12:00 p.m.-2:30 p.m.
Executive Committee Meeting
Location: Concerto B
2:00 p.m.-6:00 p.m.
Registration/Information Desk Open
Location: Ballroom Registration
5:00 p.m.-10:00 p.m.
Council Meeting and Dinner
Location: Concerto A
7:00 a.m.-7:00 p.m.
Registration/Information Desk Open
Location: Ballroom Registration
4:00 p.m.-9:30 p.m.
Exhibit Hall Open
Location: Symphony Ballroom I
8:30 a.m. - 5:30 p.m.
*ASA Andrology Lab Workshop "Restoring Strict Morphology Relevance: A Consensus Workshop"
Location: Concerto A
8:30 a.m. - 8:35 a.m.
Welcome from ALW Committee
Erma Z. Drobnis, PhD
University of Missouri School of Medicine; Reproductive Medicine & Fertility
8:35 a.m. - 8:45 a.m.
Purpose of the Workshop and Ground Rules
8:45 a.m. - 8:55 a.m.
Connecting Devices and Data Input
Anna-Marie Bort, MLT, (ASCP)CME
Fertility Solutions, Inc.
8:55 a.m. - 9:20 a.m.
Classification I: Participants
9:20 a.m. - 9:50 a.m.
Classification II: Participants
9:50 a.m. - 10:20 a.m.
Classification III: Participants
10:20 a.m. - 10:35 a.m.
10:35 a.m. - 10:55 a.m.
WHO's on Fifth: How Morphology Lost Predictive Value
Susan Ann Rothmann, PhD, HCLD
Fertility Solutions, Inc.
10:55 a.m. - 11:10 a.m.
Results of Classification Sessions
11:10 a.m. - 11:30 a.m.
Susan Ann Rothmann, PhD, HCLD
Fertility Solutions, Inc.
11:30 a.m. - 12:00 p.m.
Anna-Marie Bort, MLT, (ASCP)CME
Fertility Solutions, Inc.
12:00 p.m. - 1:30 p.m.
LAB SCIENCE FORUM LUNCHEON "Discordance and the lab: Evaluating and processing semen from virus-infected patients"
Location: Concerto B
Erma Z. Drobnis, PhD
University of Missouri School of Medicine; Reproductive Medicine & Fertility
1:30 p.m. - 1:50 p.m.
Algorithm Worked Examples
Anna-Marie Bort, MLT, (ASCP)CME
Fertility Solutions, Inc.
1:50 p.m. - 2:00 p.m.
2:00 p.m. - 2:40 p.m.
Classification I: Participants
2:40 p.m. - 3:20 p.m.
Classification II: Participants
3:20 p.m. - 3:30 p.m.
3:30 p.m. - 4:10 p.m.
Classification III: Participants
4:10 p.m. - 5:30 p.m.
Results of Classification
5:30 p.m. -
12:00 p.m. - 1:00 p.m.
Industry Satellite Symposia Lunch
Location: Concerto D
1:00 p.m. - 4:00 p.m.
ASA Clinical Symposium: Sexual Function and Fertility in the Spinal Cord Injured Male
Location: Symphony Ballroom III/IV
1:00 p.m. - 2:45 p.m.
Infertility and Ejaculatory Dysfunction in Spinal Cord Injured Men
Nancy L. Brackett, PhD, HCLD
Univ of Miami Miller School of Medicine
Emad Ibrahim, MD
Lois Pope Life Center
Charles M. Lynne, MD
University of Miami
1:00 p.m. - 1:15 p.m.
Effect of Spinal Cord Injury on Erection and Ejaculation
Emad Ibrahim, MD
Lois Pope Life Center
1:15 p.m. - 1:29 p.m.
Assisted Ejaculation Procedures in Spinal Cord Injured Men
Emad Ibrahim, MD
Lois Pope Life Center
1:30 p.m. - 1:44 p.m.
Causes of Abnormal Semen Quality in Spinal cord Injured Men
Nancy L. Brackett, PhD, HCLD
Univ of Miami Miller School of Medicine
1:45 p.m. - 1:59 p.m.
Assisted Reproduction in Spinal Cord Injured Men
Charles M. Lynne, MD
University of Miami
2:00 p.m. - 2:30 p.m.
VIDEO " Penile Vibratory Simulation and Electroejaculation Procedures in Spinal Cord Injured Men"
Charles M. Lynne, MD
University of Miami
2:30 p.m. - 2:45 p.m.
2:45 p.m. - 3:00 p.m.
3:00 p.m. - 3:20 p.m.
Medical Management of Erectile Dysfunction in the Spinal Cord Injured Men
Alan Scott Polackwich, Jr., MD
Mount Siani Medical Center
3:20 p.m. - 3:40 p.m.
Surgical Management of Erectile Dysfunction in the Spinal Cord Injured Men
Doron Sol Stember, MD
Beth Israel Medical Center
3:40 p.m. - 3:45 p.m.
3:45 p.m. - 4:00 p.m.
Costs, Billing, Reimbursement - Practical Aspects of Integrating SCI Patients into a Clinical Practice
Dana Alan Ohl, MD
University of Michigan
6:00 p.m. - 6:10 p.m.
Welcome and Opening Remarks
6:10 p.m. - 6:30 p.m.
Updates from NIH, NICHD & NIEHS
Daniel S. Johnston, PhD
NIH, Contraception Research Branch
Stuart B. Moss, PhD
National Institutes of Child Health and Human Development
Thaddeus T. Schug, PhD
National Institute of Environmental Health Sciences
6:30 p.m. - 6:50 p.m.
*ANDROLOGY Journal Award
6:50 p.m. - 7:45 p.m.
EMIL STEINBERGER MEMORIAL LECTURE "Making a Good Genome for the Sperm"
Mary Ann Handel, PhD
The Jackson Laboratory
7:45 p.m. - 8:00 p.m.
*Distinguished Andrologist Award
8:00 p.m. - 9:30 p.m.
ASA Welcome Reception
Location: Symphony Ballroom I
6:30 a.m.-8:00 a.m.
Past President's Breakfast
Location: Concerto D
7:00 a.m.-6:00 p.m.
Registration/Information Desk Open
Location: Ballroom Registration
7:00 a.m.-4:00 p.m.
Exhibit Hall Open
Location: Symphony Ballroom I
7:00 a.m.-8:00 a.m.
Continental Breakfast in Exhibit Hall
Location: Symphony Ballroom I
8:00 a.m. - 9:00 a.m.
LECTURE I: The importance and Strategy for Placing Male Reproductive Health in the Centre Stage in the Political and Research Agenda
Christopher Barratt, PhD
University of Dundee
9:00 a.m. - 9:15 a.m.
*Distinguished Service Award
9:15 a.m. - 10:45 a.m.
SYMPOSIUM I - New Insights into ART
Peter Chan, MD
McGill University Health Center
9:15 a.m. - 9:45 a.m.
Large Scale Studies on Controversy on ICSI Offspring Outcomes
Mary Croughan, PhD
University of California, Office of the President
9:45 a.m. - 10:15 a.m.
Implications of the Aging Male Gamete on ART Outcome
Gianpiero D. Palermo, MD, PhD
Weill Cornell Medical, Reproductive Medicine
10:15 a.m. - 10:45 a.m.
Autism and Other Developmental Disorders in ART
Sven Sandin, PhD
Mount Sinai School of Medicine
10:45 a.m. - 11:00 a.m.
Break & Networking in Exhibit Hall
11:00 a.m. - 12:30 p.m.
*Poster Session I
Location: Symphony Ballroom II
DIFFERENTIAL TOLEROGENIC CAPACITY OF THE EPIDIDYMIS AND TESTIS IN MICE WITH CONDITIONAL DELETION OF TGFBR2 IN DENDRITIC CELLS.
Fernando Pierucci-Alves¹, Monica T. Midura-Kiela², Sherry D. Fleming³, Bruce D. Schultz¹ and Pawel R. Kiela4
¹Kansas State University, Dept of Anatomy & Physiology; ²University of Arizona, Dept of Pediatrics; ³Kansas State University, Division of Biology; 4University of Arizona, Depts of Pediatrics and Immunobiology
Presented By: Fernando Pierucci-Alves
Sperm are immunogenic and peripheral tolerance mechanisms are necessary for reproductive success. Initial data revealed prominent physiological signaling by transforming growth factor beta (TGFβ) in murine epididymis, where large networks of dendritic cells (DCs) and macrophages exist. This study’s overarching hypothesis is that TGFβ-signaling in epididymal DCs maintains immunotolerance to sperm in the epididymis and disruption of this signaling pathway breaks sperm tolerance through impaired or insufficient regulatory T cell (Treg) function. In male mice with DC-specific TGFβ receptor 2 deletion (Tgfbr2ΔDCFoxP3GFP-KI), we detected severe epididymal leukocytosis with sperm granulomas, antisperm antibodies but no apparent testicular pathology at the histological level. To further these observations, we quantified leukocytes (CD45+) and Tregs (FoxP3/GFP+) in epididymides, testes and kidneys from 4 Tgfbr2ΔDCFoxP3GFP-KI males and control littermates by flow cytometry. We used kidneys as a sperm-free organ of the genitourinary tract, and as an additional control. Compared to controls, the epididymis of Tgfbr2ΔDCFoxP3GFP-KI mice had 3.4 times more infiltrating CD45+ leukocytes (P<0.05), while the Tgfbr2ΔDCFoxP3GFP-KI testis and kidney exhibited 1.2- and 1.5-fold increase in infiltrating leukocytes (P>0.05), respectively. Tregs were 5-6 times more abundant in epididymis and kidney of Tgfbr2ΔDCFoxP3GFP-KI mice, while the testis exhibited 53 times more Tregs – compared to controls. These data suggest the epididymis is most susceptible to inflammation when the DC/TGFβ-signaling axis is disrupted, and that the testis maintains alternative robust immunosuppressive mechanisms to fend off autoimmune responses. Additional studies are addressing how loss of TGFβ-signaling disrupts epididymal and testicular DC tolerogenic capacities and testing whether there is differential induction and function between epididymal and testicular Tregs. [Supported by P20GM103418 (K-INBRE); Johnson Cancer Research Center]
HUMANIN TRANSGENIC MICE ARE PROTECTED FROM CYCLOPHOSPHAMIDE-INDUCED MALE GERM CELL APOPTOSIS
YanHe Lue MD¹, Hemal Mehta MS², James Hoang BS¹, Kelvin Yen PhD², Junxiang Wan PhD², Ronald Swerdloff MD¹, Pinchas Cohen MD² and Christina Wang MD¹
¹Division of Endocrinology, LABioMed at Harbor-UCLA; ²USC Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA
Presented By: YanHe Lue MD
Humanin (HN) is a cytoprotective peptide encoded by a mitochondrial gene. We have previously demonstrated that the pharmacological administration of HN or its analogue HNG protects male germ cells against cyclophosphamide (CP)-induced apoptosis in mice. To examine the role of endogenous HN in the cytoprotection of male germ cells from chemotherapy, we generated HN transgenic (HNt) mice expressing a CMV-promoter driven humanin transgene. After genotyping by PCR, 1) groups of 7 adult (5-10 month-old) HNt and age-matched wildtype (WT) mice were used for the characterization of male reproductive phenotype, and 2) groups of 6 adult HNt and age-matched WT mice were treated with a single-dose of CP injection (i.p. 200mg/kg) to examine male germ cell apoptosis (quantified as apoptotic index (AI): the number of apoptotic germ cells/100 Sertoli cells). The plasma testosterone (T) was measured by RIA. HNt mice were viable, fertile and smaller in size (BW:28.5±2.2g) with an average of 18% decrease in body weight (BW) as compared to WT (BW:34.9±10.3g) mice. The testis weight (TW:88±10.1mg, p=0.007) in HNt mice was significantly lower than WT (TW:105.2±9.3mg) mice. There was no difference in cauda epididymal sperm count between HNt (1.3±0.07 million/mg cauda) and WT (1.3±0.03 million/mg cauda) mice. Testis histological examination in HNt mice showed normal histology with the baseline germ cell apoptosis rates reminiscent of WT levels. HNt mice have similar plasma T levels (0.6±0.4ng/ml) as WT (0.7±0.5ng/ml) controls. Two days after CP treatment, there were no marked changes in body and testis weight, and plasma T levels. The germ cell apoptosis rate in WT mice was significantly (p<0.001) increased at spermatogenic stages I-III (AI:46.1±4.6), VII-VIII (AI:20.6±0.9) and XI-XII (AI:56.9±4.8) as compared to non-treated WT mice (stages I-III AI: 9.5±2.1; VII-VIII:2.5±0.6; XI-XII:17.5±1.8). In HNt mice, CP treatment significantly increased germ cell apoptosis at stages XI-XII (AI: 23.7±2.9; p=0.03), but not at stages I-III (AI:14.9±2.3) and VII-VIII (AI:4.8±1.1) as compared to baseline levels of HNt mice (stages I-III AI:8.3±1.8; VII-VIII:3.8±0.8; XI-XII:13.3±3.2), suggesting that male germ cells in HNt mice were partially resistant to CP-induced apoptosis. Thus, we conclude that HN 1) is cytoprotective hormone; and 2) mimics the effects of caloric−restriction on metabolism and chemotherapy−protection.
PATERNAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS ALTERS THE SPERM PROTEOME AND INDUCES NEGATIVE PREGNANCY OUTCOMES TRANSGENERATIONALLY
Janice Bailey PhD, Nancy Coté PhD, Clotilde Maurice PhD, Florence Roux-Dalvai MSc, Arnaud Droit PhD and Mathieu Dalvai PhD
Presented By: Janice Bailey PhD
BACKGROUND: The Arctic food chain is contaminated with persistent organic pollutants (POPs). As a consequence, there are major health discrepancies between Inuit and non-Aboriginal Canadians, including adverse pregnancy outcomes or disruption of the immune system. Although healthy pregnancies are multifactorial, it possible that POPs exposure contributes to these adverse outcomes. Moreover, the paternal influence of contaminant exposure on his offspring has not been well-investigated. We hypothesized that early paternal exposure to Arctic POPs affects his sperm proteome and that of his offspring and future generations.
METHODS: Sprague-Dawley females (F0) were gavaged with an environmentally-relevant concentration of an Arctic POPs mixture or corn oil (Control) and mated to untreated males. F1 fathers were mated to untreated females to generate F2 litters; F2 development was followed until PND 90. F3 and F4 generations were similarly produced. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labelling, 2D−LC−MS/MS and immunoblotting analyses were used to identify differentially expressed sperm proteins from all generations.
RESULTS: POPs-exposed F1 males had fewer motile sperm and decreased epididymal sperm counts (P=0.0001). F2 sons from the POPs lineage were subfertile (P=0.02) and their F3 POPs grandsons had fewer pups/litter (P≤0.0001). For F1, F2 and F3 males, 7, 20 and 36 proteins were differentially expressed compared to the control lineage, some of which are implicated in motility, apoptosis or male infertility (SOD1, VDAC2, CS, SLCA3, IZUMO) and may explain the observed subfertility. Importantly, 3 differentially expressed sperm proteins were conserved between the F1 and F3 males and 6 between the F2 and F3 males, showing a transgenerational effect of POPs. Bioinformatics and GO analyses showed that 45-50% of these proteins are implicated in the respiratory chain. Surprisingly, a majority are also part of the same biological pathways as brain disorders. Finally, by converting the rat ID in human genes ID for human diseases identification, we observed a role of certain differentially expressed proteins in infection processes and reduced prostate cancer occurrence.
CONCLUSIONS: POPs exposure induces proteome changes in sperm that can be observed through three generations. The proteins and pathways identified have launched new hypotheses on POPs effects and in our understanding of the adverse health effects observed in Inuit populations.
DEVELOPMENT OF SPERM IN VITRO FROM SPERMATOGONIAL CELLS OF PREPUBERTAL CANCER PATIENTS
Mahmoud Huleihel PhD, Maram Abofoul-Azab PhD¹, Joseph Kapelushnik MD², Haim Pinkas MD³ and Eitan Lunenfeld MD4
¹The Center of Advanced Research and Education in Reproduction , The Shraga Segal Dep. of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.; ²Dep. of Pediatric Oncology and Hematology, Soroka Medical Center, Beer-Sheva, Israel; ³Male Infertility & Sperm Bank, Helen Schneider Hospital for Women, Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel.; 4Fertility and IVF Unit, Dep. OB/GYN, Soroka Medical Center and The Center of Advanced Research and Education in Reproduction , Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
(Presented By: Mahmoud Huleihel PhD)
Introduction: Childhood cancer estimated to affect 0.1% of prepubertal boys. About 80% will survive the disease due to the progress in anti-cancer treatments. Some of these cure patients will become azoospermic. Prepubertal males do not produce spermatozoa; therefore, the only suggested option of their fertility preservation is testicular tissue/cells cryopreservation before aggressive anti-cancer treatments. None of the published in vitro methodology could induce differentiation of human spermatogonial stem cells (SSCs) to sperm cell. Using three-dimensional (3D) culture systems we were able to induce proliferation and differentiation of isolated SSCs to meiotic and postmeiotic stages (mouse and monkey) and the generation of sperm (mouse). Objectives: To examine the presence of spermatogonial cells (SCs) in chemotherapy-treated prepubertal cancer patient males and the possibility to induce those cells into complete spermatogenesis in vitro. Methods: Testicular biopsies were obtained from eight prepubertal patients; seven from chemotherapy-treated cancer patient and one from a patient with β-thalassemia major. Testicular cells were enzymatically isolated and cultured in a methylcellulose culture system (MCS)-enriched with specific growth factors for a period of 5-15 weeks. The presence of premeiotic, meiotic and postmeiotic cells in MCS was examined by immunofluorescence staining (IF) and/or PCR analysis. Results: We observed biologically active SCs in testicular biopsies from prepubertal cancer patient males who had already received substantial chemotherapy. Isolated testicular cells cultured in MCS developed into colonies which contained premeiotic (OCT4,PLZF,VASA,SALL4,GFR-a,CD9,a-6-INTEGRIN,c-KIT), meiotic (LDH, BOULE, CREM-1) and postmeiotic (PROTAMINE, ACROSIN) cells, as were confirmed with IF/PCR analyses. In addition, we were able to identify sperm-like cells in MCS. Conclusions: We demonstrated for the first time, the presence of biologically active spermatogonial cells in testicular biopsies of chemotherapy-treated prepubertal cancer patient males, and the feasibility of their development in MCS to each stage of spermatogenesis including sperm. Should this system be further validated and improved for the production of fertilization competent gametes, then it may assist in future therapeutic strategies for infertility of cancer patient boys and non-obstructive azoospermic patients where no sperms were found in their testicular biopsies.
REGULATION OF CYP26B1 EXPRESSION IN THE SPERMATOGONIAL STEM CELL NICHE
Parag Parekh PHD¹, Thomas Garcia PHD¹,², Reham Waheeb DVM, PHD³, Vivek Jain MS¹,², Gunapal Shetty PHD¹, Marvin Meistrich PHD¹, Marie-Claude Hofmann PHD¹ and Pooja Gandhi MS¹
¹University of Texas MD Anderson Cancer Center; ²University of Houston Clear Lake; ³University of Alexandria, Damanhour
Presented By: Parag Parekh PHD
Cytochrome P45026B1 (CYP26B1) regulates the concentration of all-trans-retinoic acid (RA) and plays a key role in germ cell differentiation by controlling local distribution of RA. Interestingly, little is known about the mechanisms of Cyp26b1 gene regulation. In Sertoli cells, it is maintained by SF1 and SOX9 during gonad development and throughout life but inhibitors that would balance its expression, possibly accounting for the pulses of RA in the adult seminiferous epithelium, are not known. Our previous data from Sertoli-cell specific NOTCH gain- and loss-of-function mouse models indicated that expression of Cyp26b1 is inversely correlated to NOTCH pathway activity. We hypothesized that 1) Spatiotemporal Cyp26b1 downregulation is directly dependent on canonical NOTCH signaling; and 2) A subset of premeiotic germ cells is responsible for Cyp26b1 downregulation through the NOTCH ligand JAG1. Germ cell-Sertoli cell co-cultures experiments demonstrated that JAG1, mainly expressed by Aundiff spermatogonia, activated NOTCH signaling in primary Sertoli cells and induced the transcriptional repressors and canonical NOTCH target genes Hes/Hey. Upregulation of Hes/Hey gene expression by JAG1 was associated with significant decreases in Cyp26b1 expression, while simultaneous downregulation of Hes/Hey by RNAi led to significant increases. Further, Luciferase and ChIP-PCR assays demonstrated that HES/HEY directly bind to the Cyp26b1 promoter to downregulate its expression. Investigation of stage-specific NOTCH activity using transgenic mice, together with qPCR analysis of Hes/Hey and Cyp26b1 expression, indicated lowest expression of Cyp26b1 at stages VI-VIII of the seminiferous epithelium, when NOTCH activity and RA production are highest. To elucidate which germ cells activate NOTCH signaling in Sertoli cells in vivo, we performed germ cell depletion experiments using moderate doses of busulfan. We found that elimination of undifferentiated spermatogonia will downregulate NOTCH signaling and upregulate Cyp26b1 expression in Sertoli cells. In conclusion, we believe that NOTCH signaling, induced by JAG1-expressing Aundiff in Sertoli cells, is a mediator of germ cell differentiation by controlling Cyp26b1 expression and possibly RA pulses.
Supported by NIH R01HD081244
HIGH-RESOLUTION PHENOTYPING OF SPERMATOGENIC DEFECTS USING SINGLE-CELL SEQUENCING
Min Jung, Jannette Rusch PhD, Abul Usmani PhD and Don Conrad PhD
Department of Genetics, Washington University in St. Louis
Presented By: Min Jung
Introduction: RNA sequencing of testis tissue provides great promise for improving the description of molecular defects in men with gonadal dysfunction. With thoughtful interpretation, gene expression data could be useful for classifying patients into a diagnostic hierarchy, defining i) disrupted cell types, ii) disrupted pathways, and, iii) in conjunction with genome sequencing data, disrupted gene(s) and causal genetic variants. However, the cellular diversity of testis severely limits the utility of expression measurements made on bulk tissue. Thus, the application of single-cell RNA-sequencing on male germ cells represents an amazing new set of scientific opportunities for research in male reproductive biology and translational medicine
Objectives: We aim to develop a single-cell framework that utilizes large-scale single-cell expression profiles from normal and disease models to elucidate the fundamentals of spermatogenesis and to phenotype spermatogenic defects.
Methods: Using Drop-seq, we generated single-cell expression measurements on over 30,000 cells from wild-type mice and over 20,000 cells from mouse mutants with infertility of mechanisms known (e.g. MLH3 deficiency) and unknown (spontaneous infertility after a transgene insertion). We constructed a pipeline for interpreting the single-cell data using publicly available single-cell computational tools such as RaceID2, Waterfall, and Monocle2, and an in-house cell-type assigner algorithm.
Results: We find that the increased resolution of single cell expression profiling reveals novel cell-type specific markers, 5 of which we have confirmed with immunofluorescence staining. As part of the framework, we developed a cell-type identifier that provides automated assignments of cells to 4 stages of germ cells and 3 types of somatic cells. Our cell-type assigner algorithm has 96% accuracy when benchmarked with data from flow-sorted germ cells. Finally, unsupervised ordering of male germs on a developmental timescale depicts the genetic landscape of both normal and infertile mice, inferring the cell-types and pathways that are dysregulated in the germ cell development of disease models.
Conclusions: Our single-cell framework has great potential for expanding our ability to dissect pathophysiology in tissues with extensive cellular heterogeneity and decrypt the spermatogenic failure in more patients.
ISOTRETINOIN IMPROVES TOTAL MOTILE SPERM COUNT IN SOME MEN WITH IDIOPATHIC OLIGOASTHENOZOOSPERMIA
John Amory, Kevin Ostrowski MD¹, John Gannon MD², Kathryn Berkseth MD¹, Faith Stevison MS¹, Nina Isoherranen PhD¹, Charles Muller PhD¹ and Thomas Walsh MD, MSE¹
¹UW; ²Intermountain Health
Presented By: John Amory
Introduction: There is no effective medical therapy for men with infertility due to idiopathic oligoasthenozoospermia (IOA). As men with IOA have lower intratesticular concentrations of 13-cis-retinoic acid, we hypothesized that men with IOA may exhibit improved sperm counts during treatment with 13-cis-retinoic acid (isotretinoin).
Methods: We conducted a single-arm, pilot study to determine impact of therapy with isotretinoin on sperm indices in 20 infertile men with IOA. Men were between 21 and 60 years of age without identifiable hormonal or genetic abnormalities and with total motile sperm counts of less than 10 Million on two occasions. All men received isotretinoin 20 mg by mouth twice daily for 20 weeks and had semen analyses, routine blood counts, chemistries and examinations every four weeks during treatment.
Results: Twenty men enrolled in the study and 16 completed all study procedures. All men experienced dry facial skin and chapped lips during treatment, which resolved after Isotretinoin discontinuation. There were no significant laboratory abnormalities noted in any subject, and no subject experienced worsening mood. Mean (SD) total motile sperm count increased from 3.2 (3.1) Million at baseline to 8.5 (13) Million after twenty weeks of therapy (p=0.005 c.f. baseline). Eight of sixteen men (50%) men achieved a total motile sperm concentration of greater than 10 million, and nine men (56%) had a greater than a five-fold increase in total motile sperm count by the end of treatment. The remaining seven men had either minimal or no apparent response to therapy. No significant improvement in sperm motility was observed. Six clinical pregnancies (three spontaneous, 3 via ART) occurred, four of which, including all three of the spontaneous pregnancies, were observed in men with improvements in sperm counts on therapy.
Summary: Approximately 50% of men with idiopathic IOA experienced an increase in total, motile sperm count with isotretinoin therapy. Treatment was generally well tolerated.
Conclusions: Isotretinoin therapy of men with IOA is feasible and is associated with significant improvements in sperm production in a subset of men. Randomized, placebo-controlled studies of isotretinoin therapy for infertile men with IOA are warranted, using live birth as the outcome measure.
Funding: The Eunice Kennedy Shriver National Institute of Child Health and Human Development supported this work through grant K24 HD082231 to J. Amory.
GNRH-ANTAGONIST TREATMENT BEFORE ALLOGENEIC SPERMATOGONIAL STEM CELL TRANSPLANTATION ENHANCES SPERMATOGENEIC RECOVERY IN RHESUS MONKEYS
Gunapala Shetty PhD¹, Jennifer Mitchell VMD¹, Zhuang Wu MD¹, Truong Lam BS¹, Lorraine Hill DVM¹, Ramesh Tailor PhD¹, Karen Peters BS², Kyle Orwig PhD² and Marvin Meistrich PhD¹
¹University of Texas M.D. Anderson Cancer center; ²Magee-Womans Research Institute, University of Pittsburgh School of Medicine
Presented By: Gunapala Shetty PhD
Cancer treatment regimens can completely kill the stem spermatogonia in children and young patients in which case cryopreservation of spermatogonia before therapy and subsequent autologous transplantation may be the only option for restoring male fertility. Previous studies suggested that cancer treatment regimen can cause functional damage in the somatic environment of the testis, which can at least partially be alleviated by hormone suppression treatments.
To optimize conditions for the restoration of spermatogenesis by spermatogonial stem cell (SSC) transplantation, we combined transplantation with the hormone suppression during different times relative to transplantation. We irradiated the testes of 16 pubertal rhesus monkeys with 7 Gy and 4 months later transplanted cryopreserved testicular cells containing SSCs, allogeneically to one of the testes. Six of these irradiated monkeys were treated with GnRH-antagonist (GnRH-ant) starting 2 months after irradiation until the time of transplantation (GnRH-pre group). Five of the monkeys received GnRH-ant treatment starting 3 months after irradiation and lasting until 1 month after transplantation (pre+post group). This GnRH-ant treatment suppressed serum testosterone levels to an about 25% of the pre-treatment values of about 2.5 ng/ml. Five of the monkeys received sham-injections instead of GnRH-ant.
At 9 months after transplantation the transplanted testes in control group showed average weights that were 5% greater than those of the sham-transplanted testes. However in the pre-GnRH-ant group the increase was consistently around 10%, whereas there was no difference in the pre+post GnRH-ant group. Similarly the average numbers of cauda epididymal sperm derived from the transplanted testes in the control group were 2.6-fold higher than those from the sham-transplanted testes. However in the pre-GnRH-ant group the ratio of sperm in the transplanted to untransplanted side was 4.0, whereas in the pre+post-GnRH-ant group the ratio was only 1.4-fold. The results suggested that the hormone suppression prior to transplantation has a beneficial effect on the recovery of sperm from the transplanted SSCs, but hormone suppression after transplantation does not augment the recovery, and rather could be detrimental instead. The tubule differentiation indices indicating the testicular spermatogenic potential in the testes will be discussed. This work was supported by the grant 1P01HD075795-01 from NIH/NICHD.
SPERM MITOCHONDRIAL COPY NUMBER AND DELETIONS: ASSOCIATIONS WITH URINARY-ISOPROSTANE AND PHTHALATE METABOLITES IN MALE PARTNERS UNDERGOING ASSISTED REPRODUCTIVE TECHNOLOGIES (ART)
Alexandra Olmsted¹, Haotian Wu MS¹, Rahil Tayyab PhD², Cynthia Sites MD² and J.Richard Pilsner MPH, PhD¹
¹University of Massachusetts Amherst, Department of Environmental Health; ²Baystate Medical Center, Department of Obstetrics and Gynecology
Presented By: Alexandra Olmsted
Phthalates, a chemical class of plasticizers, are ubiquitous in the environment and recognized as endocrine disrupting compounds (EDCs). Recent data suggest that oxidative stress is a potential mediator for the associations between phthalate exposure and poor male reproductive health. Mitochondria are implicated in the production of excess oxidative stress and sperm mitochondrial copy number (MtCopy) and deletions (MtDeletions) have been linked with male infertility. However, little is known about the relationship of these mitochondrial biomarkers in sperm with phthalate exposure and oxidative stress.
To examine associations of MtCopy and MtDeletions with concentrations of urinary phthalate metabolites and isoprostane in male partners undergoing ART.
A total of (n=96) sperm samples were collected from male partners undergoing ART at Baystate Medical Center, in Springfield, MA from 2014 to 2016 as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Seventeen urinary phthalate metabolites (n=50) were analyzed by the Centers for Disease Control using tandem mass spectrometry. 15F2t-Isoprostane (n=90) was measured using a competitive enzyme-linked immonsorbent assay. A triplex Taqman qPCR method was developed for relative quantification of genomic DNA, MtCopy and MtDeletions. Multivariable linear or logistic regression was employed to examine associations controlling for age, BMI, batch and male infertility status, as defined by abnormal semen quality parameters.
Quartiles of MtCopy and MtDeletions were positively associated with the odds of male infertility (p for trend < .0001 and 0.007, respectively). Urinary metabolites of DiBP, MiBP, and MHiBP displayed an inverse borderline association with MtCopy (β=-0.86; p =0.07 and β=-0.99; p = 0.06, respectively); whereas, urinary MEHP concentrations were positively associated with MtDeletions (β = 0.06; p = 0.004). Urinary isoprostane levels were inversely associated with MtCopy but did not reach statistical significance (β = -0.13; p = 0.07).
These results suggest that certain phthalate metabolites and a known biomarker of oxidative stress may be associated with MtCopy and MtDeletions. Current research is investigating these relationships in a larger sample size.
EFFECTS OF LONG-TERM TESTOSTERONE THERAPY (TTH) WITH TESTOSTERONE UNDECANOATE INJECTIONS (TU) ON ANTHROPOMETRIC AND METABOLIC PARAMETERS IN HYPOGONADAL MEN AND AN UNTREATED CONTROL GROUP: REAL-LIFE REGISTRY DATA FROM A UROLOGY/ANDROLOGY OFFICE
Farid Saad DVM, PhD¹,²,³, Aksam Yassin MD, PhD4, Gheorghe Doros PhD5 and Abdulmaged Traish PhD6
¹Bayer AG, Berlin, Germany; ²Dresden International University, Berlin, Germany; ³Gulf Medical University, Ajman, UAE; 4Institute of Urology and Andrology; 5Boston University School of Public Health; 6Boston University School of Medicine
Presented By: Farid Saad DVM, PhD
Introduction and Objectives:
Although TTh is considered life-long treatment, very few groups have reported long-term data. The present ongoing registry has been established in 2004 to monitor effectiveness and safety of TU.
Of 505 hypogonadal men (mean age 59.0±9.5 years) with total testosterone ≤12 nmol/L and symptoms, 321 received TU 1000 mg every 12 weeks following an initial 6-week interval for a maximum of 12 years (T-group). 184 men had opted against TTh and served as controls (CTRL). Mean follow-up was 8.3±3.5 years in the T-group and 5.5±1.6 years in CTRL.
In the T-group, weight decreased from 99.4±13.4 to 87.5±5.9 kg at 12 years. In CTRL, weight increased from 91.4±10.5 to 96.5±12.4 kg at 8 years (p<0.0001 for both).
In the T-group, waist circumference decreased from 107.2±9.6 to 93.6±3.1 cm at 12 years. In CTRL, waist circumference increased from 99.8±9.1 to 104.7±8.3 cm at 8 years (p<0.0001 for both).
BMI decreased from 31.5±4.3 to 27.6±2.1 kg/m2 at 12 years in the T-group and increased from 29.2±3.2 to 30.7±4.0 kg/m2 at 8 years in CTRL (p<0.0001 for both).
In the T-group (29.3% had type 2 diabetes), HbA1c decreased from 6.5±1.2 to 5.5±0.4% at 12 years. In CTRL (28.4% with T2DM), HbA1c increased from 6.0±0.7 to 6.2±0.8% at 8 years.
All lipids (mmol/L) improved in the T-group at 12 years: total cholesterol from 6.7±1.3 to 5.1±0.7; HDL from 1.1±0.3 to 1.5±0.3; LDL from 4.0±0.8 to 2.8±0.7; triglycerides from 2.9±1.0 to 2.0±0.4; Non-HDL cholesterol from 5.6±1.4 to 3.6±0.9. In CTRL, lipids worsened at 8 years: total cholesterol from 6.4±1.5 to 6.8±1.4; HDL from 1.4±0.4 to 1.2±0.3; LDL from 3.3±0.9 to 4.0±0.6; triglycerides from 2.2±1.0 to 2.8±0.7; Non-HDL cholesterol from 5.0±1.6 to 5.6±1.4.
Blood pressure (BP; mmHg) improved in the T-group at 12 years: systolic BP from 136.4±13.2 to 118.3±4.1; diastolic BP from 81.4±8.8 to 72.9±3.9. In CTRL, BP increased at 8 years: systolic BP from 128.7±11.2 to 132.1±9.5; diastolic BP from 84.3±8.0 to 87.3±5.7.
Adherence to testosterone was 100% as all injections were administered in the office and documented. There were 24 deaths (7.5%) in the T-group and 27 deaths (14.7%) in CTRL.
Conclusions: Very long-term TTh with TU resulted in sustained improvements in anthropometric and metabolic parameters. All components of the metabolic syndrome improved, thus potentially reducing cardiometabolic risk. These effects may have contributed to the observed reduced incident all-cause mortality.
IMPLICATIONS OF ANDROGEN RECEPTOR ACTIVATION ON THYROID CANCER PHENOTYPE
Anvita Gupta BE¹, Melanie Jones PhD², Timmy O'Connell MS¹, Monica Schwarcz MD¹, Dorota Halicka MD, PhD¹, Jiangwei Li PhD¹, Zbigniew Darzynkiewicz MD, PhD¹, JK Rasamny MD¹, Codrin Iacob MD³, Nina Suslina MD³, Edward Shin MD³, Augustine Moscatello MD¹, Raj Tiwari PhD¹ and Jan Geliebter PhD¹
¹New York Medical College; ²United States Military Academy Preparatory School, West Point, NY; ³New York Eye and Ear Infirmary
Presented By: Anvita Gupta BE
Introduction: Papillary Thyroid Cancer (PTC) comprises more than 90% of neoplasms in the endocrine system, with a three-fold higher incidence in women than in men. With an overall five-year survival rate of 98.1%, early stage PTC has a favorable prognosis. However, PTC exhibits increased aggressiveness with poor prognosis in men diagnosed with the disease. These striking observations led us to explore the role of androgen and androgen receptor (AR) in this disease.
Methods: Development of our model system consisted of (1) Determining levels of AR expression in PTC patient tissue samples, (2) Determining AR expression in thyroid cancer cell lines, (3) Stable transfection of 8505c PTC/anaplastic thyroid cancer cell line with AR (clone 84e7), (4) Transcriptional profiling using RNAseq on 48 hour 5α-dihydrotestosterone (DHT) treated 84E7 cells, (5) Continually activating AR in 84e7 cells with DHT for 3-6 days, followed by beta-galactosidase (SA-βGal) assays, flow cytometry, western blots and immunocytochemistry to investigate development of senescence, (6) Cytokine profiling of the senescence-associated secretory phenotype (SASP) to define the pro-tumorigenic or tumor-suppressive potential of androgen-induced senescent thyrocytes.
Results: We found a 70% decrease in median AR expression (p<0.0001) in 24 PTC patient tissue samples, compared to matched, normal thyroid tissue. Preliminary data from our lab indicate that androgen receptor (AR) acts as a negative regulator of growth as evidenced by a statistically significant 48% decrease in proliferation over 72 hours upon DHT addition to 84E7 cells. RNAseq revealed significant changes in gene expression associated with proliferation (474 genes, p=2.4E-24) and cell cycle progression (129 genes, p= 6.54E-6). AR stimulation induced senescence, evidenced by a flattened, vacuolized and granular cell morphology, and expression of SA-βGal, leading to a permanent growth arrest, without apparent cell death. Senescence was accompanied by an increase in total RNA and protein content, Reactive Oxygen Species, and markers such as p21, p27, p16, γ-H2AX, HP1- γ, and H3K9 methylation. Profiling of SASP revealed a primarily anti-inflammatory microenvironment initiated and sustained by the senescent cells.
Conclusion: Our study elucidates the induction of senescence as a novel function of AR activation in thyrocytes and may indicate a protective role of AR activation in the decreased incidence of thyroid cancer in men.
TARGETED DEGRADATION OF ANDROGEN RECEPTOR (AR) AND ITS SPLICED VARIANT AR-V7 BY THE PHYTOCHEMICAL SULFORAPHANE: NEW THERAPEUTIC OPPORTUNITY FOR CASTRATION RESISTANT PROSTATE CANCER (CRPC)
Namrata Khurana M TECH¹, Hogyoung Kim PhD², Partha K. Chandra PhD³, Sudha Talwar PhD², Pankaj Sharma PhD4, Asim B. Abdel-Mageed PhD5, Debasis Mondal PhD6 and Suresh C. Sikka PhD5
¹PhD Student; ²Post Doc,Dept. of Urology,Tulane University School of Medicine; ³Post Doc,Dept. of Pharmacology,Tulane University School of Medicine; 4Professor,Amity Institute of Biotechnology, Amity University, Noida, U.P. , India.; 5Professor,Dept. of Urology,Tulane University School of Medicine; 6Research Associate Professor,Dept. of Pharmacology,Tulane University School of Medicine
Presented By: Namrata Khurana M TECH
Introduction: Androgen deprivation therapy (ADT) suppresses the growth of prostate cancer (PC) expressing full length androgen receptor (AR-fl). However, castration resistant prostate cancer (CRPC) recurs due to the induction of ligand-independent AR splice variants, particularly AR-V7. Strategies to suppress both AR-fl and AR-V7 levels are critically needed for treating CRPC. Our previous study demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, enhances the rate of degradation of AR-fl in several PC cell lines, e.g. LNCaP and C4-2B. SFN has been shown to inhibit the chaperone activity of heat-shock protein 90 (HSP90) and induce the potent antioxidant transcription factor, nuclear factor erythroid-2-like 2 (Nrf-2). We hypothesize that the combined exposure of SFN with clinically approved drugs that inhibit HSP90 (e.g. Ganetespib) (G) and activate Nrf-2 (e.g. Bardoxolone-methyl) (BM) would suppress AR-fl and AR-V7 for treating CRPC.
Methods: The current study was conducted in AR-V7- expressing CRPC cell line, CWR22RV1 (22RV1). Effect of drug(s) on cell viability and migration were monitored by MTT and migration assays, respectively. AR protein expression and its subcellular localization were measured by immunoblot analysis (IB) and immunofluorescence microscopy (IFM), respectively. Proteasomal activity was monitored by proteasomal assay.
Results: 22RV1 cells were significantly (p<0.05) more resistant to the potent anti-androgen, enzalutamide (ENZ) compared to LNCaP and C4-2B cells. Co-exposure to SFN (5-25 μM) significantly (p<0.01) enhanced the efficacy of ENZ. The immunoblot studies showed that SFN decreases the half-life of both AR-fl and AR-V7 proteins (p<0.01), possibly by increased protein ubiquitination and proteasomal activity. The IFM analysis showed that SFN treatment down-regulated both cytosolic and nuclear AR levels. Co-exposure of SFN with physiologic doses (<1 uM) of G and BM caused rapid degradation of both AR-fl and AR-V7, decreased cell viability and further augmented the efficacy of ENZ in 22RV1 cells.
Conclusion: Our findings suggest that the multimodal actions of SFN can cause rapid decrease in both AR-fl and AR-V7 protein levels and further implicate that its combination with G and BM is an effective adjunct to current ADT in CRPC patients, especially those expressing AR-V7.
EFFECTS OF TESTOSTERONE REPLACEMENT THERAPY ON METABOLIC DISORDERS IN PATIENT WITH TYPE 2 DIABETES MELLITUS AND ANDROGEN DEFICIENCY.
Shota Janjgava MD, PhD¹, Elene Giorgadze MD, PhD² and Lasha Uchava MD, PhD²
¹National Institute of Endocrinology; ²National Institue of Endocrinology
Presented By: Shota Janjgava MD, PhD
Over the past few decades, obesity and Diabetes mellitus has become a global health challenge. Multiple epidemiological studies have shown that low testosterone levels are associated with and predict the future development of T2D and the metabolic syndrome.
Aim of study: The aim of study was to show the influence of testosterone replacement therapy on obesity, HbA1c level, arterial hypertension and dyslipidemia with patient diabetes mellitus and Androgen deficiency.
Materials and Methods: 125 male patient with diabetes mellitus was screened, 85 subjects with 41-65 years and BMI 27,0 – 48,0 kg/m2 were randomized In placebo-controlled study, who underwent a routine physical examination and choose free testosterone examination. According to the laboratory and clinical condition we divided patients into two groups. 1) First group treatment group 2) Second group placebo group. In the first group we used diet, physical activity (Lifestyle intervention implies reduced calorie diet (The reduction of daily calorie intake in 800-1200 calorie, it was selected individually), patient’s antidiabetic therapy and testosterone replacement therapy (TRT), (testosterone undecanoate 250 mgr/ml intra- muscular 3 months 1 time). In second group we used diet, physical activity (Lifestyle intervention implies reduced calorie diet (The reduction of daily calorie intake in 800-1200 calorie, it was selected individually), patient’s antidiabetic therapy and placebo.
Results: After six months of treatment we repeated the diagnostic assessments: We had some positive results cholesterol, triglyceride and LDL levels decreased, and HDL increased both of group but better results was in first group which was clinically significant. Free testosterone level increased in all groups but the best results was in I group which was clinically significant where was used of testosterone undecanoate. HbA1c decreased in both group but in I group we had the best result. BMI decreased in both groups but more reduction was in I group. leptin level after treatment was approximately same in both groups, but compared best results was achieved in I group, also blood pressure were reduced in both group, where we found alike results .
Conclusion: Our study demonstrated that it is possible to break into this vicious circle by raising testosterone levels in diabetic men and low testosterone level. In addition to traditional CV risk factors, novel risk factors are also inversely related to testosterone levels.
GASTRIC DISORDERS, HIGHER STRESS AND IRRITABILITY ARE ASSOCIATED WITH MALE INFERTILITY
Dayane Reis BSc student¹,²,³,4, Juliana R Pariz MSc, PhD student¹,²,³,4,5, Victória Coutinho BSc student¹,²,³,4, Rosa Alice C Monteiro BSc¹,5 and Jorge Hallak MD; PhD¹,³,4,5
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Methodist University of Sao Paulo, Brazil; ³Dept. of Urology, FMUSP, Brazil; 4Reproductive Toxicology Unit, Dept. of Pathology, FMUSP, Brazil; 5Oswaldo Cruz German, Brazil
Presented By: Dayane Reis BSc student
Introduction: Several lifestyle habits are being related or associated with male fertility. The association of gastric disorders, stress and a higher irritability degree, very common nowadays with male fertility is seldom analyzed in the initial anamnesis.
Objective: To correlate gastritis, burning, reflux and higher irritability behavior with hormonal, seminal and sperm functional parameters.
Methods: Medical records of 432 patients (22 to 69 y.o.), between 2000 and 2016, in which anamnesis revealed clinical data and self-report of gastritis, gastric burning, reflux and irritability, were correlated with semen analyzes, sperm functional tests and sex hormones profile. The Pearson's correlation coefficient test (p <0.05) was used to evaluate the correlations between the study data.
Results: We observed negative correlation between gastritis and non-progressive spermatozoa (-0.134; p=0.006) and the presence of anti-spermatozoa antibodies (-0.189; p=0.008); and a positive correlation with spermatozoa with abnormal morphology (0.191, p<0.001), round cell concentration (0.312, p=0.001) and reactive oxygen species (ROS) in seminal plasma (0.644, p=0.032). When evaluating burning sensation in stomach, there was positive correlation with seminal ROS (0.853; p=0.002) and IGBP-3 (1,000; p=0.0003). In addition, irritability showed a positive correlation with seminal pH (0.148, p=0.006), spermatozoa with abnormal morphology (0.124, p=0.012), spermatozoa with normal morphology (Kruger) (0.126, p=0.012), and TSH; p=0.016). Of the patients evaluated with gastric burning, 31.12% use some type of medication.
Conclusion: Our results indicate that patients who cannot handle stress and present with a higher irritability degree and gastric disorders are related to changes in semen parameters that may impair male fertility. Self-medication, indiscriminate use of omeprazole, poor diet and stress, have negative effects on male fertility, contributing to the increasing incidence of male infertility. Therefore, we recommend that a complete anamnesis is performed when investigating the male in order to stablish the proper diagnosis of the cause of infertility to counsel for proper treatment and restoration of fertility potential without the indiscriminate use of assisted reproductive technologies.
Financial support: Androscience/FAPESP
Keywords: Male infertility, Irritability, gastric disorders, gastritis, semen, sperm.
Ethics Committee Approval: FMUSP Ethics Committee n°859215/2014
THE INFLUENCE OF WINE CONSUMPTION IN MALE FERTILITY POTENTIAL: INITIAL REPORT
Victória Coutinho BSc student¹,²,³,4, Juliana Pariz PhD student¹,²,³,5,6, Dayane Reis BSc student¹,²,³,4, Rosa Monteiro BSc¹,6 and Jorge Hallak MD;PhD¹,³,5,6
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Methodist University of Sao Paulo, Brazil; ³Department of Urology, FMUSP, Brazil; 4Reproductive Toxicology Unit, Department of Pathology, FMUSP, Brazil.; 5Reproductive Toxicology Unit, Department of Pathology, FMUSP, Brazil; 6Oswaldo Cruz German Hospital, Brazil.
Presented By: Victória Coutinho BSc student
Introduction: Male fertile potential is determined by the homeostasis of a complex system involving hormonal balance, sperm production, environmental factors and lifestyle habits. High alcohol consumption has been associated with a variety of diseases, including male infertility, which occurs due to adverse effects in hypothalamic-pituitary-gonadal axis, influencing the activity of Sertoli and Leydig cells and consequently on spermatogenesis.
Objectives: To correlate wine consumption with hormonal, semen and sperm functional parameters in an infertile population.
Methods: Medical records of 432 patients (22 to 69 y.o.), between 2000 and 2016, in which anamnesis contained clinical data on self-reported wine consumption (time in years and quantity in glasses mL/week; n=52), semen analysis, sperm function tests (ROS and DNA fragmentation by SCSA) and sex hormone profile. Azoospermic patients were excluded. The Pearson's correlation coefficient test (p <0.05) was used to evaluate the correlations between the study data.
Results: We observed a mean of 3.02 wine doses per week and mean of 10.35 years of consumption in the study group and a negative correlation with progressive spermatozoa (-2.34; p = 0.043). We found a positive correlation between time of consumption (years) with higher LH (0.962, p = 0.038) and androstenedione levels (0.845, p = 0.008).
Conclusion: Moderate wine consumption for a long period of time, was associated with changes in semen parameters that may influence male fertility potential and should be critically investigates in the anamnesis of the infertile male.
Financial support: Androscience/FAPESP
Ethics Committee Approval: FMUSP (n°859215/2014)
ARE RESPIRATORY ALLERGIES RELATED TO MALE FERTILITY?
Dayane Reis BSc student¹,²,³,4, Juliana Pariz PhD student¹,²,³,4,5, Victória Coutinho BSc student¹,²,³,4, Rosa Alice Monteiro BSc¹,5 and Jorge Hallak MD;PhD¹,³,4,5
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Methodist University of Sao Paulo, Brazil; ³Dept. of Urology, FMUSP, Brazil; 4Reproductive Toxicology Unit, Dept. of Pathology, FMUSP, Brazil; 5Oswaldo Cruz German, Brazil
Presented By: Dayane Reis BSc student
Introduction: Respiratory allergies are achieving epidemic proportions, affecting approximately 30% of the population in urban areas of industrialized countries. Respiratory allergies, rhinitis and sinusitis have been frequently reported by men in the fertility evaluation. Studies have already described ultra-structural changes in nasal tissue, but the association between these allergies and male fertility is still unknown.
Objective: To evaluate the effect of respiratory allergies, rhinitis and sinusitis on sperm quality, sperm functional parameters and hormonal profile.
Methods: 432 patients (22 to 69 y.o.), between 2000-2016, in which anamnesis revealed clinical data and self-report of respiratory allergies, sinusitis and rhinitis, were correlated with semen analyzes, sperm functional tests and sex hormones profile. The Pearson's correlation coefficient test (p <0.05) was used.
Results: We observed a negative correlation between respiratory allergies and progressive motility (-0.124, p=0.010), total motility (-0.147, p=0.002), grade A motility (-0,129, p=0.008), sperm vitality (-0.956; p<0.001) and LH (-0.475; p=0.046); and positive correlation with immotile spermatozoa (0.136, p=0.005) and T4 (-0.576, p=0.005). When evaluating sinusitis, there was a negative correlation with normal morphology sperm (-0.106, p=0.033), sperm vitality (-0.956, p<0.001), sperm creatine kinase activity (-1,000, p=0.019), LH (-0.455, p=0.058) and TSH (-0.733, p=0.025); and positive correlation with spermatozoa with abnormal sperm morphology (0.100; p=0.033). In addition, rhinitis presented a positive correlation with seminal ROS (0.937, p<0.001), estradiol (0.307, p=0.015), prolactin (0.325, p=0.011), and IGFBP-3 (1,000; p<0.001).
Conclusion: Our results indicate that respiratory allergies, rhinitis and sinusitis are related to changes in parameters that determine the male fertile pattern. This initial study allows us to suggest two mechanisms of influence of respiratory allergies in male fertility: (I) ultrastructural modifications in microtubules, dinein sheaths and ciliary membrane observed in patients with chronic respiratory disorders are similar to those observed in spermatozoa of asthenozoospermia and teratozoospermia of infertile patients; (II) hormonal and sperm function changes may be related to medications often used for patients with chronic respiratory allergies.
Financial support: Androscience/FAPESP
Ethics Committee Approval: FMUSP n°859215/2014
NEW INSIGHTS INTO THE PANDEMIC OF LOW VITAMIN “D” LEVELS AND ITS ASSOCIATION WITH SEMEN QUALITY AND HORMONAL LEVELS IN FERTILE AND INFERTILE MALE SUBJECTS
Inari Ciccone BSc; MSc student¹,²,³, Juliana R Pariz MSc; PHD Student¹,²,³,4, Elaine Costa MD; PHD¹,²,5,4 and Jorge Hallak MD, PHD¹,²,³,4
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Dept. of Urology, USP, Brazil; ³Reproductive Toxicology Unit, Dept. of Pathology, USP, Brazil; 4Oswaldo Cruz German Hospital, Brazil; 5Reproductive Toxicology Unit, Dept. of Endocrinology, USP, Brazil
Presented By: Inari Ciccone BSc; MSc student
Background: Vitamin D is a versatile signaling molecule, that targets also male reproductive organs, in addition to the classic effects on bone, calcium and phosphate homeostasis. Accumulating evidence from animal and human studies suggests that it is involved in many functions in both genders. Objective: To evaluate the influence of vitamin D status on semen quality and hormonal profile in male fertility and infertility.
Patients and Methods: We evaluated 100 men (aged 18 to 50 y.o.) from a private andrology reference medical clinic. Infertility (n=70) and fertility (n=30) groups. According to vitamin D level status all of them were classified in sufficient (group 1 - 25OHvitaminD ≥ 30ng/ml), insufficient (group 2 - 25OHvitaminD from 21 to 29ng/ml) and deficient (group 3 - 25OHvitaminD ≤ 20ng/ml). Blood samples were collected to analyze serum vitamin D, LH, FSH, estradiol, total and free testosterone, prolactin and SHBG levels. Semen was analyzed according to WHO guidelines, strict criteria and sperm functional tests were performed (ROS, CK, beads). In addition, karyotype, frequency of varicocele, smoking, alcohol ingestion, and body composition were considered. Statistical analysis was performed by SPSS program version 19.0 (SPSS Inc., Chicago, IL). T-test was used for unpaired samples and Spearman test for correlation analysis. Statistical significance was considered with P value < 0, 05. Results: According to vitamin D status, patient distribution was: Infertile: group 1 - 28.5% (20/70), group 2 - 43% (30/70) and group 3 - 28,5% (20/70). Fertile: group 1 - 34% (10/30), group 2 - 40% (12/30) and group 3 - 26% (8/30). Sperm motility was significantly lower in group 3 in comparison to group 1 in infertile patients. Regarding fertile men, we found higher sperm volume in group 3 than the group 1, a significant reduction of sperm concentration and worse morphology by strict criteria in group 3 in comparison to group 1. Hormonal levels were similar in all vitamin D groups. Conclusion: Our results demonstrated that sufficient vitamin D levels has a positive influence on spermatogenesis and semen quality, suggesting that vitamin D replacement should highly be concerned on male fertility treatment and that low vitamin D have reached epidemic proportions in industrialized cities, even in Brazil.
|Poster #18|HEAT SHOCK PROTEINS 70 AND 90 ALPHA ARE INCREASED IN SEMEN OF SMOKERS
Mariana Antoniassi BSc, MSc, Larissa Belardin BSc, MSc, Rhayza Andretta BSc, MSc, Jheysson Moura BSc and Ricardo Bertolla DVM, PhD
Department of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University
Presented By: Mariana Antoniassi BSc, MSc
INTRODUCTION: Heat shock proteins (HSPs) are a family of proteins that function as molecular chaperones by stabilizing new proteins to ensure correct folding or by helping to refold proteins that were damaged by cellular stress. Production of high levels of heat shock proteins can also be triggered by exposure to different kinds of environmental stress conditions, such as infection, inflammation, and exposure to toxins (smoking, metals). In this study we wished to verify if smoking alters seminal HSP expression levels, and if this is potentiated by the presence of varicocele.
METHODS: To verify the levels of HSP, we recruited 15 controls (healthy adult men without varicocele, non-smokers), 17 smokers without varicocele, and 15 smokers with varicocele. Semen was collected and an aliquot was submitted to standard semen analysis, as per the World Health Organization (2010). The remaining volume was centrifuged at 800 x g for 30 minutes to separate the supernatant seminal plasma, which was frozen without cryoprotectants and kept at -20 C. At the time of analysis, the seminal plasma was thawed and centrifuged at 16,100 x g for 1 hour at 4 C to remove cellular debris. The total amount of proteins in each sample was quantified using a BCA protein assay. Quantification was performed by measuring absorbance at 540 nm wavelength, using a microplate reader. An aliquot of each sample was diluted in Milli Q water (1:250) and a volume corresponding to 4 ug/mL of protein was used for the analysis. The HSP Magnetic Bead Kit was used to detect changes in HSP27 (Total), HSP27 (pS78/pS82), HSP60, HSP70 (HSP72), and HSP90 alpha in human seminal plasma using the Luminex® system. Univariate statistics were performed using a Kruskal-Wallis test followed by Games-Howell post-hoc test (p<0.05).
RESULTS: Seminal plasma results are presented in table 1. The levels of HSP 70 and HSP 90 alpha were increased in smokers and decreased in control and smokers with varicocele.
CONCLUSION: The semen of smokers seems to have an inflammatory state characterized by increased levels of HSP 70 and HSP 90 alpha. These proteins works as molecular chaperones, providing sperm protection against stress, such as cytokines and other inflammation molecules.
SPERM DNA INTEGRITY IN ADULT SURVIVORS OF PAEDIATRIC LEUKEMIA AND LYMPHOMA
Hermance Beaud MSc¹, Oceane Albert PhD², Bernard Robaire PhD³, Marie Claude Rousseau PhD¹, Peter Chan MD, CM, MSc4 and Geraldine Delbes PhD¹
¹INRS-Institut Armand-Frappier, Laval, Canada; ²Department of Pharmacology and Therapeutics, McGill University, Montreal, (Quebec) Canada; ³Department of Pharmacology and Therapeutics, McGill University, Montreal, (Quebec) Canada. Department of Obstetrics & Gynecology, McGill University, Montreal, QC, H4A 3J1, Canada.; 4Division of Urology, McGill University Health Center, Montreal, (Quebec) Canada.
(Presented By: Hermance Beaud MSc)
Quality of life of young cancer survivors is an important healthcare issue. Chemotherapeutic drugs are well-known toxicants to the male gonad. Prospective studies on adult cancer survivors show that although sperm production may recover, DNA damage and chromatin abnormalities can persist years post-chemotherapy. Such biomarkers of sperm damage have been correlated to male infertility and poor embryo development. In the case of childhood cancer, it has been shown that survivors are more likely than siblings to report low sperm count and to use assisted reproductive technologies. However, it is still unclear if the sperm produced many years after remission of cancer are damaged. To address this issue, we investigated the long-term impact of the chemotherapeutic treatment of childhood acute lymphoblastic leukemia and lymphoma, two malignancies with highest incidence in children, on male reproductive health and sperm quality. Men 19-40 years were recruited into 3 groups: diagnosed before (ages 4-13, n=7) or after puberty (ages 14-18, n=6), or without history of cancer (control, n=12). All cancer subjects had been in remission for at least 3 years and had no co-morbidity related to fertility. Interestingly, we observed that survivors were significantly overweight compared to controls and that this difference was particularly significant in the group of men diagnosed after puberty. Average levels of circulating hormones (LH, FSH, testosterone, estradiol), sperm concentrations, and progressive motility showed no difference among groups. However, 5 survivors of lymphoma (38.5%) were azoo/oligospermic, including 3 diagnosed pre-puberty and 2 after puberty. For non-azoospermic participants, the DNA fragmentation index (DFI) and the high DNA stainability were evaluated using the SCSA and the percentage of tail DNA was obtained using the HT-COMET assay. Interestingly, the %DFI correlated significantly with the % tail DNA (r=0.73; p=0.0002). Nevertheless, none of these parameters were different among groups, suggesting that sperm quality is not impaired in cancer survivors after protracted time periods. Although limited by the small number of subjects, these data suggest that survivors of childhood cancer, independently of the age of diagnosis, have a higher risk of infertility due to no or low sperm count and that when sperm are present, chances of DNA and chromatin abnormalities appear similar to those seen in the general population. Funded by The Cole Foundation.
ASSOCIATIONS OF PHENOLS AND PARABENS WITH SPERM GENOME-WIDE DNA METHYLATION
Haotian Wu¹, Molly Estill², Stephen A. Krawetz², Cynthia Sites³, Tayyab Rahil³ and J. Richard Pilsner¹
¹UMass Amherst; ²Wayne State University School of Medicine; ³Baystate Medical Center
Presented By: Haotian Wu
Background - Phenols and parabens are diverse and ubiquitous families of endocrine disruptors with widespread human exposure around the world. Previous epidemiologic studies report that select compounds within these chemical families may be associated with adverse male reproductive endpoints. Given the emerging evidence that the sperm epigenome during spermatogenesis is responsive to environmental conditions and can affect offspring health and development, we examined the associations between eleven urinary phenols and parabens and sperm DNA methylation.
Methods - A total of 48 male participants were recruited from the Baystate Medical Center’s IVF clinic from 2014 to 2015 as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Seven phenols and four parabens were measured from spot urine samples by the CDC. Sperm DNA methylation was assessed with the HumanMethylation 450K array. We used the minfi package to correct for technical variation in background signals, remove probes below the background fluorescence level, and adjust for differences in Type I and Type II probes. Batch effects were corrected via ComBat and sex chromosomes were removed by DMRcate. The resulting beta-values were analyzed using Aclust and CpGassoc, corrected for false-discovery rate.
Results - After pre-processing, 6479 clusters were constructed with a mean of 3.5 CpG sites per cluster. Adjusting for male age, BMI, infertility diagnosis, and current smoking, urinary concentrations of bisphenol-A, propyl paraben and, 5-dichlorophenol were associated with one differentially methylated region(s) (DMR) each, butyl paraben and triclocarban were associated with 2 DMRs and methyl paraben were associated three DMRs. Of the nine unique clusters (one cluster was identified by both Methyl- and Propyl- Paraben), six are entirely within genes or span the transcription start site. These affected genes are all protein-coding and have known functions related to DNA double strand repair, internal cell motility, brain development, and vasoconstriction in vas deferens.
Discussion - To our knowledge, this is the first study to associate paternal phenol and paraben exposures with sperm DNA methylation in humans. Future analyses will further investigate biological pathways of the identified regions using ontological analyses and whether these sperm DNA methylation patterns are linked to early life development.
EFFECTS OF PRENATAL EXPOSURE TO DI-N-BUTYL PHTHALATE ON THE DEVELOPMENT OF ADULT LEYDIG CELLS IN RAT DURING PUBERTY
Linxi Li PhD¹, Guoxin Hu PhD², Xiaomin Chen PhD¹, Huitao Li Master¹ and Ren-Shan Ge MD¹
¹The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University; ²School of Pharmaceutical Sciences of Wenzhou Medical University
Presented By: Linxi Li PhD
Introduction: Fetal exposure to di-n-butyl phthalate (DBP) causes the adult disease such as lower testosterone production and infertility. However, the mechanism is still unknown. The objective of the present study is to determine how DBP affects the involution of fetal Leydig cells during neonatal period and how this event causes the delayed development of the adult Leydig cells during puberty.
Methods: The pregnant Sprague-Dawley dams were randomly divided into 3 groups and were gavaged with 0 (corn oil, the vehicle control), 100 or 500 mg/kg DBP from gestational day 12 to 21. The blood and testes were collected from male pups at postnatal day 4, 7, 14, 21, 28, and 56. Serum testosterone concentrations were assessed and the mRNA levels of Leydig cell- or gonadotroph cell-specific genes were measured.
Results: Prenatal exposure to DBP caused the aggregation of fetal Leydig cells, which slowly disappeared when compared to the control. This effect was associated with the reduction of testicular testosterone secretion and down-regulation of the mRNA levels of Leydig cell biomarkers including Scarb1, Star, Cyp11a1, Hsd3b1, Hsd11b, and Hsd17b3.
Conclusion: We demonstrated that the increasing aggregation of fetal Leydig cells with the increasing doses of DBP delayed their involution, thus leading to the delayed development of the adult Leydig cells.
|Poster #22|AMELIORATIVE EFFECTS OF VITAMIN C ON THE HEMATOLOGY, SEMEN QUALITY AND HORMONAL PROFILE OF TEDDY GOAT BUCKS IN ARSENIC TOXICITY
muhammad zubair PhD
University of Poonch rwawalakot Azad Kashmir
Presented By: muhammad zubair PhD
Ameliorative effects of vitamin C on the hematology, semen quality and hormonal profile of Teddy goat bucks in arsenic toxicity
1Muhammad Zubair, 2Maqbool Ahmad, 3Muhammad Kashif Saleemi 3Shafia Tahseen Gull and 2Huma Jamil
1Faculty of veterinary Sciences, University of Poonch Rawalakot Azad Kashmir, Pakistan
2Department of Theriogenology, University of Agriculture Faisalabad Pakistan
3Department of Pathology, University of Agriculture Faisalabad Pakistan
Corresponding Author; firstname.lastname@example.org
The present environmental study was conducted to investigate the toxic effects of arsenic on male reproductive functions of Teddy bucks, and to examine whether these toxic effects are ameliorated by vitamin C. For this purpose, 12 adult Teddy bucks were randomly divided into three treatment groups viz; A (control), B (sodium arsenite), C (sodium arsenite + vitamin C), with four animals in each group. Sodium arsenite and vitamin C was fed with 5 and 200 mg/kg BW/day respectively. These treatments continued for 12 weeks. Semen quality parameters (volume, sperm motility, sperm count acrosomal integrity, membrane functional integrity and DNA integrity) was evaluated on weekly. Hematological variables (red blood cells count, hemoglobin, packed cell volume and white blood cells), serum concentrations of male sex hormones (testosterone, LH, FSH) and cortisol were recorded fortnightly. The data were subjected to two-way analysis of variance, followed by Duncan test for multiple mean comparisons. There was significant reduction (P<0.05) in semen quality parameters, male sex hormones and hematological variables in arsenic treated animals as compared to control. Co-administration of vitamin C with sodium arsenite ameliorated these toxic effects of arsenic on these parameters. It was concluded that the use of the vitamin C has the protective effects against the arsenic toxicity.
Key Words; Semen, hormones, blood, teddy bucks
IS IT POSSIBLE FOR SMOKING-ABSTINENCE TO REVERSE THE NICOTINE-INDUCED ALTERATIONS IN THE EPIDIDYMIS?
Panagiota Tsounapi PhD¹, Masashi Honda PhD², Fotios Dimitriadis PhD³, Katsuya Hikita PhD², Nikolaos Sofikitis PhD, DMSci4 and Atsushi Takenaka PhD²
¹Division of Urology Tottori University Faculty of Medicine; ²Division ofUrology Tottori University Faculty of Medicine; ³Department of Urology Aristotle University; 4Department of Urology University of Ioannina School of Medicine
Presented By: Panagiota Tsounapi PhD
Introduction and objectives
Available data indicate that up to 13% of infertility is attributed to cigarette smoking. In the present study, nicotine was selected as a major addictive substance of tobacco smoke and the effects of nicotine-induced alterations in oxidative stress (OS) in the epididymis and the testis were examined. Furthermore, the impact of nicotine-abstinence was investigatd alleviate these changes.
Adult Wistar rats were treated orally with nicotine (15 mg/kg). One group was administered nicotine for 10 weeks (Nico-group) and another group was administered nicotine for 7 weeks followed by 3 weeks of abstinence (Abst-group). The Control group had free access to fresh water. Tissue levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were evaluated both in the testis and the epididymis. Additionally, cotinine levels in the urine, serum and seminal vesicular fluid (SVF) were evaluated. Furthermore, testosterone was measured in the urine samples. Finally, immunohistochemistry was performed for OS-markers and Cytochrome P450 2A6 (CYP2A6) in epididymal tail samples.
Nicotine treatment significantly increased MDA levels both in the testis and epididymis in Nico-group compared to Abst and Control groups. TAC was significantly decreased in both epididymis and testis in Nico group compared to Abst and Control groups. Cotinine concentrations in urine, serum and SVF were significantly increased in Nico-group compared to Abst group. Testosterone levels in the urine in Nico group were significantly lower compared to Control-group, while there was no significant difference between Control and Abst-group, neither between Nico and Abst groups. OS-markers had stronger expression in epididymal samples from Nico-group compared with Abst and Control groups. CYP2A6 which is the primary enzyme responsible for the oxidation of nicotine and cotinine was expressed and localized in the epithelial cells of the epididymis in Nico-group.
The present data show that the harmful effects of nicotine in the testis and epididymis can be reversed by abstinence. It is possible that additional treatment with an antioxidant reagent may enhance the antioxidant defenses of testis and epididymis, and further ameliorate the cigarette smoke-induced oxidative stress in both testicular and epididymal tissue. The present data may help clinicians to advice patients, especially those who attend assisted-reproductive programs, to quit smoking.
PURINERGIC MODULATION OF V-ATPASE-DEPENDENT PROTON SECRETION IN EPIDIDYMAL CLEAR CELLS
Maria Agustina Battistone PhD¹, Flavia Gombar Master², Maria Alejandra Peralta Bachelor¹, Nicolas DaSilva PhD¹, Dennis Brown PhD¹ and Sylvie Breton PhD¹
¹Program in Membrane Biology/Division of Nephrology. Massachusetts General Hospital. Harvard Medical School; ²Laboratory of Morphometry, Metabolism & Cardiovascular disease Department of Anatomy - IBRAG - UERJ
Presented By: Maria Agustina Battistone PhD
The epididymis maintains an optimum environment for sperm maturation. Elaborate communication networks between the different epithelial cell types (clear cells (CCs), principal cells (PCs) and basal cells) are important to establish an acidic luminal environment, which is required to maintain sperm dormant before ejaculation. CCs secrete H+ via the vacuolar H+-ATPase (V-ATPase) located in their apical membrane. This process is regulated by the cAMP/PKA and calcium/PKC pathways, which induce apical V-ATPase accumulation following redistribution of recycling sub-apical vesicles. We showed that PCs secrete ATP, which is hydrolyzed into adenosine by ectonucleotidases (EctoNs).
Immunofluorescence (IF) showed expression of the adenosine receptors, A1, A2A and A2B, and the ATP receptor P2X4 in the apical membrane of CCs. A2A and A2B increase cAMP, and P2X4 increases calcium, and we, therefore, studied their role in the regulation of V-ATPase in CCs. IF and Western blot following cell fractionation showed V-ATPase membrane accumulation when mouse epididymides were perfused in vivo with adenosine or an A2B agonist (BAY-60-6583; 100 µM), and with the non-hydrolysable ATP analog ATPγS or a P2X4 agonist (ivermectin; 100 µM). No effect was detected using an A2A activator, compared to control. These results indicate that V-ATPase-dependent H+ secretion in CCs is activated by adenosine via A2B and by ATP through P2X4.
Moreover, we detected a 3-fold increase in luminal adenosine concentration (p<0.03) and a 50% reduction in ATP concentration (p<0.05) after perfusion at 7.8 versus the control pH of 6.6, suggesting an increase in ATP hydrolysis by EctoNs at alkaline pH. Indeed, specific EctoN inhibitors (AMPCP and POM-1, 10 µM) decreased luminal adenosine levels measured at pH 7.8 (33 and 47% vs control, p<0.03). Perfusion of the cauda at alkaline pH 7.8 induces V-ATPase-dependent H+ secretion by CCs, which restores the pH towards 6.6. EctoN inhibitors partially prevented this recovery (control: 7.2±0.05, AMPCP: 7.5±0.03, POM-1: 7.6±0.04, p<0.01). These data suggest that ATP and adenosine are luminal modulators of H+ secretion by CCs and that the production of adenosine is itself modulated by luminal pH.
Altogether, our study reveals novel mechanisms by which CCs respond to luminal agonists via purinergic regulation, and provides new frameworks for the discovery of potential diagnostic targets of male infertility, and the development of novel methods for male contraception.
MAZ HAPLOINSUFFICIENCY ALTERS BACULUM MORPHOGENESIS IN MICE
Marisol O'Neill MS, Gene Huang MD, Meade Haller PhD and Dolores J. Lamb PhD
Baylor College of Medicine
Presented By: Marisol O'Neill MS
Introduction and objectives: Hypospadias is a common congenital genitourinary (GU) anomaly affecting 1 in 125 males. Using array comparative genomic hybridization and the DECIPHER database, we identified myc-associated zinc finger (MAZ), which is highly expressed in the genital tubercle, as a candidate gene for GU development. MAZ copy number variants (CNVs) were identified in 6.5% of patients GU anomalies. Of the patients with Lower GU defects, micropenis and hypospadias were common in patients with duplications while cryptorchidism and hypospadias were common in patients with deletions suggesting MAZ may be a dosage sensitive gene. We hypothesized that MAZ haploinsufficiency would affect penile morphology in mice. Due to differences in anatomy of the murine and human penis, hypospadias in mice has been reported as a spectrum of morphometric changes not limited to defects of the urethral meatus. Using micro computed tomography (micr-CT), we phenotyped penises from Maz+/Δ and wild type mice.
Methods: Murine Penises were fixed overnight in formalin followed by dehydration in 70% ethanol. To allow visualization of penises by micro-ct, fixed penises were submerged 0.1N iodine overnight. Stained penises were briefly rinsed with PBS to remove any loose iodine then embedded in 1% agarose. Imaging was performed using a SkyScan 1272 X-Ray Microtomograph using a 0.5 μm aluminum filter and 5 μm pixel size. Images were reconstructed using NRecon software; visualization and measurements of specimen were performed in 3D Slicer v4.
Results obtained: Maz Δ/Δ mice are perinatal lethal therefore only Maz+/Δ mice were examined. Five wild type and seven Maz +/Δ adult penises were measured for changes in total penis length, MUMP length, and baculum length and width. While there was no difference in the total penis (5.65 ± 0.11 mm vs 5.74 ± 0.19 mm; p = 0.48) or MUMP (1.71 ± 0.06 mm vs 1.72 ± 0.05 mm; p=0.62) lengths of Maz+/Δ mice, the baculum had a 3% shorter length (4.12 ± 0.09 mm vs 3.99 ± 0.10 mm; p = 0.04) and 31% smaller baculum shaft width (0.35 ± 0.03 mm vs 0.24 ± 0.06 mm; p = 0.004) compared to wild type mice.
Conclusions: Baculum shaft width and length were significantly smaller in Maz+/Δ mice when compared to wild types. Baculum shaft width has been reported to be associated with reproductive success in male mice; further studies are required to confirm this association.
Funding: NIH grants T32DK007763 and R01DK078121 to DJL
CRITICAL ROLE OF REGULATORY SPERMATOZOAL TRANSCRIPTS IN EARLY EMBRYONIC DEVELOPMENT
VIDHU DHAWAN MD, MANOJ KUMAR MSc PhD, DIPIKA DEKA MD, NEENA MALHOTRA MD, NEETA SINGH MD, VATSLA DADHWAL MD and RIMA DADA MD
AIIMS, NEW DELHI, INDIA
Presented By: VIDHU DHAWAN MD
Introduction: The knowledge of molecular and cellular events underlying fertilization and early embryonic development has increased substantially over years. A plethora of studies have been conducted in recent years to assess the role of paternal factors for successful fertilization and embryonic development. The presence of RNA in sperm has contraindicated the previously held belief that paternal contribution is limited to one copy of the genome. Also the defects in sperm chromatin integrity are a potential cause of pregnancy losses.
Objectives: The present study was designed with an aim to evaluate the expression pattern of spermatozoal FOXG1B, RPS6, RBM9 and RPL10A in male partners of couples experiencing early pregnancy loss in both spontaneous as well as assisted conceptions. Seminal oxidative stress and DNA Fragmentation Index (DFI) was also assessed.
Methods: Semen sample was from male partners of couples experiencing recurrent pregnancy loss (RPL) (n=75), recurrent implantation failures (RIF) (n=30) and 30 fertile controls. Semen analysis was assessed by WHO (2010) criteria. Gene expression analysis was done by q-PCR analysis and the relative quantification of target genes was calculated after normalization to β-actin with 2Ct method. Reactive oxygen species (ROS) levels (RLU/sec/million sperm) were assessed by luminol-dependant chemiluminescence. The Sperm chromatin structure assay (SCSA) was performed by flow cytometry to determine DFI.
Results: The average DCt of FOXG1, RPS6, RBM9 and RPL10A genes was found to be 3.62, -0.58, 1.5, 3.25 in RPL patients and 5.41, 4.6, 4.3, 6.7 in RIF patients. However, the average DCt of healthy fertile controls was found to be 3.94, 2.2, -0.06 and 2.5. The mean ROS level in the cases was seen to be higher (>25) in 32% of RPL patients (356.9 ± 137.8) and 75% RIF patients (142.78 ± 75.65) with respect to controls (26.7 ± 9.8). The While the odds of occurrence of RPL was 0.77 times higher (p=0.587). The mean DFI of male partners of couples with RPL (38.29 ± 9.0) and RIF (41.3 ± 5.1) was significantly (P < 0.0001) higher as compared to controls (27.4 ± 6.4).
Conclusion: Sperm transcript dysregulation along with oxidative DNA damage are one of the main causes of early pregnancy loss. Regulation of oxidative stress and DNA damage following adoption of various complementary and alternative medicine therapies may help in normalization of transcript dysregulation.
RETROSPECTIVE STUDY ON 1578 CASES OF PATIENTS WITH AZOOSPERMIA WHO HAVE TAKEN PRIOR DIAGNOSTIC TESTICULAR BIOPSY.
xunbin Huang, wenjing Li BS, na Fang Bs and congcong Cao Bs
Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology
Presented By: xunbin Huang
Prior diagnostic testicular biopsy (PDTB) is usually considered the golden standard for the diagnosis of azoospermia and which has been widely performed in clinical work for decades. With the development of non-invasive detections and the Microdissection Testicular Sperm Extraction (micro-TESE), preoperative application of testicular biopsy should be significantly limited. This study attempts to take a retrospective analysis on the patients with azoospermia who have had PDTB and to reevaluate its clinical value. A retrospective analysis of 1578 patients with azoospermia who have taken PDTB was made on their clinical data (including ages, illness years, BMI, penis length, testicular volume, serum FSH, LH, PRL, T, E2, seminal pH and semen volume, chromosomal karyotypes, Y chromosome microdeletions and cell-free seminal mRNA). The results shown that FSH, LH, testicular volume, seminal pH, semen volume and cell free seminal mRNA (cfs RNA) were useful factors that distinguish the nonobstructiveazoospermia (NOA) and the obstructive azoospermia (OA); and the non-invasive detections (chromosomal karyotype, Y chromosome microdeletion, cfs-mRNA ) could partially substitute PDTB; Any histopathologic types of testis have certain levels of sperm retrieval rates (SRR), PDTB may mislead the results of sperm retrieval; the limitation and non-necessity of the PDTB were elaborated and a new therapeutic strategy of step-by-step micro-TESE were introduced. In conclusion, preoperative use of non-invasive detections combined with step-by-step micro-TESE can avoid unnecessary damage induced by PDTB to the patients with azoospermia.
ANALYSIS OF THE CELLULAR AND NUCLEAR INTEGRITY OF SPERM FROM PATIENTS WITH VARICOCELE
Viviane Paiva Santana MSc, Cristiana Libardi Miranda-Furtado PhD, Daiana Cristina Chieli Pedroso MSc, Matheus Credendio Eiras, Marilda Hatsumi Yamada Dantas and Rosana Maria Reis MD, PhD
Department of Gynecology and Obstetrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil
Presented By: Viviane Paiva Santana MSc
Varicocele has a negative impact on seminal parameters and deleterious effects on sperm DNA fragmentation (SDF) and epigenetic changes. SDF and oxidative stress appear to be related to reduced sperm DNA methylation and decreased telomere content, which may lead to genomic instability. The aim of the present study was to verify the effect of varicocele on seminal quality, SDF, global DNA methylation and telomere length in varicocele. This is a case-control study with 40 volunteers, 20 men with varicocele (8 grade II and 12 grade III) and 20 men without the disease. The seminal quality was evaluated by spermogram and the SDF was analyzed by Halosperm G2® kit. Some samples were selected by the DNA quality, the sperm DNA methylation was evaluated by ELISA (Enzyme-Linked Immunosorbent Assay) and telomere content by real-time polymerase chain reaction (qPCR). The comparisons were performed using the non-parametric Mann-Whitney test (p <0.05). There were no differences in mean age between the groups (p> 0.05). There was a decrease in sperm concentration (p = 0.0033), progressive motility (p = 0.0041) and morphology (p = 0.0015) in varicocele group compared to the controls. Even without significant differences, a higher percentage of DNA fragmentation was observed in the varicocele group (Varicocele: 37 ± 2; Control: 26 ± 1) (p> 0.05). In the patients with the disease, despite to have lower percentage of sperm DNA methylation (49.74 ± 20.75) in relation to the control group (64.66 ± 17.08), we did not observe statistical difference (p> 0.05). No difference was observed in telomere content between the groups (p> 0.05). When varicocele group was separated according to degree (II and III), no differences were observed in any of the variables (p> 0.05). We showed that varicocele is associated with decreased in seminal quality and may be related to a higher percentage of DNA damage, regardless of the degree of disease. However, an increasing of sample size is necessary to confirm the results. Regarding the telomere length, a larger sample may answer the actual impact of the disease, and its relation to the sperm DNA fragmentation. However, this is the first study that sought to evaluate the effect of the disease on this parameter.
IMPACT OF INCREASED SEMINAL 8-HYDROXY-2’-DEOXYGUANOSINE LEVELS ON INCREASED RISK OF CHILDHOOD CANCER RETINOBLASTOMA
Shilpa Bisht MSc, Bhavna Chawla MS, Manoj Kumar MSc, PhD and Rima Dada MD, PhD
All India Institute of Medical Sciences, New Delhi, India
Presented By: Shilpa Bisht MSc
Introduction: Oxidative stress (OS) has been implicated in a wide array of diseases and pathophysiological conditions such as neurodegenerative disorders, inflammatory diseases and cancer. OS is the major cause of sperm DNA damage as it induces peroxidative damage to sperm plasma membrane and DNA fragmentation in sperm nuclear and mitochondrial genome. OS induced sperm DNA damage is associated with pathologies such as male factor infertility, recurrent pregnancy loss (RPL), and high frequency of childhood morbidities and childhood diseases such as complex polygenic diseases, dominant genetic disorders, neuropsychiatric debility and childhood cancers. OS induces production of mutagenic oxidative base adduct 8-OHdG (8-hydroxy-2’-deoxyguanosine) in sperm DNA. 8-OHdG has the ability to pair with adenine and results in G:C to T:A transversions which results in single and double strand DNA breaks in the sperm DNA and therefore, increased mutation rate. Retinoblastoma (RB) is the most common childhood intraocular cancer and the exact etiology for RB causation is not known in unilateral sporadic RB cases.
Objectives: The present study was planned with an aim to determine 8-OHdG levels in seminal plasma of fathers of children affected with unilateral sporadic RB which may predispose the sperm to develop de novo germline mutations and may be the underlying cause for the development of unilateral sporadic RB.
Methods: The present study is a case-control study. Semen sample were obtained from 60 fathers of children with RB and 60 fathers of healthy children followed by separation of seminal plasma. 8-OHdG levels were determined in the seminal plasma of cases and controls by ELISA (DNA/RNA Oxidative Damage EIA kit from Cayman Chemical, Ann Arbor, MI).
Results: The mean ages of cases and controls were 32.17±11.2 years and 29.5±4.54 years respectively. 8-OHdG plasma levels were found to be significantly higher (p value= 0.019) in fathers of RB patients (7281±28 pg/ml) as compared to the fathers of healthy children (5871±39 pg/ml).
Conclusion: Increased levels of 8-OHdG (a promutagen) were detected in seminal plasma of fathers of children with RB which may predispose to mutation in germline. Persistence of these mutagenic bases post fertilization increases incidences of mutation in embryo and thus, may be underlying cause for the development of unilateral sporadic RB in children.
Financial Funding: Indian Council of Medical Research, New Delhi, India.
SIMPLE AND HIGHLY EFFICIENT POLYETHYLENIMINE TRANSFECTION PROTOCOL FOR TRANSIENT TRANSFECTION IN MOUSE SPERMATOGONIAL STEM CELLS
Chatchanan Doungkamchan MD¹, Yi Sheng MD², Meena Sukhwani PhD²,³ and Kyle E. Orwig PhD¹,²,³
¹Molecular Genetics and Developmental Biology Graduate Program, Magee-Womens Research Institute, University of Pittsburgh School of Medicine; ²Magee-Womens Research Institute, Pittsburgh; ³Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
Presented By: Chatchanan Doungkamchan MD
In this study, we aimed to develop a simple transient transfection protocol for mouse spermatogonial stem cells (mSSCs) to facilitate downstream gene editing studies. Polyethylenimine (PEI) is a cationic transfection reagent that has been widely used to transiently transfect mammalian cells, but has not been tested in mSSCs. In this study, we developed a modified PEI protocol that allows simple, efficient, low toxicity transient transfection in mSSCs.
To assess transfection efficiency using PEI compared to Lipid-based reagent, established mSSC cultures from EF1a-EGFP mice were passaged; replated into 24-well plates; expanded until 80% confluent; and transfected with a chicken β-actin (CAG)-mCherry reporter plasmid. The transfection efficiency and cell viability were evaluated 48 hours after transfection by flow cytometry. Lipid-based reagent transfection was done using Superfect (Qiagen) according to manufacturer’s protocol. PEI transfection protocol was done by separately mixing 1 μg plasmid DNA with 10 μL of 50 mM sodium chloride (NaCl); and 10 μL of PEI with 5 μL NaCl. The mixtures were allowed to equilibrate for three minutes before the PEI/NaCl mixture was added into DNA/NaCl mixture and incubated for 30 minutes. The mixture was then mixed with 350 μL Iscove's Modified Dulbecco's Medium (IMDM) media and added to the mSSCs culture for six hours before replacing transfection media with 1 mL of supplemented IMDM media. To improve transfection efficiency, we modified PEI protocol (mPEI) by replacing NaCl with plain IMDM media.
Transfection efficiency with PEI (46.90%±2.54) was significantly higher than Superfect (1.92%±0.15, p<0.0001). The viability after PEI transfection (55.50%±5.97) was significantly higher than Superfect (37.86%±1.72, p=0.0116). The transfection efficiency was improved further using the modified PEI protocol (65.40%±0.90, p=0.0023) without decreasing viability (58.23%±3.06, p=0.7048). To test the long-term survival and proliferation in vitro, the mCherry-positive cells from modified PEI protocol were sorted and cultured for at least 3 passages. Transplant experiments are underway to test the stem cell function of PEI transfected, FACS-sorted mSSCs.
We developed a transient transfection protocol for mSSCs using PEI (mPEI) that is simple, cost-effective, highly efficient and feasible in most labs. This work was supported by discretionary funds to KEO.
SPERM DNA METHYLATION ALTERATIONS AND EPIGENETIC VARIABILITY IN TOBACCO SMOKERS
Emma James BS¹, Timothy Jenkins PhD², David Alonso BS³, James Hotaling MD, MS4, Douglas Carrell PhD5 and Kenneth Aston PhD²
¹Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, Utah, USA; ²Andrology and IVF Laboratories, University of Utah Department of Surgery, Salt Lake City, Utah, USA; ³Department of Psychology, University of Utah, Salt Lake City, Utah, USA; 4Urology, University of Utah Department of Surgery, Salt Lake City, Utah, USA; 5Andrology and IVF Laboratories Department of Surgery, Department of Obstetrics and Gynecology, Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, USA
Presented By: Emma James BS
Introduction and objectives
The numerous health consequences of tobacco smoke exposure have been thoroughly characterized, and effects on male gametes in the context of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking confers increased risk for a number of conditions in offspring. The mechanism for this increased risk has not been elucidated; therefore this study seeks to determine whether mediation occurs, at least in part, through epigenetic modifications transmitted through sperm.
In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium HumanMethylation450 beadchip. We investigated two models of DNA methylation alterations: (1) consistently altered methylation at specific CpGs or within specific genomic regions using tools for differential methylation analysis available in R and (2) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke using mean centralization as a method to determine variability.
We identified 141 significantly differentially methylated CpGs associated with smoking, 74% of which displayed hypomethylation, with the remaining 26% being hypermethylated. The differentially methylated CpGs were not associated with a specific biological pathway or GO term, however there was a significant bias in the genomic context of altered CpGs. Significant enrichment of differentially methylated CpGs occurred at shores and significant depletion was found at CpG islands. In addition, we identified a pattern of increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers.
Altered sperm DNA methylation resulting from paternal lifestyle factors is a plausible mechanism for phenotype modification in offspring. Our finding is consistent with the broad range of offspring health disparities associated with pre-conception paternal smoke exposure and warrants further investigation to identify specific mechanisms by which sperm DNA methylation perturbation confers risk to offspring health. Controlled, multigenerational animal studies are required to assess the transmission of altered sperm DNA methylation patterns across generations.
IN VITRO CULTURE OF KLINEFELTER MOUSE SPERMATOGONIAL STEM CELLS
Guillermo Galdon MD¹, Nima Pourhabibi Zarandi MD², YanHe Lue MD, PhD³, Ronald Swerdloff MD³, Stanley Kogan MD, FACS¹,4,5, Hooman Sadri-Ardekani MD, PhD¹,4,5 and Anthony Atala MD¹,4,5
¹Wake Forest Institute for Regenerative Medicine ; ²wake Forest Institute for Regenerative Medicine ; ³Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute; 4Department of Urology; 5Wake Forest School of Medicine
(Presented By: Guillermo Galdon MD)
Introduction: Klinefelter Syndrome (KS) is characterized by masculine phenotype, supernumerary X chromosomes and a dramatic loss of spermatogonial stem cells (SSC) starting at the onset of puberty. In order to study this process and explore possible therapies, our current method of SSC isolation and propagation have been adapted to KS (41,XXY) mouse model aiming to expand these cells in vitro and overcome the in vivo loss of SSC.
Material and Methods: Putative SSCs were isolated and cultured from testes of normal (40, XY) mice aged 1-day old and 3-day old. The propagation of the cells was optimized comparing different culture medias, culture surfaces and seeding concentrations. Propagated cells were characterized using SSC specific markers assessed by Q-PCR, Digital-PCR and Flow Cytometry analyses. Histological images were used to examine the evolution of cells morphology in culture. The optimized SSC isolation, culture and evaluation system established from normal mouse was then applied to 3-day old KS mouse testicular cells.
Results: The presence of SSC population was demonstrated in normal and KS cultured testicular cells by qPCR, and FACS. Quantification of undifferentiated spermatogonia by using Digital-PCR showed >15% ZBTB16 (PLZF) positive cells in culture. Preliminary data culturing KS mouse testicular cells showed a viable culture of slowly growing cells up to 60 days. Ongoing work is focusing on optimization of culture system and full characterization of cultured KS testicular cells as well as testing their transplantation efficacy to restore fertility.
Conclusions: This work overcomes the initial quiescent stage of neonatal germ cells loss in KS mouse testis to successfully expand them in vitro. Extension of this novel method may lead to new therapeutic options for KS patients.
PRENATAL AND POSTNATAL GENETIC DIAGNOSIS OF 45,X/46,XY MOSAICISM AND ITS CLINICAL IMPLICATIONS: A 20-YEAR STUDY
Mahmoud Aarabi MD PhD¹, Urvashi Surti PhD², Selma F. Witchel MD³, Francis Schneck MD4, Aleksandar Rajkovic MD PhD5 and Svetlana Yatsenko MD²
¹Medical Genetics & Genomics Laboratories, Magee-Womens Hospital of UPMC and Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA; ²Medical Genetics & Genomics Laboratories, Magee-Womens Hospital of UPMC and Departments of Obstetrics, Gynecology and Reproductive Sciences, Pathology, Human Genetics, University of Pittsburgh, Pittsburgh, PA; ³Pediatric Endocrinology, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA; 4Pediatric Urology, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA; 5Medical Genetics & Genomics Laboratories, Magee-Womens Hospital of UPMC and Departments of Obstetrics, Gynecology and Reproductive Sciences, Pathology, Human Genetics, University of Pittsburgh, and Magee-Womens Research Institute, Pittsburgh, PA
Presented By: Mahmoud Aarabi MD PhD
The prognosis and clinical management of patients with 45,X/46,XY mosaicism differ from non-mosaic 45,X (Turner syndrome). Phenotypes range from apparently normal males, infants with genital ambiguity, or female external genitalia with an increased risk for gonadoblastoma. Gender assignment and prediction of reproductive potential for individual patients can be challenging in this mosaic genetic condition. To ascertain long term clinical outcomes, we compared cytogenetic findings and clinical manifestations and performed genotype/phenotype correlation in 50 cases diagnosed with 45,X/46,XY mosaicism at the University of Pittsburgh Medical Center between 1996-2016. All patients had a 45,X cell line along with a cell line containing one or two copies of normal or isodicentric Y chromosome as confirmed by fluorescence in situ hybridization (FISH) or microarray studies. In 43 patients with available clinical data, 13 (30.2%) resulted in spontaneous miscarriage or pregnancy termination; in 30 patients phenotype range included normal males (n=4, 9.3%), infertile males (n=4, 9.3%), infants with ambiguous genitalia (n=13, 30.2%) and females with premature ovarian insufficiency or Turner syndrome (n=9, 21%). In patients with cytogenetic studies involving multiple tissues, the proportion of each cell line was compared in cultured and uncultured peripheral blood samples, buccal cells and urinary epithelial cells. Gonadal tissue was assessed when available following prophylactic gonadectomy performed due to high risk of gonadal neoplasia. Interestingly, the level of mosaicism varied among different tissues of the same patient. Such discrepancy was also observed in prenatal vs. postnatal samples of affected newborns. This cytogenetic variation may contribute to the phenotypic heterogeneity among individuals with 45,X/46,XY mosaicism. We hypothesize that an individual’s reproductive phenotype reflects the gonadal cytogenetic findings rather than the peripheral blood karyotype. In our study, the 45,X/46,XY mosaic levels in urine samples appeared to correlate with percentage of abnormal cells in gonadal tissues. Thus, a noninvasive alternative method, direct genetic analysis of urinary epithelial cells may be helpful since urinary tract cells share similar embryonic origins with gonads. Further research will elucidate the consequences of the tissue-specific differences in the relative proportions of cells with different chromosome or genetic composition.
WHOLE EXOME SEQUENCING IDENTIFIES GENES AND PATHWAYS WITH POTENTIAL INVOLVEMENT IN PEYRONIE’S AND DUPUYTREN’S DISEASES
Alexander W. Pastuszak MD, PhD, Yofre Cabeza-Arvelaiz PhD, Suman Maity PhD, Cristian Coarfa PhD, Larry I. Lipshultz MD and Dolores J. Lamb PhD
Baylor College of Medicine
Presented By: Alexander W. Pastuszak MD, PhD
Background: Peyronie’s disease (PD) is inherited in a subset of men and has a co-prevalence of ~20% in men with Dupuytren’s disease (DD), a related fibrotic diathesis. Recent forward-genetic screening for genetic factors with potential involvement in PD and DD identified several candidate genes involved in fibrosis and inflammation. Here, we examine nucleotide-level alterations with potential roles in pathogenesis of PD and DD in a father-son duo using whole-exome sequencing.
Methods: Whole-exome genomic data was generated at the RNA and Genomic Profiling Sequencing Core (https://www.bcm.edu/garp/), and mapped using BOWTIE2 to the human genome build UCSC hg19; single nucleotide variants (SNVs) were inferred using the GATK platform, annotated using the annovar software, and filtered for novel, non-synonymous SNVs. Enriched pathways were determined using the Gene Set Enrichment (GSEA) method, and the gene set collection from the Molecular Signature Database (MSigDB).
Results: Whole-exome sequencing identified 95/117 unique SNVs in each sample, with 150 SNVs shared between the two samples. Further analysis identified 150 nonsynonymous shared SNVs. Pathway analysis revealed enrichment in known PD and DD pathogenic pathways including collagen formation / ECM organization (COL1A2, CRTAP), regulation of cell proliferation (SPEG, QSOX1, FGR1OP, LRP5), and the inflammatory response (HLA-DRB5, KDM6B). Several pathways not previously implicated in PD and DD were identified, including chromosomal rearrangement (FGFR1OP, COL1A2, AUTS2, AFF1, SHANK3), EGF-like domain-containing genes (MUC3A, SNED1, CD93, FAT2, SSPO, LRP5, MUC4), and maintenance of GI epithelium (MUC2, MUC6, MUC4). SNVs in disease-associated genes, including osteoporosis and Parkinson’s disease, as well as SNVs in genes involved in head and neck, GU, GI, neurologic, and lung malignancies, were also identified. Neither of the two family members have reported any of the listed conditions to date.
Conclusions: In addition to pathways that can affect fibrosis, men with a genetic predisposition to PD and DD exhibit genetic alterations in essential cellular functions and disease-related pathways, including malignancies. This is the first study to genetically link fibrotic diatheses to other health conditions, and future work should focus on confirming these relationships. Moreover, men with PD or DD may warrant additional follow-up after diagnosis and treatment of these conditions.
CHARACTERIZATION OF THE PARTIAL AZFC Y-CHROMOSOME MICRODELETIONS IN SUBFERTILE AND INFERTILE MEN
Phil Bach MD¹, Anna Mielnik MS², Alexander Bolyakov MS², Ryan Flannigan MD², Peter Schlegel MD² and Darius Paduch MD, PhD²
¹Weill Cornell Medicine; ²Department of Urology, Weill Cornell Medicine
Presented By: Phil Bach MD
Introduction and Objectives: Deletions in the AZF region of the Y-chromosome, also known as Y-chromosome microdeletions (YCMD), are responsible for approximately 10% of male infertility cases. Well characterized YCMD include AZFa, AZFb, AZFb+c, AZFc, and AZFa+b+c, which result in varying degrees of spermatogenesis dysfunction. In particular, deletions in the AZFa and AZFb regions are associated with azoospermia with poor prognosis for testicular sperm retrieval. On the other hand, deletions in the AZFc region can present with severe oligospermia or azoospermia and have more promising prognoses for testicular sperm retrieval. Recently, partial deletions have been described in the AZFc region. We aim to characterize the frequency and presenting phenotypes for these partial AZFc YCMD (b2/b3, b1/b3, and gr/gr).
Methods: A retrospective review of all YCMD testing performed on consecutive subfertile and infertile men was conducted. Patients with b2/b3, b1/b3, and gr/gr AZFc YCMD were identified and their charts analyzed for sperm concentration and hormone levels.
Results: A total of 3,195 subfertile or infertile men were screened for YCMD between 1997 and 2016. A total of 446 patients (14%) had YCMD identified. Amongst YCMD identified, the frequencies of b2/b3, b1/b3, and gr/gr AZFc YCMD were 2.7%, 1.1%, and 26%, respectively. While we did not have enough clinical data to analyze patients with b1/b3 AZFc YCMD, 89% of patients with b2/b3 AZFc YCMD presented with azoospermia while the remaining 11% presented with cryptoozospermia. Of patients with gr/gr AZFc YCMD, 81% presented with azoospermia, 6% presented with cryptoozospermia, 5% with oligospermia, and only 6% with normoozospermia. While serum testosterone levels were normal amongst men with b2/b3 and gr/gr AZFc YCMD (401+/-139ng/dl and 344+/-149ng/dl, respectively), FSH levels were markedly elevated in both groups (20+/-9mIU/mL and 22+/-16mIU/mL for b2/b3 and gr/gr, respectively).
Conclusions: Partial AZFc deletions, especially the gr/gr deletion and, to a lesser extent, the b1/b3 and b2/b3 deletions, are commonly seen YCMD in subfertile and infertile men. The overwhelming majority of patients with b2/b3 and gr/gr AZFc deletions present with severe dysfunction of spermatogenesis (either azoospermia or cryptoozospermia). More work is required to further characterize these partial AZFc YCMD in order to better understand their impact on testicular sperm extraction results and reproductive outcomes.
LOSS OF TRANSLATIONAL SUPPRESSION OF PRM1/2 VIA YBX2 IS A CULPRIT OF EARLY AND LATE MATURATION ARREST IN HUMANS.
Ryan Flannigan MD¹, Anna Mielnik MSc¹, Alex Bolyakov MSc¹, Phil Bach MD¹, Peter Schlegel MD¹, Jen Grenier PhD², Andrew Grimson PhD² and Darius Paduch MD PhD¹
¹Weill Cornell Medical College; ²Cornell University
Presented By: Ryan Flannigan MD
Introduction: YBX2 protein binds to Y-box promotors and mRNAs regulating translation of important mRNAs in pachytene spermatocytes and round spermatids. CDK1 phosphorylation of YBX2 enhances binding to RNAs. Here, YBX2 binding provides temporal translational suppression of PRM1/2 and SMCP, as they expressed only as mRNAs in spermatocytes but not expressed as proteins until spermatids. Loss of YBX2 activity leads to male infertility due to loss of translational suppression. Our study aim was to analyze expression of known YBX2 promoter binding elements among well-defined histological categories of male infertility: Sertoli cell only syndrome, maturation arrest (MA)(early and late) and hypospermatogenesis.
Methods: RNAseq was performed using human testis samples from men with nonobstructive azoospermia (50) and 10 from normal controls. SYCP3 was used as a marker for spermatocyte quantity and CLGN for spermatids. Phantom5 was used to predict enhancers and inhibitors of YBX2 expression. Statistical analysis was performed using JMPgenomics. FDR<0.001 was used to identify differentially expressed genes.
Results: Expression of YBX2 was 207x less in men with eMA and lMA as compared to controls. Expression of PRM1/2 was 1.99E+10 and SMCP 2.33E+10x lower in MA than in controls. Both eMA and lMA have similar numbers of SYCP3 positive spermatocytes and CLGN positive spermatids as seen in normal testis (fold ratio 0.78). Thus, decreased expression of PRM1/2 and SCMP in eMA and lMA is not due to the loss of spermatocytes or spermatids. Expression levels of CDK1 were similar between MA and NL testes. Multifactorial regression analysis demonstrated a decrease in YBX2 expression in MA is due to decrease in COMP levels (p<0.0001). No additional YBX2 promoter regulators: CTCFL, EZH2, KDM5A, PPARG, CTBP1, E2F4, and USF1 were statistically different.
Conclusions: Decrease in YBX2 expression in men with eMA and lMA leads to release of translational suppression and loss of mRNAs typically sequestrated by YBX2: PRM1/2 and SMCP. This finding has significant therapeutic implications; increasing YBX2 levels could restore PRM1/2 expression and potentially direct arrested spermatids toward maturation.
TESTICULAR PATHOLOGY IS NOT ALTERED IN OBESE INFERTILE MEN WHO PRESENTSEMEN ANALYSES, SPERM FUNCTIONAL TESTS, ELECTRON MICROSCOPY AND TESTIS HISTOLOGY IN OBESE INFERTILE PATIENTS
Caroline Ranéa BSc¹,²,³, Juliana Risso Pariz MSc, PhD student¹,4,²,³,5, Rosa Alice Casemiro Monteiro BSc¹,5, Inari Ciccone BSc, MSc student¹,6,7, Elaine Maria Frade Costa MD, PhD¹,³,5,8, Hector E Chemes MD, PhD¹ and Jorge Hallak MD, PhD¹,²,³,5
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Dept. of Urology, FMUSP, Brazil; ³Reproductive Toxicology Unit, Dept. of Pathology, FMUSP, Brazil; 4Methodist University of Sao Paulo, Brazil; 5Oswaldo Cruz German, Brazil; 6Dept. of Urology, USP, Brazil; 7Reproductive Toxicology Unit, Dept. of Pathology, USP, Brazil; 86Dept. of Endocrinology, USP, Brazil
Presented By: Caroline Ranéa BSc
Introduction: It is estimated that one-third of adult men around the world are obese and one-third are overweight, routinely diagnosed by body mass index (BMI). There is a strong relationship between obesity and male infertility; however, its physiological mechanisms are not well elucidated.
Aims: To evaluate the effect of obesity (BMI and body fat percentage evaluation) in seminal and functional parameters, testicular histology and hormonal profile.
Methods: We included data from 83 medical records of infertile patients aged 21 to 45 y.o., classified according to body fat percentage (BFP) according to bioimpedance values [eutrophic≤19% (n=27), high>19%(n=56)] and BMI [eutrophic (n=34; 18.5Results: Grade A motility decreased in overweight and obesity groups when compared to the eutrophic group. Grade C motility increased in overweight and obesity groups, compared to the control group. We observed an increase in percentage of anti-sperm antibodies in the overweight group (p<0.05). Patients with BFP>19% had a reduction in progressive motility and reduced sperm maturation by CK activity (p<0.05). We did not observe significant alterations in the hormonal profile, testis histology and maturation of sperm chromatin in patients with excess of fat tissue.
Conclusion: Excessive body fat has a negative effect on the final steps of spermatogenesis, demonstrated by reduced total progressive motility, increased forms of immature sperm and high anti-sperm antibodies.
Financial support: Androscience/CNPq - PIBIC
Keywords: Male infertility, body mass index, body fat, semen, sperm.
Ethics Committee Approval: FMUSP Ethics Committee (n°859215/2014)
IMPROVING POST-THAW SPERM CRYOSURVIVAL RATES IN THE ANDROLOGY LAB: CHOOSING THE BEST PROCESSING TECHNIQUE PREVIOUS TO THE CRYOPRESERVATION PROCESS IS CRUCIAL
Beatriz de Campos BSc student¹,², Juliana Pariz PhD student¹,²,³,4,5, Rosa Monteiro BSc¹,5 and Jorge Hallak MD;PhD¹,³,4,5
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Methodist University of Sao Paulo, Brazil; ³Dept. of Urology, Universidade de São Paulo, Brazil; 4Reproductive Toxicology Unit, Dept. of Pathology, Universidade de São Paulo, Brazil; 5Oswaldo Cruz German Hospital, Brazil
Presented By: Beatriz de Campos BSc student
Introduction: Seminal processing before cryopreservation is not always used, but depending on initial semen characteristics may be a crucial to improve post-thaw cryosurvival rates.
Objective: To determine the optimal pre-freeze semen processing method to improve post-thaw sperm quality.
Methods: Ninety-one normozoospermic men (19 to 68 y.o.) had semen samples cryopreserved from 2002-2016. After routine initial analyses, samples were processed by three different methods: simple centrifugation, density gradient using Isolate® or simple washing and were subsequently cryopreserved with Test Yolk Buffer (TYB®), serum substitute (SSS®) and / or human serum albumin (BSA®) by the liquid vapor freezing method. After 24 hours, a small sample was evaluated. The one-way ANOVA test was used for statistical analysis and adopted p <0.05.
Results: TYB samples were used as control group, with total motility = 72.82±8.64%, progressive motility = 53.51±12.93%, and cryosurvival (CS) = 18.29±16.64%. The association of density gradient processing and TYB + SSS showed an increase in grade B motility (49.33±11.19%, p=0.013) and in CS (36.51 ± 31.42%, p=0.009) when compared to control group. When evaluating the simple wash + SSS, a reduction of total motility (62.00 ± 5.70%, p=0.019), progressive motility (43.00 ± 15.24%, p = 0.094), grade A motility, p=0.040), grade B (35.00±16.20%, p=0.227), and in CS (8.10±6.59%, p=0.376) was observed. and increase in sperm motility (38.00±5.70%, p=0.018).
Conclusion: We conclude that the density gradient pre-freeze seminal processing associated with TYB+SSS resulted in better post-thaw sperm quality samples and may be considered as standard part of the protocol in normozoospermic patients who seek cryopreservation for several reasons.
Financial support: Androscience
Keywords: Male infertility, cryopreservation, seminal processing, semen, sperm.
Ethics Committee Approval: FMUSP Ethics Committee (n° 031/13)
|Poster #39|DOES DETECTION OF DDX4 MRNA IN CELL FREE SEMINAL PLASMA REPRESENT A RELIABLE NOT N-INVASIVE GERM CELL MARKER IN PATIENTS WITH NON-OBSTRUCTIVE AZOOSPERMIA ?
Rania Abdelmaksoud MD¹, Wafaa Abdallah MD² and Doaa Hashad MD³
¹Department of Dermatology, Venereology and Andrology, Faculty of Medicine, University of Alexandria; ²Professor of Dermatology, Venereology and Andrology; ³Associate Professor of Clinical Pathology, Faculty of Medicine, University of Alexandria
Presented By: Rania Abdelmaksoud MD
The present study aimed to investigate the potential application of DDX4 gene expression in cell free seminal mRNA as a non-invasive biomarker for the identification of the presence of germ cells in men with non-obstructive azoospermia and to correlate this factor with testicular biopsy. Male reproductive organs-specific genes were chosen; DDX4 which is a germ cell-specific gene, and transglutaminase 4, which is a prostate specific gene that was used as a control gene. Thirty-nine azoospermic males and twenty-eight normospermic fertile males (serving as a control group) participated in the study. Histopathological examination of testicular biopsies categorized azoospermic males into 20.5% with maturation arrest, 17.9 % with incomplete Sertoli Cell Only Syndrome and 61.5 % with completed Sertoli Cell Only Syndrome.
Using real time-Polymerase chain reaction, positivity for DDX4 gene was detected in 17 out of 39 males with NOA which was due to maturation arrest in 35.3% (n=6/17) of cases, while it was due to incomplete sertoli cell only in 23.5% (n=4/17) and due to complete sertoli cell only in 41.2% (n =7/17). The study recommends joint utilization of molecular transcripts as non-invasive biomarkers and histopathological examination of testicular biopsies in management of cases with azoospermia of the non-obstructive type .
POSITIVE EFFECT OF MELATONIN AND CAFFEINE SUPPLEMENTATION IN STRUCTURAL AND FUNCTIONAL CHARACTERISTICS IN PRE-FREEZE AND POST-THAW SEMEN SAMPLES
Juliana R Pariz MSc, PhD student¹,²,³,4, Priscilla R Costa PhD5, Dayane G Reis BSc student¹,²,³, Victória S Coutinho BSc student¹,²,³, Donald P Evenson PhD6 and Jorge Hallak MD, PhD¹,²,³,4
¹Androscience – High Complexity Clinical and Research Andrology Laboratory, Brazil; ²Dept. of Urology, USP, Brazil; ³Reproductive Toxicology Unit, Dept. of Pathology, USP, Brazil; 4Oswaldo Cruz German Hospital, Brazil; 5Department of Immunology, Universidade de São Paulo, Brazil; 6SCSA Diagnostics, United States of America
Presented By: Juliana R Pariz MSc, PhD student
Introduction: Cryopreservation process can damage spermatozoa and impair structural and functional characteristics. Plasma, nuclear membranes and cellular organelles can suffer from freeze and thaw process.
Objective: To evaluate the effect of melatonin (MEL) and caffeine (CAF) supplementation in structural and functional characteristics in pre-freeze and post-thaw seminal samples.
Methods: Twenty-six semen samples from men between 22 and 54 years-old. All samples were normozoospermic according to WHO criteria. Samples were cryopreserved using Human Tubal Fluid modified without any supplement or with MEL 2mM. After thawing, samples were analyzed as they were cryopreserved or supplemented also with CAF 2mM. Samples were incubated for 15 minutes before final analysis. At the end of the experiments, we obtained five groups: pre-freeze samples (Group I), post-thaw samples without any supplementation (Group II), post-thaw samples supplemented with MEL (Group III), CAF (Group IV) and MEL+CAF (Group V). Sperm count, motility, hyperactivity, reactive oxygen species (ROS), mitochondrial activity and DNA fragmentation (SCSA) were evaluated by Student´s T test and one-way analysis of variance (p<0.05).
Results: Pre-freeze and post-thaw results in non-supplemented samples: progressive motility (51.92vs7.27%; p<0.001). High mitochondrial activity sperm (25.30vs8.30%; p<0.001), sperm vitality (78.33vs41.67%; p<0.001), sperm hyperactivation (8.43vs0.69%; p=0.002). No statistical differences in ROS, SCSA were observed. Supplementation with CAF+MEL (Group V), improved progressive motility (16.47vs7.27%; p=0.017), motility grade b (15.38vs7.27%; p=0.025) and high mitochondrial activity sperm (16.86vs8.30%; p=0.05); reduction of lower mitochondrial activity sperm (10.24vs18.15%; p=0.018) when compared with samples without supplementation. In groups III and IV, were only one supplement was added, either CAF or MEL, no differences were noticed.
Conclusion: Cryopreservation has negative effects in sperm quality in normozoospermic samples. ROS and sperm DNA damage in pre-freeze and post-thaw samples did not show differences. Samples supplemented with CAF+MEL improved significantly post-thaw progressive motility and mitochondrial activity and could be a new resource in andrology.
Financial support: Androscience/Capes/SCSA Diagnostics
Keywords: Cryopreservation, sperm, caffeine, melatonin, ROS, SCSA.
Ethics Committee Approval: FMUSP 031/13
|Poster #41|VITAMIN E REDUCES INTRACELLULAR SUPEROXIDE ANION ACTIVITY IN CRYOPRESERVED HUMAN SEMEN
Bruna Lima BSc, Luana Adami BSc, Larissa Belardin MSc, Fátima Okada PhD, Ricardo Bertolla DVM, PhD and Deborah Spaine PhD
Universidade Federal de São Paulo
Presented By: Bruna Lima BSc
INTRODUCTION AND OBJECTIVE: Despite the long-term benefits of sperm cryopreservation in order to preserve male fertility, this technique still brings adverse effects to cells. The mechanism behind these modifications may be related to excessive reactive oxygen species (ROS) generation. While ROS are necessary for physiological sperm functions, unbalanced levels lead to important alterations, such as sperm DNA fragmentation. Aware of importance of balance between ROS and antioxidants levels, there is evidence that antioxidant supplementation protects sperm from cryodamage. Specifically, vitamin E is a potent chain-breaking lipophilic antioxidant. Thus, this study aimed to verify the possible protectant effect of vitamin E in human sperm during cryopreservation.
METHODS: A prospective paired study was carried out including semen from 21 adult men. Fresh samples was analysed according to World Health Organization (2010) guidelines. The remaining semen volume was divided into two aliquots; one cryopreserved using a standard Test-Yolk Buffer, and one using the same buffer supplemented with 40µM alpha-tocopherol acetate. After a three-day storage period in liquid nitrogen, the cryopreserved samples were thawed and evaluated for mitochondrial activity, acrosome integrity, DNA fragmentation, intracellular superoxide anion activity, vitality and motility. Both groups were compared using a paired Student’s t test or a Wilcoxon test, when appropriate. An alpha of 5% was adopted.
RESULTS: Results are presented in table 1. No differences were observed in mitochondrial activity, acrosome integrity, DNA fragmentation, vitality and motility. Vitamin E-supplemented pairs presented lower intracellular superoxide anion levels.
CONCLUSION: Vitamin E is able to improve intracellular oxidative stress in cryopreserved human semen. Because excessive superoxide activity may increase ROS levels, which in turn would lead to downstream alterations to sperm mitochondria, DNA, and acrosome integrity, and because tests were carried out immediately post-thaw, it may be that our analysis detected, by a sensitive method, initial alterations before they effected cellular alterations.
SUPPORT: Urological Research Center - Sao Paulo Federal University
|Poster #42|SEMEN CRISP3 LEVELS IN THE ADULT VARICOCELE.
Larissa Belardin MSc, Mariana Camargo PhD, Paula Intasqui MSc, Paula Del Giudice PhD, Mariana Antoniassi MSc, Renato Fraietta MD; PhD and Ricardo Bertolla DVM; PhD
Universidade Federal de São Paulo - UNIFESP
Presented By: Larissa Belardin MSc
Introduction: Varicocele is the most prevalent treatable cause of male infertility, and its surgical treatment (varicocelectomy) has been shown to improve male fertility. We have demonstrated a higher expression of Cysteine-rich secretory protein 3 (CRISP3) in the seminal plasma of adolescents with varicocele. Because CRISP3 is related to fertilization and with inflammation and cancer, we hypothesized that CRISP3 in varicocele may be playing an inflammatory role. We aimed to verify seminal levels of CRISP3 in adults with and without varicocele, and before and after varicocelectomy. Methods: This prospective study included 83 adults, divided into two sub-studies to verify: (I) the effect of varicocele by comparing controls without varicocele (C, n=25) to men with varicocele (V, n=36), and (II) the effect of varicocelectomy by comparing men before (PRE) and six months after (POST) varicocelectomy (n=22). After seminal (WHO, 2010) and sperm functional analysis, 50 µg of seminal plasma total protein were utilized for Western blotting analysis for quantification of CRISP3. DJ-1 [PARK7] was used as a loading control. Groups were compared by an unpaired Student’s T or Mann-Whitney test (sub-study I), and by a paired Student’s T or Wilcoxon test (sub-study II) (p<0.05). Results: Results are presented in Table 1. For sub-study I, varicocele was associated to lower mitochondrial activity (DAB IV) and to increased seminal CRISP3 levels (nearly 100-fold increased in the unglycosylated form [29kDa], and a 7-fold increased in the glycosylated form [31kDa]). Varicocelectomy leds to an increase in sperm with normal morphology and mitochondrial activity (DAB I). CRISP3 levels decreased, both in the unglycosylated (5.6-fold) and the glycosylated (4.3-fold) forms. Conclusion: In the presence of varicocele, there is a marked increase in seminal CRISP3 levels, more so in the unglycosylated form. Surgical intervention (varicocelectomy) decreases CRISP3 levels and improves semen quality.
Financial support: São Paulo Research Foundation (process number: 2016/05487-3).
|Poster #43|THE EFFECT OF TESTICULAR GERM CELL TUMORS ON FUNCTIONAL ASPECTS OF SPERM AND OXIDATIVE STRESS OF SEMINAL PLASMA
Maria Beatriz Ribeiro de Andrade MSc, Paula Intasqui MSc, Larissa Belardin MSc, Danielle Tibaldi MSc, Ricardo Pimenta Bertolla DVM; PhD and Deborah Montagnini Spaine PhD
Sao Paulo Federal University
Presented By: Maria Beatriz Ribeiro de Andrade MSc
INTRODUCTION AND OBJECTIVE: Testicular germ cell tumors (TGCT) can affect spermatogenesis and lead to alterations in semen quality and to sperm functional traits. This may be due to changes in tumor cells metabolism or to a local inflammatory process. Therefore, we wished to verify if men with testicular tumors present alterations in semen quality, sperm functional traits, and in seminal plasma oxidative stress.
MATERIALS AND METHODS: A prospective study was carried out including 24 patients with TGCT who provided semen sample before orchiectomy, of which 14 non-seminoma and 10 seminoma. A control group was comprised of 17 normozoospermic men. Men with clinical varicocele, smoking, with systemic diseases (such as cancer and endocrinopathies and their treatments), endocrine disorders, genetic syndromes, or history of genitourinary disorders were excluded from the control group. Following semen analysis, an aliquot was used for analysis of (i) sperm DNA fragmentation (alkaline Comet assay, classified as low [Class I] to high [Class IV] DNA fragmentation); (ii) acrosome integrity (PNA-FITC); and (iii) mitochondrial activity (Grade I [all mitochondria active] to IV [all mitochondria inactive]. Seminal plasma MDA levels were measured as markers of oxidative stress. Groups were compared using one-way ANOVA followed by a Bonferroni post-hoc test, or Kruskal-Wallis, followed by a Games Howell post-hoc test, when appropriate (p<0.05).
RESULTS: Results are presented in table 1. A decrease in sperm morphology (%) (p= 0.012), mitochondrial activity (p˂0.00001) and sperm concentration (p=0.00002) were observed in the non-seminoma group when compared to controls. A decrease in sperm morphology (p=0.009), mitochondrial activity (p˂0.00001), non-progressive motility (p=0.010) and sperm concentration (p=0.001) were observed in the seminoma group when compared to controls.
CONCLUSIONS: Based on our findings, a testicular germ cell tumor is associated to a decrease in sperm morphology, sperm mitochondrial activity, and sperm concentration. This demonstrates important alterations to spermatogenesis before removal of the affected testis.
Acknowledgements: M. B. Ribeiro de Andrade received a scholarship from CAPES.
|Poster #44|TRANSGENDER SPERM CRYOPRESERVATION: TRENDS AND FINDINGS IN THE PAST DECADE
Kai Li MD¹, Dayron Rodriguez MD¹, Scott Gabrielsen MD, PhD¹, Amy Blanchard², Grace Centola PhD² and Cigdem Tanrikut MD¹
¹Massachusetts General Hospital; ²New England Cryogenic Center
Presented By: Kai Li MD
Introduction and Objective:
Awareness and acceptance of transgenderism has increased in the last two decades. The 2001 World Professional Association for Transgender Health’s Standards of Care advocates discussion of reproductive issues with transgender patients prior to initiation of hormonal therapy. To date, there is limited literature regarding the incidence and semen characteristics of transgender individuals banking sperm. We sought to assess transgender sperm cryopreservation compared to the non-transgender population in the last 10 years. We also compared semen parameters between the two populations.
We performed a retrospective analysis of sperm cryopreservation performed at a single center from 2006 through 2016. We analyzed 194 transgender samples and 2327 non-transgender samples for a total of 84 unique transgender bankers and 1398 unique non-transgender bankers. Bankers who preserved multiple samples had the collective semen parameters averaged and the mean used for statistical analysis. Semen samples were analyzed according to WHO 4th and 5th edition guidelines based on year of sample production. Linear regression was used to compare the annual incidence of cryopreservation from 2006-2016 of transgender versus non-transgender. Semen parameters were compared using Student’s T-test.
The number of transgender individuals pursuing sperm cryopreservation increased relative to non-transgender individuals from 2006 to 2016. The trajectory of the two groups was significantly different (Figure 1, p<0.001). There were no significant differences in ejaculatory volume, total sperm count, percent motility, or total motile sperm between the two groups.
This is the largest report to date on the incidence of transgender sperm cryopreservation and comparison of semen characteristics. The incidence of sperm cryopreservation by transgender individuals has increased in the last decade, paralleling the increase in awareness and acceptance, and may reflect increased discussion between transgender individuals and medical professionals. There were no significant differences in semen parameters.
|Poster #45|NORMAL PREOPERATIVE FOLLICLE-STIMULATING HORMONE LEVEL IS ASSOCIATED WITH IMPROVEMENT IN SEMEN PARAMETERS FOLLOWING MICROSURGICAL VARICOCELECTOMY
Lunan Ji MD, Samuel Shabtaie, Nachiketh Soodana Prakash MD and Ranjith Ramasamy MD
University of Miami
Presented By: Lunan Ji MD
Introduction: We investigated whether preoperative follicle−stimulating hormone (FSH) level is associated with changes in post−operative semen parameters following microsurgical varicocelectomy.
Methods: We identified 37 men who had undergone microsurgical varicocelectomy between August 2015 and June 2016. We compared semen parameters in men based on their pre−operative FSH level, normal <10 mIU/ml (n=25) and abnormal ≥10 mIU/ml (n=12). We compared varicocele grade, testis volume, prevalence of bilateral disease, pre−operative, and post−operative semen parameters (at 3 months) between men with normal and abnormal FSH.
Results: The age, varicocele grade, pre−operative testosterone levels were similar between men who underwent microsurgical varicocelectomy with normal and high FSH. Men with higher FSH had higher rates of bilateral disease. Men with FSH <10 mIU/mL had higher increases in absolute total sperm count (20.4M vs. 0.8M, p=0.002), sperm concentration (5.2M/mL vs. 1.4M/mL, p=0.05), and total motile count (5.1M vs. 1.4M, p=0.02) post−operatively compared to those with abnormal FSH. As expected, testis volume was smaller in the men with high FSH (12 cc vs. 14 cc, p=0.004). Change in motility was not significantly different between men with abnormal and normal FSH.
Conclusions: Our study suggested an association between men with normal FSH levels (<10 mIU/ml) and significant improvements in total sperm count, sperm concentration, and total motile count among those who underwent microscopic varicocelectomy. Normal FSH levels can suggest preserved spermatogenesis and therefore a significant benefit can be expected after varicocele repair.
EFFECT OF SPERM MORPHOLOGY ON INTRAUTERINE INSEMINATION PREGNANCY SUCCESS: A SYSTEMATIC REVIEW AND META-ANALYSIS
Taylor P. Kohn MPhil¹, Jaden R. Kohn BS¹, Nancy Brackett PhD², Charles Lynne MD² and Ranjith Ramasamy MD²
¹Baylor College of Medicine, Houston, TX; ²Department of Urology, University of Miami Miller School of Medicine, Miami, FL
Presented By: Taylor P. Kohn MPhil
Introduction and Objective: The World Health Organization (WHO) 2010 guideline defines >4% normal sperm morphology, by the Kruger strict criteria, as the lower limit of normal. While initially described as a strong predictor of intrauterine insemination (IUI) success, several recent publications have demonstrated no relationship between sperm morphology and IUI pregnancy success rates. To assess the available evidence, we performed a systematic review and meta−analysis to determine the effect of abnormal sperm morphology on pregnancy success for couples undergoing IUI.
Materials and Methods: We performed a systematic search of MEDLINE, EMBASE, and The Cochrane Library for studies evaluating semen morphology using Kruger's strict criteria and IUI success rates (measured by clinical pregnancies per cycle of IUI) published through November 2016. Studies were eligible for inclusion if they assessed IUI pregnancy success rate for percent sperm morphology >4% and ≤4% or percent sperm morphology ≥1% and <1%. Studies were assessed both overall and by WHO guideline Eras: pre−1999, 2000−2010, and post 2010. Estimates were pooled using random−effects meta−analysis.
Results: Data were extracted from 22 observational studies involving 41,585 cycles. Twenty studies reported sperm morphology >4% and ≤4% and nine studies reported sperm morphology ≥1% and <1%. When assessing the 4% threshold, a statistically significant difference was evident overall (>4%: 14% vs. ≤4%: 12%, p = 0.03). However, no significant difference was seen in IUI success rate when the 1% sperm morphology threshold was assessed: (<1%: 14% vs. ≥1%: 14%, p = 0.97). When assessed by the different WHO criteria a new trend was apparent: a significant difference in pregnancy rate was seen between couples with >4% normal sperm morphology compared to those with ≤4% in the pre−1999 Era (>4%: 14% vs. ≤4%: 8%, p = 0.006). Whereas, no significance was seen in the 2000−2010 Era (>4%:16% vs. ≤4%: 14%, p = 0.3) or in the post−2010 Era (>4%:12% vs. ≤4%: 11%, p = 0.1).
Conclusion: Abnormal sperm morphologies according to Kruger’s criteria are not predictive of diminished IUI pregnancy rates. Further, IUI pregnancies can be equally achieved in even men with <1% normal sperm morphology as compared to men with >1%. Thus, couples with abnormal sperm morphologies should not be excluded from a trial of IUI as long as the other semen parameters are within normal limits.
A SYSTEMATIC REVIEW OF THE EFFICACY AND SAFETY OF TRANSURETHRAL SURGERY FOR EJACULATORY DUCT OBSTRUCTION-RELATED INFERTILITY
Clark Judge BA and Peter Stahl MD
Presented By: Clark Judge BA
Objective: Our goal was to evaluate the efficacy and safety of transurethral surgery for EDO-related infertility.
Methods: Pubmed, Embase, and MEDLINE databases were searched for studies evaluating efficacy and safety of transurethral surgery for EDO-related infertility. We analyzed all studies of transurethral surgery in infertile men with complete or partial EDO. The impact of transurethral surgery on semen quality, pregnancy rates, and complications was analyzed by structured data synthesis, as heterogeneity of data reporting precluded meta-analysis.
Results Obtained: We identified 22 cohort studies, 8 case series, and 10 case reports that described outcomes of transurethral surgery for 482 infertile men with either complete or partial EDO. Surgical treatments used transurethral resection of the ejaculatory ducts (TURED) (399/482, 83.8%), transurethral incision of the ejaculatory ducts (TUIED) (11/482, 2.3%), unspecified TURED/TUIED (49/482, 10.2%), transurethral dilation of the ejaculatory ducts (21/482, 4.4%), and transurethral laser treatment (2/482, 0.4%). Sperm were detectable in the ejaculate postoperatively in 64.9% (244/376) of azoospermic patients treated surgically for complete EDO. The rate of achieving a normal semen analysis (SA) after surgery for complete EDO amongst men for whom full SA data were reported was 35.3% (54/153), and the aggregate reported spontaneous pregnancy rate was 19.0% (56/294). In non-azoospermic men with partial EDO, the aggregate reported rates of semen quality improvement, achievement of normozoospermia, and spontaneous pregnancy were 77.4% (82/106), and 53.1% (17/32), and 29.6% (29/98), respectively. Aggregate data derived from studies that reported postoperative adverse events revealed significant complications in 14.0% (42/299). The most common reported complications were watery ejaculate (3.3%), epididymitis/orchitis (2.3%), prolonged hematuria requiring catheterization (2.3%), conversion to azoospermia (1.7%), and hematospermia (1.3%).
Conclusions: This systematic review demonstrated that transurethral surgery appears to be an effective treatment for complete or partial EDO that results in improved semen quality for the majority of patients. Spontaneous pregnancies have been reported in 19-30% of cases, and the risk of postoperative complications appears to be acceptably low. These data are valuable for counseling patients on treatment options for EDO-related infertility.
|Poster #50|POLICY ON POSTHUMOUS SPERM RETRIEVAL: SURVEY OF 75 MAJOR ACADEMIC MEDICAL CENTERS
Nicholas Waler¹ and Ranjith Ramasamy MD²
¹University of Miami Miller School of Medicine; ²Department of Urology, University of Miami, Miami, FL
Presented By: Nicholas Waler
Introduction and Objectives
Very few studies have addressed attitudes on posthumous sperm retrieval due to the ethical and legal ramifications of the use of gametes after death. We evaluated the presence and content of a policy on posthumous sperm retrieval at the 75 major academic medical centers in the U.S.
We surveyed the 75 major academic medical centers as ranked for research in 2016 by U.S. News & World Report using a questionnaire based telephone/web survey. We gathered data on presence and content of posthumous sperm retrieval policies. If not published, we contacted the legal counsel for the medical center, the ethics and compliance offices, the Urology Department, as well as the infertility center for each institution, in that order.
Out of the 75 major academic medical centers, we gathered data on posthumous sperm retrieval from 30 (40%). Of the 30 institutions, 10 (33%) had policies regarding posthumous sperm retrieval, 19 (60%) did not have a policy, and one center remained undisclosed. Five of the 19 medical centers without policies have discussed development of a policy but did not formalize it due to lack of legal guidance as a barrier to policy adoption. Out of the 10 centers that had a policy, four required prior written consent, while six allowed for verbal or inferred consent from the surviving life partner.
Very few, about 1/3, of the surveyed academic medical centers have policies on posthumous sperm retrieval. Medical centers can adopt individualized policies based on guidelines published by the American Society for Reproductive Medicine.
|Poster #51|SERUM METABOLOMIC PROFILING IDENTIFIES CHARACTERIZATION OF NON-OBSTRUCTIVE AZOOSPERMIC MEN
Zhe Zhang MD, Yuzhuo Yang MD and Hui Jiang MD
Peking Univeristy Third Hospital
Presented By: Zhe Zhang MD
Male infertility is considered a common health problem, and non-obstructive azoospermia with unclear pathogenesis is one of the most challenging tasks for clinicians. The objective of this study was to investigate the differential serum metabolic pattern in non-obstructive azoospermic men and to determine potential biomarkers related to spermatogenic dysfunction. Serum samples from patients with non-obstructive azoospermia (n = 22) and healthy controls (n = 31) were examined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Serum metabolomic profiling could differentiate non-obstructive azoospermic patients from healthy control subjects. A total of 24 metabolites were screened and identified as potential markers, many of which are involved in energy production, oxidative stress and cell apoptosis in spermatogenesis. Moreover, the results showed that various metabolic pathways, including D-glutamine and D-glutamate metabolism, taurine and hypotaurine metabolism, pyruvate metabolism, the citrate cycle and alanine, aspartate and glutamate metabolism, were disrupted in patients with non-obstructive azoospermia. Our results indicated the serum metabolic disorders may contribute to the etiology of non-obstructive azoospermia. This study suggested serum metabolomics could identify unique metabolic patterns of non-obstructive azoospermia and provide novel insights into the pathogenesis underlying male infertility.
PENILE PROSTHESIS COMPLICATIONS: A DESCRIPTIVE STUDY OF THE DETROIT AFRICAN AMERICAN PATIENT POPULATION.
Mohammed Zaher DO¹, William Ducomb Medical Student², Maha Husainat Research Assistant¹, Ibraheem Malkawi Research Assistant¹ and Mazen Abdelhady MD¹
¹Detroit Medical Center; ²Michigan State University
Presented By: Mohammed Zaher DO
Introduction: Failure and infection rates of inflatable penile prosthesis (IPP) implantation have been vastly studied with the largest studies showing reoperation rates ranging from 6-7%. In our previous study, we found a revision rate of 17% of which 70% of the failures were African American (AA) patients. The IPP mechanical failure and infection rate is not well researched in the AA population. In this study, we further evaluated the failure and infection rate of IPP implantation in this possibly high risk AA population. Our objective in this study was to determine which variables had an impact on penile implants in AAs and if the AA population had a higher failure rate.
Methods: This was a retrospective chart review of men ages 18 and older that had an IPP implanted at the Detroit Medical Center between 2000-2016. Variables included: all races, reason for IPP placement, type of IPP, IPP failure, reason for failure, and co-morbidities
Results: A total of 150 patients were included, 93 were AA. The average age of our patients at time of surgery was 66 years (range 39-94). The average age of patients at time of IPP failure was 68 years (range 47-90). 39 had a failed IPP (26%), of those, the majority of the race being AA (69%, n=27). The most common reason for insertion of an IPP was organic erectile dysfunction (ED) (74%, n=111), while the most common cause for failure was IPP malfunction (67%, n=26). Other reasons for IPP failure were erosions (23%, n=9) and infections (10%, n=4), of which 78% (n=7) and 50% (n=2) were AA, respectively. Of the 39 patients who failed their first IPP, 36% failed again (n=14); of these patients majority were AA (71%, n=10) (p <0.05).
Conclusion: In our study, African Americans had an overall higher IPP mechanical failure as well as secondary revision rates compared to other races. These higher rates found in our AA patient population could be secondary to their high incidence of existing co-morbidities. Our research suggests that when considering an IPP placement for erectile dysfunction, the surgeon should take race and co-morbidities into account when discussing with patients the possible complications and failure rate.
SOCIOECONOMIC DISPARITIES IN THE TREATMENT OF ERECTILE DYSFUNCTION: A SYSTEMATIC REVIEW
Denise Asafu-Adjei MD, MPH¹, Mofan Gu MPH², Matthew Pagano MD¹, Ifeanyi Onyeji MD¹ and Peter Stahl MD¹
¹Columbia University Medical Center; ²Columbia University Mailman School of Public Health
Presented By: Denise Asafu-Adjei MD, MPH
Introduction and Objectives:
Previous literature suggests that socioeconomic disparities exist between the rate and types of treatment for erectile dysfunction (ED). The objective of this study was to identify the various socioeconomic factors that contribute to these disparities, examine the mechanisms by which these factors interact, and propose ways to diminish the impact of these disparities as it pertains to patient outcomes.
A systematic literature search via PubMed was conducted, using a combination of keywords including socioeconomic status (SES) as well as treatment terms for ED. Articles were initially screened based on title and abstract, but were eliminated based on established exclusion criteria. The goal was to obtain articles with primary data and quantitative analysis that would allow for adequate extraction of data. A total of 1246 articles were obtained from PubMed and information was extracted from 15 articles for systematic review. The data were not amenable to meta-analysis and therefore a structured data analysis was performed.
Six studies addressing treatment-seeking behavior showed that patients with higher income and educational status were more likely to seek active treatment for ED. Eight studies focused on ED treatment rates and the data suggest that men with higher incomes are more likely to seek treatment for ED. In terms of treatment type disparities, there is literature to suggest that African-American and Hispanic men are more likely to be treated with penile prosthesis than Caucasian men. Additionally, men with Medicaid or self-pay patients are more likely to be treated with semi-rigid rather than inflatable prostheses, as compared to men with commercial insurance.
There is a signal in the published literature that ED treatment rates and the types of treatments provided vary according to socioeconomic factors. It remains unknown whether the observed differences are due to patient choice, physician bias, differences in ED severity, and/or SES factors. However, the significant findings across multiple studies warrant further examination into the root causes of these published trends. In the future, greater patient and physician education and awareness may be necessary to address these disparities and implement standard-of-care algorithms across patient groups and institutions.
TESTICULAR SPERM RETRIEVAL IN LATE ADOLESCENTS (AGED 15-19 YEARS) WITH NON-MOSAIC KLINEFELTER SYNDROME AND AZOOSPERMIA
Han-Yu Weng, Yung-Ming Lin PhD and Yu-Sheng Cheng
National Cheng Kung University Hospital
Presented By: Han-Yu Weng
Introduction and Objectives: It has been shown that most men with Klinefelter syndrome (KS) are born with spermatogonia and the germ cells appear to undergo apoptosis during puberty. To date, controversy exist in the age of fertility preservation in adolescents with non-mosaic KS. WHO identifies adolescence as the period that occurs after childhood and before adulthood, from ages 10 to 19. Previous report revealed extreme low sperm retrieval rate (SRR) in early adolescents. The objective of this study is to retrospectively analyze the clinical characteristics and SRR of late adolescents (age 15-19 years) with non-mosaic KS in our clinic and to review the literature.
Methods: Three late adolescents who were referred for hypogonadism and fertility counselling underwent microsurgical testicular sperm extraction (microTESE). Their medical history, physical examination findings, testicular volume, serum hormone parameters, microTESE outcome were analyzed. Then, a comprehensive literature searches for systematic review using MEDLINE and PubMed was conducted. Exclusion criteria includes mosaic KS, aged 14 years or younger, aged 20 years or older, and no SRR as primary outcome.
Results obtained: Increased serum FSH and LH levels as well as decreased testosterone levels were noted in our three patients. All three patients had bilateral testicular atrophy (less than 5 ml for each testicle). Spermatozoa were retrieved in two patients (66.7%). Two patients with complete Sertoli cell-only syndrome and one patient with hypospermatogenesis were noted in testicular histopathology. The literature search identified a total of 89 scientific papers. Eight publications and 85 late adolescents with age 15-19 years were enrolled in final analysis. A total of 88 late adolescents underwent testicular sperm retrieval, and 42 met with success (SRR: 47.7%).
Conclusions: Testicular SRR in late adolescents is not superior to those who undergo the procedure later in their life.
|Poster #55|ANXA7 AND PSMA5 ARE SEMINAL BIOMARKERS OF SPERM ACROSOME INTEGRITY.
Paula Intasqui MSc, Larissa Belardin MSc, Mariana Antoniassi MSc, Mariana Camargo PhD, Daniel S. Zylbersztejn PhD and Ricardo P. Bertolla PhD
Department of Surgery, Division of Urology, Sao Paulo Federal University
Presented By: Paula Intasqui MSc
INTRODUCTION AND OBJECTIVE: The sperm acrosome is important for fertilization and, thus, for male fertility. Among proteins involved in acrosome formation (spermiogenesis) and reaction (capacitation), two proteins have been recently highlighted. Annexin A7 (ANXA7) is a Ca2+-dependent phospholipid binding protein with GTPase activity that facilitates vesicle transport and membrane fusion. It is important for acrosome reaction and sperm-zona pellucida binding. Proteasome subunit alpha type-5 (PSMA5) protein is part of the proteasome 20S core, and recent studies have demonstrated that this complex is important for acrosome formation, selection of sperm with intact acrosome, and exocytosis. Therefore, we aimed to evaluate expression of these proteins in seminal plasma and correlations with acrosome integrity in ejaculated sperm.
METHODS: A cross-sectional study was performed including 155 normozoospermic men. After semen analysis (WHO 2010 guidelines), sperm acrosome integrity was evaluated using PNA-FITC staining. Samples were then divided according to acrosome integrity into control (> 82% sperm with intact acrosome, top 15%, n=23) and study (< 67% sperm with intact acrosome, bottom 15%, n=19) groups. The remaining semen volume was centrifuged for seminal plasma separation, in which ANXA7 and PSMA5 expression was evaluated by Western blotting. Data were normalized by a loading control. Groups were compared by an unpaired Student’s t test or by the Mann Whitney test.
RESULTS: The low acrosome integrity group presented decreased percentage of sperm with intact acrosomes (64% ± 3.36 vs. 85.5% ± 4.01, p<0.001), as expected, as well as decreased sperm morphology (6.0% ± 2.04 vs. 9.0% ± 6.13, p=0.031) and percentage of sperm with active mitochondria (8.5% ± 3.81 vs. 9.5% ± 9.78, p=0.021). ANXA7 and PSMA5 expression data are presented in Figure 1. ANXA7 was significantly underexpressed, whereas PSMA5 was overexpressed in the study group. Moreover, PSMA5 negatively correlated with the percentage of intact acrosome (p=0.025, r=-0.180, Spearman’s correlation).
CONCLUSION: ANXA7 and PSMA5 are seminal biomarkers of acrosome integrity and damage, respectively.
SUPPORT: FAPESP (2014/11493-0, 2014/17185-6).
|Poster #56|EFFECTS OF VARICOCELE IN SPERM CAPACITATION
Rhayza Andretta PhD candidate¹, Larissa Belardin MSc¹, Letícia Castro DVM, MSc², Jheysson Moura BSc¹, Renato Fraietta DM, PhD¹, Fatima Okada PhD¹ and Ricardo Bertolla DVM, PhD¹
¹Federal University of Sao Paulo ; ²University of Sao Paulo (USP)
(Presented By: Rhayza Andretta PhD candidate)
Introduction: Varicocele is defined as an abnormal dilation of the testicular veins in the pampiniform plexus with retrograde blood flow in the internal spermatic veins as a result of incompetent or absent valves. It is considered one of the main causes of male infertility affecting 15-25% of the adult male population, and is responsible for the alterations to semen quality, leading to failures in the processes associated with fertilization, such as sperm capacitation. Sperm capacitation is characterized by physiological changes that occur mainly in the female reproductive system, rendering sperm functionally competent to fertilize the oocyte. Many changes occur during capacitation, such as increased plasma membrane fluidity, increased protein phosphorylation, changes in motility pattern, and acrosome reaction. However, it is not known if varicocele may affect these mechanisms. Objectives: To evaluate the effect of varicocele on sperm capacitation. Methods: Men were divided into 2 groups: controls without varicocele (n=17) and varicocele (grades II and III, n=32). Semen was collected by masturbation after 2-5 days of ejaculatory abstinence. One aliquot was used for seminal analysis (World Health Organization, 2010). The remaining volume was submitted to a discontinuous density gradient (45 to 90%) and centrifuged at 300 g for 20 minutes. The pellet was then washed in human tubal fluid (HTF). An aliquot was used to evaluate motility by computer-assisted sperm analysis (IVOS). Capacitation was then induced using HTF supplemented with 1% bovine serum albumin (BSA) for 2 hours at 37o C, 5% CO2. Following capacitation induction, the following measures were assessed: motility (IVOS), capacitation assay (chlortetracycline), intracellular superoxide anion activity, and mitochondrial activity. Groups were compared using a Student’s t test or a Mann-Whitney test. Results: Results are presented in table 1. Men with presented lower ejaculate volume. After capacitation, the varicocele group presented lower sperm motility and mitochondrial activity when compared to controls. Conclusion: Our data suggest that varicocele may exert negative effects to sperm capacitation. Financial support: CAPES.