Oral Communications Basic Science II

Kathryn Lye1 , Paula Mackie1 , Farwah Iqbal1,2, Peter Szaraz1,2, Shlomit Kenigsberg1 , Andrée Gauthier-Fisher1 , Clifford L. Librach1, 2, 3, 4, 5
1 CReATe Fertility Centre, 2 Department of Obstetrics and Gynecology, 3 Department of Physiology, 4 Institute of Medical Sciences, University of Toronto, 5 Department of Gynecology, Women’s College Hospital, Toronto, ON

Introduction: Several studies suggest that the regenerative activity of mesenchymal stem cells (MSCs) is predominantly mediated through paracrine effects, rather than direct differentiation.   It’s recently been shown that MSCs secrete extracellular vesicles (EVs) which could mediate much of these paracrine effects through intercellular transfer of their contents. Our objectives here were: to determine whether FTM HUCPVCs, a source of MSCs with superior regenerative potential, secrete EVs in vitro; to optimize a method for efficient isolation of EVs from conditioned media (CM); and to investigate the angiogenic properties of FTM HUCPVC-derived EVs.

Methods: EVs were isolated from CM of FTM HUCPVCs and fibroblast controls cultured in EV-depleted media by ultracentrifugation (UC) or by sucrose cushion UC. The presence, size and morphology of the FTM HUCPVC-derived EVs were determined by transmission electron microscopy (TEM). To visualize the uptake of EVs by target cells, rat endothelial progenitor cells (EPCs) were treated with FTM HUCPVC-derived EVs pre-labeled with PKH26 dye, and imaged using fluorescence microscopy.  To investigate paracrine angiogenic properties of FTM HUCPVCs in vitro, rat aortic rings were exposed to FTM HUCPVCs seeded onto transwell inserts for 4 days. The mean radial network growth and number of loops formed were quantified under bright field microscopy with ImageJ software.

Results: EVs were effectively isolated from FTM HUCPVC CM using UC, with or without, a sucrose cushion. UC with a sucrose cushion significantly reduced contaminating proteins in the EV fraction. From TEM, isolated EVs were 30 to 200nm, had a cup-shaped morphology, and included  CD9+ve and CD9-ve EVs. The uptake of PKH26-labeled EVs derived from both FTM HUCPVCs and fibroblasts was observed in EPCs.   Aortic rings treated with FTM HUCPVCs in a transwell system showed increased mean radial network growth (148.1 ± 18.8 vs 118.7 ± 24.2, P<0.05) and mean number of loops (190.4 ± 22.8 vs 90.1 ± 24.5, P<0.001) when compared to untreated networks.

Conclusions: FTM HUCPVCs secrete EVs in vitro. UC with sucrose cushion isolated the purest EV fraction of the methods tested.  FTM HUCPVC-derived EVs are uptaken by EPCs in culture.  FTM HUCPVCs display paracrine angiogenic properties.  Experiments utilizing purified FTM HUCPVC-derived EVs are in progress to determine whether they mediate some of these angiogenic and regenerative properties.

Kathleen Hammond
University of Cambridge, Cambridge, United Kingdom

Introduction: In Canada, there is little regulation and oversight of the care provided by fertility clinics and agencies to egg donors and intended parents in the practice of egg donation. Anecdotal reports on egg donation in Canada (e.g. Motluk, 2010) have highlighted troubling issues with the quality of care received by egg donors and intended parents from Canadian clinics and agencies. This paper provides data on: (i) those aspects of care that are most important to egg donors and intended parents in the course of donation, and (ii) egg donors’ and intended parents’ experiences with and evaluations of the care they received from Canadian fertility clinics and agencies.

Methods: Twenty intended parents and 16 egg donors participated in this study. All had received part, or all, of their treatment in Canada. All participants took part in in-depth semi-structured interviews regarding their experience with egg donation in Canada. Resulting data was analyzed through grounded theory and narrative summary analysis for re-occurring themes.

Results: The three aspects of care that most affected egg donors’ overall evaluation of care were: (i) how well clinic or agency staff informed them about donation, (ii) accessibility of the clinic or agency, and (iii) the physical care they received. Intended parents were most concerned with: (i) whether clinics and agencies followed through with promises, (ii) management of the egg donation process, (iii) their interactions with clinic and agency staff, and (iv) the physical care provided to egg donors. For egg donors, quality of care made the difference between them feeling like a “means to an end”, or the “important one.” For intended parents’ quality of care made the difference between them feeling as though they received “conveyor belt” or “compassionate” care. Nine egg donors and 10 intended parents reported poor overall care.

Conclusions: Through the care they provide, fertility clinic and agency staff play an important role in the physical and emotional experience of egg donation for egg donors and intended parents. There is the possibility of conflicts of interest on the part of fertility clinic and agency staff. The findings of this study suggest that the business interests of clinics and agencies might, at times, be interfering with the provision of high quality care. This study highlights the need for greater regulation and oversight of the care provided by fertility clinics and agencies in Canada. It highlights particular aspects of care that should be the focus of these initiatives.

References: Motluk, A., 2010. The Human Egg Trade: How Canada’s fertility laws are failing donors, doctors, and parents. The Walrus, pp.30–37.

Paula Mackie1, Simon Alfred1, Buddy Bao1, Melissa Filice1, Tigran Harmandayan1, Ekaterina Shlush1, Hanna Balakier1, Sergey Moskovtsev1,2, Clifford L. Librach1,2,3
1Create Fertility Centre, Toronto, ON; 2Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON; 3Department of Gynaecology, Women’s College Hospital, Toronto, ON

Introduction: Selection of the best embryo for implantation is a key issue with IVF.   Aneuploidy accounts for up to 70% of failed implantation or early pregnancy loss. Half of the embryonic genome is derived from sperm which are the product of several meiotic divisions and recombination events to form haploid cells from diploid progenitors. Errors during these events could lead to spermatozoa with a chromosomal imbalance. When ICSI is utilized, sperm selection is critical and traditionally based solely on morphology.  Preselection of optimal sperm subpopulations may also be useful for standard IVF.   FISH has been used to investigate sperm aneuploidy but is limited to a few select chromosomes. More recently, advanced techniques such as array CGH have been attempted but, unlike next generation sequencing (NGS), analysis is limited to chromosomal losses or gains. Here we have developed a method for whole genome analysis (WGA) of single sperm by NGS as a tool for identifying and analyzing sperm-selection markers. We recently observed reduced CD127 expression on the surface of sperm from asthenozoospermic patients. Our aim was to utilize NGS to determine if there is a relationship between CD127 expression and sperm aneuploidy.

Materials and Methods: Institutional REB approval was obtained. Excess semen donated from normozoospermic samples (n=2) was centrifuged through an 80% density gradient. Pelleted sperm was incubated with anti-CD127-PE and sorted by flow cytometry. DNA from CD127+/- sorted populations (n=20) was isolated and amplified using the SurePlex™ whole genome amplification kit, including a decondensation step using Proteinase K and DDT (1 mM). Libraries were constructed, sequenced, and alignment files analyzed in R using the CNAnorm package. Validation was performed using FISH (n=960 spermatozoa).

Results:  We successfully sequenced DNA from 40 spermatozoa. Aneuploidy rates were lower in the CD127+ (15%) compared to CD127- (35%) sperm but did not reach statistical significance, possibly due to the small sample size. FISH results on the same samples showed significantly (P<0.01) higher aneuploidy in the CD127- population for chromosomes analyzed (13, 18, 21, X/Y).

Conclusions:  Single sperm whole genome analysis by NGS appears to be an important method for validating putative markers of sperm quality (eg. CD127). These findings are vital to consider when attempting to develop improved methods of sperm selection for IVF or ICSI.

Jenna Haverfeld1,2, Nicola L Dean3, Diana Nöel3, Veronique Paradis3, Isaac Jacques Kadoch2,3, Greg FitzHarris1,2
1CHUM Research Centre, University of Montreal, Montreal, QC; 2Department of Obstetrics and Gynaecology, University of Montreal, Montreal, QC; 3Clinique de Procréation Assistée du CHUM, Montreal, QC

Human eggs feature high rates of aneuploidy, which is the underlying cause of age-associated infertility, spontaneous abortions and birth defects. The origin of egg aneuploidy is poorly understood. Here we use low-damage 4D long-term confocal imaging to examine chromosome dynamics during anaphase in human oocytes. To do this, germinal vesicle stage oocytes were collected, with appropriate consent, from patients undergoing standard intracytoplasmic sperm injection procedures, and microinjected with mRNA encoding for histone H2B tagged with red fluorescent protein (H2B-RFP). Chromosome position was recorded in real-time every 5 minutes from metaphase-I to metaphase-II. We find that the vast majority of oocytes (64.5%) feature the expected bi-directional anaphases, indicative of normal spindle structure and function. However many (45.1%) of these oocytes display chromosome segregation defects including persistent lagging chromosomes during anaphase, as recently noted in another study. Unexpectedly, the remaining oocytes (35.5%) featured three separate chromatin masses moving in appreciably different directions in anaphase, which we term tri-directional anaphases. Interestingly, in the majority (82%) of oocytes exhibiting tri-directional anaphases, two of the three chromosome masses remain inside the egg and reunite at metaphase-II, with only one of the three chromosome masses arriving in the polar body. By co-microinjecting oocytes with H2B-RFP and MAP7-GFP (to label spindle microtubules), we observed that the tri-directional divisions are attributable to transient formation of tripolar spindles specifically at anaphase. Tripolar anaphases featured extended anaphase durations (126 ± 6.5 mins) compared to oocytes with no segregation defects (57 ± 3.3 mins; P<0.01), however there was no difference in total meiosis-I duration. Our analysis thus far has revealed no correlation between the tri-directional anaphases and maternal age. We are currently performing experiments to directly test whether tripolar anaphases cause aneuploidy. We thus describe an anaphase-specific chromosome segregation defect as a mechanism that could contribute to the relatively frequent rates of aneuploidy in human eggs.

Shlomit Kenigsberg1, Isabelle Dufort4, Clifford Librach1,2,3, Marc-André Sirard4
1CReATe Fertility Centre; 2Departments of Obstetrics & Gynaecology and Physiology, University of Toronto, Toronto, ON; 3Department of Gynaecology, Women’s College Hospital, Toronto, ON; 4Département des Sciences Animales Pavillon des services, INAF, Université Laval, QC

  • Funding for this study was receive from the CFAS seed grant 2015

Introduction: The different compartments of the human ovarian follicle – the oocyte, granulosa cells (GCs) and cumulus cells (CCs) – communicate in part through the exchange of follicular fluid exosomes (FEs), which regulate their maturation during folliculogenesis. Our aim was to evaluate the regulatory effect of human FEs on gene expression of immortalized human granulosa cells in culture (KGN), in order to demonstrate the functional signaling capacity of these FEs and improve our understanding of their role(s) in follicular maturation.

Methods: This study was approved by U of T research ethical board. Patient’s Samples (n=10) were collected from young (age 25-35) fertile patients (male factor infertility) following ovum retrieval. Samples include FF and GCs from 1) lead/mature follicles (size >18mm) and 2) subordinate-immature follicles (size <15mm).  FEs were isolated from each individual follicle, and fluorescently tagged using the Syto RNASelect and DIL stain to allow tracking of both RNA and lipid materials when they are added to KGN cell cultures. Firstly, the uptake of FEs by KGN cells was assessed using confocal fluorescence microscopy to detect transfer of  FE-content to this cell type. Secondly, as a control to mimic the effects of FEs on KGN mRNAs, the cells were transfected (in triplicate) with human miR-21 mimic and inhibitor, which we found to be abundant in FF and in human FEs, and with negative Control siRNA. The Block-it fluorescent oligo was used to optimize the transfection protocol. Thirdly, KGN cells were exposed to pooled FEs from lead or subordinate follicles.  Using quantitative real-time PCR (qPCR), 5 candidate genes with specific seeds for miR-21 (TGFBR2, MARCK, PLAT, NCAPG, MEFC2) were assessed.

Results:  Positive interactions between FEs and KGN cells was obtained using lipid stained vesicles (DIL). Of the five candidate genes with specific seeds for hsa-miR-21 assessed, transfection of hsa-mir-21-5p miScript mimic in these cells resulted in a significant effect on the Non-SMC Condensin I Complex, Subunit G (NCAPG) mRNA after 24 hrs.

Conclusion:  This preliminary work indicates that follicular fluid vesicles are indeed incorporated in KGN cells in culture and therefore the KGN model could be used to assess the potential effect of follicular vesicles in the folliculogenesis process.

Megan Bowles, Sara Edsall, Jing Chen, Nadia Ouhibi, Salah Abdelgadir, Gary Nakhuda
Olive Fertility Centre, Vancouver, BC

Introduction: Multinucleation has previously been described as an indicator of reduced developmental competence and poor outcomes. It is possible that the presence of multinucleation is associated with a higher incidence of chromosome abnormalities, thereby explaining the previously described poor prognosis of such embryos. This study used time-lapse imaging to look at the prevalence of multinucleation in euploid and aneuploid embryos, as determined by comprehensive chromosomal screening (CCS), to establish whether this correlation exists.

Methods: CCS cases between March 2014 and March 2016 that had embryos cultured in the Embryoscope, were retrospectively analyzed for multinucleation at both the 2 and 4-cell stages. Trophectoderm biopsies were analyzed by Reprogenetics for 24 chromosome screening by aCGH or NGS, and then classified as aneuploid, euploid, or mosaic.

Results: Eighty one patients underwent a total of 86 CCS cycles where embryos were cultured in the Embryoscope. A total of 269 blastocysts were biopsied. Patients ranged in age from 26-44 years, with a mean of age 36.7.

Of the 269 biopsied embryos, 130 were euploid (48.3%), 111 were aneuploid (41.3%), and 28 were mosaic (10.4%).

One hundred and thirty of the 269 biopsied embryos (48.3%) exhibited multinucleation at the 2-cell stage whereas the remaining 139 (51.7%) did not. Twenty eight (10.4%) of the 269 biopsied embryos were multinucleated at the 4-cell stage, 23 (82.1%) of which also displayed multinucleation at the 2-cell stage. No multinucleation at the 4-cell stage was observed in the remaining 241 (89.6%) embryos.

It was shown that the presence of multinucleation at the 4-cell stage was correlated with multinucleation at the 2-cell stage (p=0.000). There was no statistically significant association between multinucleation at the 2 or 4-cell stage and CCS outcomes p=0.686 and p=0.298 respectively.

Discussion: This study showed that the presence of multinucleated blastomeres at the 2 or 4-cell stage is not a predictor of aneuploidy. CCS remains the gold standard for selecting embryos for transfer and as yet, multinucleation is not a tool for forecasting chromosomal abnormalities in blastocysts.