Oral Presentations Basic Science I

Peter Szaraz1,3, Alexandra Lucato1, Farwah Iqbal1,4, Matthew Librach1, Shu-Hong Li, Andrée Gauthier-Fisher1, Ren-Ke Li, Clifford L. Librach1,2,3,4,5
1Create Fertility Centre, Toronto, ON; 2Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON; 3Institute of Medical Sciences and 4Department of Physiology, University of Toronto, Toronto, ON; 5Department of Gynecology, Women’s College Hospital, Toronto, ON

Introduction: An successful candidate cell type for regenerative therapy after myocardial infarction (MI) should efficiently home to injury sites and effectively remodel extracellular matrix (ECM) at injury sites. We aimed to investigate the MMP-based ECM remodelling and homing properties of first trimester human umbilical cord perivascular cells (FTM-HUCPVCs) in vitro, using a transmembrane invasion assay, and in vivo, using a rat model of MI.

Methods: MSCs were seeded on MatrigelTM-coated trans-membrane inserts and combined with primary rat cardiomyocytes subjected to hypoxia (pO2=1%) and glucose deprivation in culture for 24h. Migration on the cardiomyocyte-facing membrane surfaces was detected by fluorescent well scans. Neutralizing antibodies against SDF1, TNFα, MMP2 and MMP15 were used to inhibit MatrigelTM processing and chemotaxis of MSCs. Animals were handled under institutionally approved protocols. MI was induced by left ventricular coronary artery ligation on 8 week old immunocompromised (Foxrnu) rats. 1 week post-MI, FTM-HUCPVCs, term-HUCPVCs or human BMSCs (n=6 per group) were injected into the injured myocardium (3X106 cells each). 2 weeks after cell implantation, animals were sacrificed. In situ gelatinase activity was assessed by fluorescent gelatinase substrate (DQ gelatin) on fixed cardiac tissue sections and the signal was quantified.  9 independent fields in 6 tissue sections per heart were analyzed. Statistical analysis was performed with ANOVA.

Results: MMP2 an MMP15 neutralizing antibodies significantly inhibited invasion of FTM-HUCPVCs towards injured cardiomyocytes in vitro (p<0.05). Serum-free culture conditions altered the trans-membrane migration of FTM-HUCPVCs in response to injured cardiomyocytes or soluble SDF1. Anti-TNFα antibody significantly inhibited trans-membrane migration of FTM-HUCPVCs expanded in either serum-containing (p<0.05) or serum-free (p<0.01) conditions. Purified TNF-α and SDF1 attracted FTM-HUCPVCs through basal membrane-like structures, however the effect was not significant.  FTM-HUCPVC-treated scar tissue displayed significantly increased gelatinase signal when compared to term-HUCPVCs and bone marrow MSCs.

Conclusion: ECM processing and homing in response to injured cardiomyocytes appeared to be mediated by MMPs as well as cytokines, including SDF1 and TNFα, and altered by xeno-free conditions. These findings suggest that FTM-HUCPVCs could be an excellent candidate for post-MI cell therapy, and that the extent of MMP activity can be controlled by culture conditions.  Our in vivo data also indicates that FTM-HUCPVCs have superior ECM processing in the scar tissue of infarcted hearts compared to older sources of MSCs.

Khaled Zohni1,2, Peter Szaraz1, Melissa Filice1, Itai Gat1,3, Andrée Gauthier-Fisher1, Clifford Librach1,4
1CReATe Fertility Centre, Toronto, ON; 2Department of Reproductive Health & Family Planning, National research centre, Cairo, Egypt; 3Pinchas Borenstein Talpiot Medical Leadership Program, Sheba Medical Centre, Tel HaShomer, Ramat Gan, Israel; Affliated to Sackler Medical School, University of Tel Aviv, Israel; 4Department of Obstetrics and Gynecology; 5Institute of Medical Sciences and 6Department of Physiology, University of Toronto, Toronto, ON; 7Department of Gynecology, Women’s College Hospital, Toronto, ON

Introduction: We have previously shown that human umbilical perivascular cells (HUCPVCs) demonstrate remarkable restorative effects on gonads exposed to cytotoxic treatment in an in vivo animal model. It is not clear whether such cells could be delivered prior to gonadotoxic chemotherapy regimens,  for fertility preservation , especially for regimens requiring repeated rounds. Our objective was to determine whether first trimester (FTM) and term HUCPVCs maintain MSC properties when exposed to the gonadotoxic chemotherapy agents.

Methods:  We treated first trimester (FTM) and term HUCPVCs, and BMSCs with moderate (150 µmol/L) and high (300 µmol/L) doses of cyclophosphamide for either 6 or 24 hours. Untreated cells served as controls. Viability was assessed using trypan blue exclusion and flow cytometry using 7 amino-actinomycin D. The proportion of cells expressing known MSCs markers was measured by flow cytometry after each treatment, as well as in exposed cells expanded for 2 additional passages. The ability of treated MSCs to differentiate into osteoblasts, chondrocytes and adipose cells in vitro was determined using differentiation assays.  Using an in vivo model, HUCPVC survival in immunocompetent CD1 mice injected with 50,000 PKH26-labeled HUCPVCs and treated with 20mg/kg busulfan 3days later, was assessed  using fluorescence microscopy on testicular tissue sections.

Results: The viability of FTM and term HUCPVCs following cyclophophophamide treatment was comparable to that of BMSCs at each dose and each timepoint (78 and 79 vs 77%, 52 and 55 vs 59% after 150 µmol/L after 6 and 24 hour exposures respectively, and 74 and 77 vs 79%, and 55 and 63 vs 59% after 300 µmol/L for 6 and 24 hours respectively).  FTM  and term HUCPVCs continued to express the MSC-associated markers, CD105, CD29 and CD44 after treatment and following 2 passages. Culture expansion was comparable between the three treated cell types and controls. All three MSC types maintained their mesenchymal lineage differentiation potential into chondrocytes, adipocytes and osteocytes after treatment with 150 and 300 µmol/L for 24 hours.   PKH26+ve cells were detected at 14 days post busulfan injection in the murine model.

Conclusion: Our findings suggest that both FTM and term HUCPVCs   remain viable and maintain their MSC properties following chemotherapy exposure both in vitro and in vivo.  Therefore, HUCPVCs are  promising cell candidates for fertility preservation strategies.

Annie Ren, Kyle Ferguson, Gordon Kirkpatrick, Tanya Vinning, Victor Chow, Sai Ma
Department of Obstetrics and Gynecology, University of British Columbia, BC

Introduction: During meiosis, homologous chromosomes pair (synapse) to aid in exchange of DNA at crossover sites along the chromosomes. Previous studies have suggested the importance of crossover distribution and frequency in chromosome segregation. In normal males, crossovers are shown to be rare near centromeres and telomeres, while frequent in subtelomeric regions.

Material & methods: Testicular tissue was obtained from 37 non-obstructive azoospermic (NOA) or obstructive azoospermic (OA) men, and 28 fertile men. Global recombination, synapsis, crossover frequency, distribution, and distance to telomeres were analyzed using immunostaining (Fig 1A). Chromosomes 13, 18 and 21 were identified by FISH (Fig 1B).

Results: Eight of 16 NOA and five of 21 OA men displayed a reduced global recombination rate (Tables 1 and 2). Seven NOA and nine OA men showed altered crossover distributions on at least one of the chromosomes studied (Figs 2 and 3). Five infertile men showed an increase in crossovers near centromeres, six men showed a decrease near subtelomeres, while five men showed an increase near telomeres (Figs 2 and 3). The mean crossover distance to telomeres was higher in the NOA and OA group than controls, on 21q and 18q respectively (Fig 4).

Conclusions: We are the first to study crossover distribution in a large cohort of infertile men, finding that NOA men may have higher risk of having altered crossover frequencies and distributions compared to OA men, which may jointly disrupt chromosome segregation. This aligns with previous data that showed higher rates of aneuploid sperm in NOA versus OA men1-4. Infertile men may display increased crossover formation near centromeres and telomeres, possibly interfering with cohesins and telomere integrity, thus causing chromosome missegregation. Notably, we observed a shift in crossovers toward the centromere on 21q in some infertile men, which is a trend implicated in maternally-derived trisomy 21. We also saw a decrease in crossovers near subtelomeres, where telomeres are thought to aid in crossover formation. Future studies linking crossover distribution to telomere integrity may offer insight for male infertility.

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()table 1 p2aP < 0.001 , bP < 0.05, recombination significantly reduced when compared with controls, Mann-Whitney Test.
cProportions of cell with unsynapsed regions were considered significantly different from controls if they were beyond the 95% CI of the control group
*Previously reported by Ferguson et al. (2009)

()table 2
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aP < 0.001 , bP < 0.05, recombination significantly reduced when compared with controls, Mann-Whitney Test.
cProportions of cell with unsynapsed regions were considered significantly different from controls if they were beyond the 95% CI of the control group
*Previously reported by Ferguson et al. (2009)

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Fig 1. Immunofluorescence and FISH analysis of pachytene spermatocytes. (A) Spermatocytes were immunostained using antibodies against SYCP3/SYCP1, MLH1 and CREST to visualize the SC (red), crossover sites (green) and centromeres (blue). A spermatocyte with 45 crossovers from patient OA20 is shown. Although patient OA20 displayed normal rates of recombination, the crossover distribution on chromosome 18 was altered. We observed an increase in crossovers near the centromere and telomere on 18q and 18p, respectively. (B) Subsequent FISH was performed to identify chromosomes 13 (green, LSI 13), 18 (blue, CEP 18) and 21 (red, LSI 21) in the previously immunostained spermatocytes.

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Fig 2. Chromosomes 21 and 18 displaying altered crossover distributions in NOA men. Chromosome arms were divided into 10% intervals, and the crossover frequency in each interval was calculated. The Y-axis represents the frequency of crossovers in each interval. The X-axis represents the relative crossover position from the centromere with the values representing the upper limit of each interval. The centromere is labeled ‘C’ with the p-arm to the left and q-arm to the right of the centromere. As crossovers in the p-arm of chromosome 21 are extremely rare, the p-arm is not shown. The black bars indicate the control group and the white bars indicate the individual NOA man. The crossover frequencies in each interval were compared to the control group and significant differences are indicated by asterisks (P < 0.05, Fisher test).

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Fig 3. Chromosomes 21, 13 and 18 displaying altered crossover distributions in OA men. The Y-axis represents the frequency of crossovers in each interval. The X-axis represents the relative crossover position from the centromere with the values representing the upper limit of each interval. As crossovers in the p-arm of chromosomes 13 and 21 are extremely rare, the p-arms are not shown. The black bars indicate the control group and the white bars indicate the individual OA man (A-I) and pooled OA group (J). The crossover frequencies in each interval were compared to the control group and significant differences are indicated by asterisks (P < 0.05, Fisher test).

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Fig 4. Average crossover distance to telomere (+SD) on chromosomes 18 and 21 in OA, NOA and control men. The absolute distance between crossover and telomere was divided by the total SC arm length in order to express the distance as a percentage. The Y-axis represents the mean distance between crossovers to the telomeres on the chromosome arms 18q-arm, 18p-arm and 21q-arm. The black bars indicate the control group, the gray bars indicate the OA group and the white bars indicate the NOA group. The average distances between crossovers to telomeres were compared to the control group and significant differences are indicated by asterisks (P < 0.05, Mann-Whitney test).

References:

1. Ma S, Ferguson KA, Arsovska S, Moens P, Chow V. Reduced recombination associated with the production of aneuploid sperm in an infertile man: a case report. Hum Reprod. 2006; 2005;21:980-985.

2. Ferguson KA, Wong EC, Chow V, Nigro M, Ma S. Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy. Hum Mol Genet. 2007;16:2870-2879.

3. Moosani N, Pattinson HA, Carter MD, Cox DM, Rademaker AW, Martin RH. Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil Steril. 1995;64:811.

4. Ushijima1 C, Kumasako Y, Kihaile PE, Hirotsuru K, Utsunomiya T. Andrology. Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridization. Human Reproduction. 2000;15:1107.

Claude Robert1, Angus Macaulay1, Alexandre Bastien1, Isabelle Gilbert1, Edward W. Khandjian2, Shlomit Kenigsberg3, Clifford Librach3,4,5
1Université Laval, Centre de recherche en reproduction, développement et santé intergénérationnelle, Laval, QC; 2Université Laval, Département de Psychiatrie et Neurosciences, Institut universitaire en santé mentale de Québec, Laval, QC; 3CReATe Fertility Centre, Toronto, ON; 4Departments of Obstetrics & Gynaecology & Physiology, University of Toronto, Toronto, ON; 5Department of Gynaecology, Women’s College Hospital, Toronto, ON

Introduction: Our project studies the role played by Fragile-X Mental Retardation protein (FMRP) in oogenesis and egg quality. In the brain, FMRP depletion results in immature dendrites leading to a reduced cellular network. FMR1 (producing FMRP) is the second leading gene for mental retardation after Down’s syndrome and associated with premature ovarian insufficiency (POI). This RNA binding protein is known for its role played in control of translation and association with stalled polyribosomes. The mechanisms through which FMRP deregulation leads to POI are still unknown. An unstable CGG stretch in the 5’ untranslated region of the FMR1 gene is influencing its expression via DNA methylation and by impairing ribosomal progression during translation. One in 259 Canadian women have a premutated allele where the CGG stretch extends to more than 54 and <200 triplet repeats. The full mutation causes a complete shutdown of the gene while the premutation is known to cause aberrant gene expression. We hypothesized that FMRP acts to develop the cellular network around the oocyte especially in cumulus cells that have cellular extensions spanning the zona pellucidae and contacting the oocyte. These transzonal projections (TZPs) have been shown to be essential for oocyte quality.

Methods: This study was approved by institutional REB. Bovine embryos and ovaries were received from a local abattoir. Follicular content, including oocytes, somatic cells (cumulus and granulosa) and IVF produced early embryos were collected. Human cumulus cells (hCCs) were pooled from oocytes obtained from four patients who underwent IVF at CReATe Fertility Centre. Using the TripleKit (Norgen), DNA, RNA and protein were isolated from each sample. qRT-PCR, Western blotting and confocal Immunofluorescence were used to quantify the FMR1 gene products. DNA sequence analyses were performed using Amplidex kits (Asuragen).

Results: Using the bovine model, we have shown that FMRP is highly abundant within the follicular cells during oogenesis, within the TZPs around oocytes and in early embryos. We also detected it in hCC samples. The repeat length and extent of DNA methylation were all within normal range.

Conclusion: The expression of the FMR1 transcript and protein was demonstrated in bovine and human transzonal projections (TZPs), cumulus cells and embryos (bovine). We aim to pursue this study using a knock-in mouse model harboring a human premutated allele.

Anja Stojsin Carter1*, Michael Neal2, Shilpa Amin2,3, Edward Hughes2,3, Mehrnoosh Faghih2,3, Megan
Karnis
2,3, Laura A. Favetta1, W. Allan King1
1Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON; 2ONE Fertility, Burlington, ON; 3McMaster University, Hamilton, ON

Introduction: Correlations between serum (SE) anti-Mullerian hormone (AMH) and ovarian response, day 3 antral follicle count (AFC), number of growing follicles at hCG trigger, and number of oocytes retrieved have been well documented. Follicular fluid (FF) AMH levels of the single largest follicle have also been correlated to fertilization and developmental potential. This study evaluated the patient SE and FF AMH levels of isolated individual ovulatory follicles compared to their corresponding oocyte and subsequent embryo development and outcome.

Material and Methods: Isolated individual FF samples (n=69) were collected at the time of oocyte retrieval from patients (n=6) that had provided informed consent. SE and FF AMH levels (pmol/L) were measured by ELISA (Roche) and compared to cycle characteristics and outcomes. Blastocyst rate (blastocyst number on Day 5 and/or 6 divided by number of oocytes retrieved) was used to measure embryo competence. Spearman correlation coefficient and Wilcox test were used for analysis and p<0.05 was considered statistically significant.

Results: FF AMH levels did not differ between mature (MII) and immature (MI and GV) oocytes (n=41 vs. n=28); normally (2PN) and abnormally (ie: 3PN, 0PN) fertilized oocytes (n=30 vs. n=11); and blastocyst and non-blastocyst group (n=17 vs. n=13). As anticipated, average FF AMH per patient was positively correlated to SE AMH levels (n=6; p=0.017). There was also a strong positive trend between SE AMH and oocyte number and AFC. However, we did not predict the finding that both the average FF AMH and SE AMH were negatively correlated with the rate of blastocyst development (p=0.036 and p=0.015 respectively) for individual patients.

Discussion: In addition to the utility of AMH in predicting ovarian reserve, this study identified systemic and individual FF AMH to be strong predictors of the overall blastocyst rate. Since AMH is involved in follicular dormancy, the relationship observed here has led us to speculate that the rate of depletion of this hormone within the individual follicle, might be indicative of oocyte competence. Overall the positive correlation of AMH to oocyte quantity and its negative correlation to quality may be clinically relevant and suggest that high oocyte yield, resulting from excessive ovarian stimulation protocols, coincides with decreasing oocyte quality and subsequent embryo development to the blastocyst.

Biao Zhang1, Reza Nosrati1, Leila Maghen2, Alexander Lagunov2, Thomas G. Hannam3, David Sinton1
1Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON; 2CCRM Toronto, Toronto, ON; 3Hannam Fertility Centre, Toronto, ON

Introduction: Sperm DNA integrity analysis is crucial to both male infertility diagnosis and assisted reproduction, due to its strong correlation with IVF outcomes and offspring health. Most validated current methods for sperm DNA integrity testing include sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. These methods inform on DNA integrity by quantifying the ratio of damaged to intact sperm DNA strands, expressed as percentage of DNA fragmentation index (%DFI). However, current methods either include an expensive flow cytometry step or long and multi-step processing, which limited their application to well-established clinics. Here, we demonstrate a simple, fast, and low-cost paper-based approach for human sperm %DFI quantification.

 

 

()Fig1Fig. 1. Schematic of the paper-based sperm DNA analysis device.

Method: Human semen samples from 20 patients were tested for DNA integrity. The performance of the paper-based device (Fig. 1) was tested in parallel with TUNEL assay, and as compared with results from SCSA. Lysed stained sperm DNA sample were applied to the paper-based device. Ion concentration polarization was used to separate intact and damaged sperm DNA in the paper-based device. Normal fit to the fluorescence intensity profiles for intact and damages DNA were used to calculate %DFI from the device.

Results: The results from both paper-based device and TUNEL assay well correlated with flow cytometry-based SCSA. Clinically, %DFI>30% is defined as the threshold for subfertility. With respect to clinical decision, the paper-based device and TUNEL assay provide 75% and 70% agreement with the flow cytometry-based SCSA, respectively. The paper-based device over-estimated DNA fragmentation from SCSA in 60% of the cases, while TUNEL assay under-estimate the %DFI in 80% of the cases.

Conclusions: Both the paper-based sperm DNA analysis device and TUNEL assay provide comparable functionality compared to the flow cytometry-based SCSA. The paper-based device measures sperm DNA fragmentation with two orders of magnitude reduced capital and operating costs as compared with SCSA. The paper-based device enables rapid, sensitive, and affordable sperm DNA integrity testing that competes directly with TUNEL assay and the gold standard method, SCSA.