRNA transcription and translation. However, common library preparation techniques for RNA sequencing selects for coding mRNAs by the presence of a poly-A tail; thus, by excluding non-PolyA ncRNAs, many biologically significant transcripts may be overlooked by using this method. The objective of this study was to evaluate differences in testicular RNA identification using 2 different methods of library preparation: polyA and ribosomal RNA depletion (RibZero) using Illumina kits. METHODS Total RNA was extracted from 3 human testis samples and processed using two different methods of library preparations: one based on polyA tags, and the other on depletion of ribosomal RNAs. The cDNA libraries were then sequenced at the same depth and annotated to known published databases. Identified transcripts were divided into two groups based on presence in one of the library preparation methods but not the other. Clinical and biological significance of identified genes was examined using the DAVID. Failure of detection of RibZero-only genes by RNAseq using polyA preparation was then confirmed by analyzing results of RNAseq in 64 testicular samples from normal patients, men with sertoli cell only (SCO), early and late maturation arrest and hypospermatogenesis. RESULTS 61 genes were detected only using polyA method with no detection in RibZero group (p=0.05): 17 of them belonged to small nuclear RNAs (snRNAs), 12 were snRNAs hosting genes, 3 humanin like proteins, and 3 were miRNA hosting genes: MIR137HG, MIR17HG, MIRLET7DHG. Deletion of MIR17HG leads to Feingold syndrome in humans and animal models. 74 genes were identified exclusively in RibZero group and not identified in the polyA group. The top 4 genes identified exclusively by RibZero were TAS2R50, MAGI1-AS, HIST1H3I, HIST1H4K. HIST1H4K was then further analyzed and its expression was highly abundant and specific to pachytene spermatocytes. Important components of the miRNA processing complex: AGO1,2, and 3 were expressed at >2x higher level (p=0.03) in ribosomal RNA library depletion preparation then polyA prep. CONCLUSIONS PolyA tail RNA enrichment method fails to adequately detect at least 7% of important RNAs in the human testis. Including ribosomal depletion RNA library preparation in addition to polyA tags enrichment is an important step to more comprehensively evaluate ncRNAs and testicular function. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e82 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Ryan Flannigan More articles by this author Anna Mielnik More articles by this author Alex Bolyakov More articles by this author Phil V. Bach More articles by this author Peter Schlegel More articles by this author Darius Paduch More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibitory effect of &lt;i&gt;Taraxacum officinale&lt;/i&gt; L (Compositae) aqueous root extract on spermatogenesis

Purpose: To investigate if T. officinale root aqueous extract has anti-spermatogenic activity similar to that of the whole plant which was shown previously to inhibit spermatogenesis. Methods: T. officinale aqueous extract was prepared by soaking 100 g of dried materials in 1 L distilled water for two days at 45 oC. Fifty adult male rats were divided into five groups and treated for 60 days. Four groups were gavaged with the whole plant or root aqueous extract in low or high doses. The male rat rats were allowed to mate with female rats. The control group received distilled water. Sperm count, motility and morphology as well as chromatin integrity were evaluated. Results: Serum testosterone level, sperm parameters, pregnancy rate and average number of fetuses per pregnant females decreased significantly in the treated groups compared to control and in the rootreceiving rats compared to the whole plant-receiving rats. Female rats which were mated with high dose root-receiving males did not deliver fetuses. Cross sections of seminiferous tubules of T. officinale treated rats showed lesions and disorganized germinal epithelium. Late spermatogenesis maturation arrest (spermatid stage) was observed in all of the treated groups except the high dose root-receiving group which showed early maturation arrest (spermatocyte stage). In addition, the mRNA level of two spermatogonial stem cell markers responsible for self-renewal and proliferation of spermatogonia increased in high dose-receiving rats. Conclusion: T. officinale root aqueous extract has inhibitory effects on spermatogenesis. Further studies are required to identify specific ingredient(s) in T. officinale that may be useful as male contraceptive(s). Keywords: Taraxacum officinale , Dandelion, GDNF family receptor alpha 1, Macrophage Colony- Stimulating Factor, Promyelocytic Leukaemia Zinc-Finger, Testosterone, Sperm count

Salvage hormonal therapy after failed microdissection testicular sperm extraction: A multi-institutional prospective study.

To validate the efficacy of salvage hormonal therapy in men with non-obstructive azoospermia at their second microdissection testicular sperm extraction. This was a multi-institutional study registered at the Japanese University Hospital Medical Information Network clinical trial center. After 1month of human chorionic gonadotropin therapy (5000IU, three times a week), patients were treated with recombinant human follicle-stimulating hormone (150IU, three times a week) and human chorionic gonadotropin for the next 3months. Three testicular samples were obtained randomly from both testes, and sent for pathological diagnosis at the first and second microdissection testicular sperm extraction. A total of 21 men, excluding those with chromosomal abnormalities, azoospermia factora or b deletions, extremely small testes (<2mL), or prior hormonal therapy, were eligible to participate based on our inclusion criteria. At the first microdissection testicular sperm extraction, 13 and six patients had Sertoli cells only and an early maturation arrest, respectively. With the second microdissection testicular sperm extraction, sperm were successfully obtained from two patients (10%). Patient age, testicular volume and hormone profiles were not associated with the results of the second microdissection testicular sperm extraction. However, the testicular histology of the two successful patients were late maturation arrest and hypospermatogenesis. Effectiveness of human chorionic gonadotropin-based salvage hormonal therapy preceding a second microdissection testicular sperm extraction seems to be limited. Non-obstructive azoospermia men who have differentiated cells in their testes are likely to respond to hormonal stimulation.

Salvage hormonal therapy after failed microdissection testiculr sperm extraction: a multi-institutional prospective study

We have reported an hCG-based salvage hormonal therapy for men with nonobstructive azoospermia (NOA) who failed to retrieve sperms by microdissection testicular sperm extraction (micro-TESE) and 20% of men could retrieve sperms at the 2nd micro-TESE (Hum Reprod 2012). To validate the efficacy of the salvage hormonal therapy, a multi-institutional prospective study has been done by recruiting NOA men who could not retrieve sperms with various histologies because our prior study included a number of patients with late maturation arrest and hypospermatogenesis.

Outcomes of microdissection testicular sperm extraction in men with early versus late maturation arrest

Maturation arrest (MA) histology on testis biopsy has been associated with worse prognosis for sperm retrieval in men with nonobstructive azoospermia (NOA). Men with early MA have worse outcomes compared to men with late MA. The objective of this study was to evaluate sperm retrieval outcomes for men with NOA and MA who underwent microdissection testicular sperm extraction (micro-TESE).

Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis

To identify potential biomarkers of azoospermia to determine a particular stage of spermatogenetic differentiation. GeneChip Human Gene 1.0 ST microarray with validation at mRNA and protein levels. Basic research laboratory. Men with various types of nonobstructive azoospermia (n = 18) and with normal spermatogenesis (n = 4). Obtaining 31 testicular biopsy samples. Gene expression analysis using the Affymetrix Human Gene 1.0 ST microarrays on 14 selected genes according to the highest fold change, verified with quantitative polymerase chain reaction and on independent set of microarray samples. Western blot and immunohistochemistry were additionally performed. The comparative analysis of gene expression profiles in the infertile and control groups resulted in the selection of 4,946 differentially expressed genes. AKAP4, UBQLN3, CAPN11, GGN, SPACA4, SPATA3, and FAM71F1 were the most significantly down-regulated genes in infertile patients. Global analysis also led to identification of up-regulated genes-WBSCR28, ADCY10, TMEM225, SPATS1, FSCN3, GTSF1L, and GSG1-in men with late maturation arrest. Moreover, the results from quantitative polymerase chain reaction and Western blot largely confirmed the microarray data. The set of selected genes can be used to create a molecular diagnostic tool to determine the degree of spermatogenic impairment for men with idiopathic nonobstructive azoospermia.

Clinical Characteristics and Reproductive Outcomes in Infertile Men With Testicular Early and Late Maturation Arrest

To compare the clinical characteristics and reproductive outcomes of nonobstructive azoospermic men with uniform early and late maturation arrest. Patients with biopsy-documented uniform maturation arrest undergoing testicular sperm retrieval and complete medical records were enrolled in the present study. Their medical history, physical examination findings, testicular volume, serum hormone parameters, genetic anomalies, sperm retrieval, and reproductive outcomes were retrospectively analyzed. In a cohort of 223 nonobstructive azoospermic men, 34 men with uniform maturation arrest (21 early maturation arrest and 13 late maturation arrest) were identified. No significant differences were seen in the age distribution, testicular volume, or hormone parameters between patients with early and late maturation arrest. Only 13 patients (38.2%) had a normal serum follicle-stimulating hormone level and normal testicular volume. Patients with early maturation arrest had a greater frequency of overall genetic anomalies, and patients with late maturation arrest had a greater frequency of previous testicular insults. The sperm retrieval and impregnation rate were nonsignificantly greater in patients with late maturation arrest. Maturation arrest has a variety of causes and presents with diverse phenotypes. Not all patients with uniform maturation arrest have a normal follicle-stimulating hormone level or testicular volume. Patients with early maturation arrest have a greater incidence of genetic anomalies and are more likely to have worse reproductive outcomes than are patients with late maturation arrest.