Friday Posters

Murid Javed, Abdulla Ahammed, Abdalla Salih, Hamoud Matraf, Hamad Sufayan
Thuriah Medical Centre, Riyadh, Saudi Arabia

Introduction: Various surgical techniques are available for sperm retrieval from men with severe male factor infertility.  Data on the successful utilization of such sperm is highly valuable especially for societies where donated sperm is not permitted. Such information will be helpful in counseling patients before deciding a surgical retrieval technique.  The purpose of this study was to analyze data from 5 surgical sperm retrieval techniques namely; TESA, TESE, Micro-TESE, PESA and MESA.

Methods: Men suffering from severe male factor infertility underwent surgical sperm retrieval either for diagnostic or therapeutic purpose at a very busy ART clinic providing full scale services to infertile couples.  Seven surgeons performed the procedures.  The technique was selected by the surgeon based on patient’s previous history and cause of azoospermia.  Data on duration of procedure, number of samples collected, quantity and quality of sperm, male and female partner’s age, sample cryopreservation, sperm utilization and various ART cycle parameters were recorded.

Results: From Jan to April 2016, 202 azoospermic men underwent any of the TESA, TESE, Micro-TESE, PESA or MESA procedure.  The purpose was either diagnostic or therapeutic. Sperm were found from 102 men (50.5%).  Mean age of the male partner from whom sperm were successfully retrieved was 38.8 ± 10.9 with a range of 25- 82 yrs.  From 102 positive samples, 52 couples utilized sperm.  Pregnancy results were available for 37 couples, from whom 15 (40%) achieved pregnancy.  Mean ± SD age of the female partner was 31 ± 6.5 with a range of 21-49 yrs.  A few selected study parameters are shown in Table 1.

Table 1: Dynamics of surgically retrieved sperm
Type Positive # (%)Negative (#)Cryo (#)Motility Seen (#)Used Fresh (#)
Diagnostic TESA13 (33)26420
Therapeutic TESA35 (95)22434
Diagnostic Micro-TESE34 (37)593090
Therapeutic Micro-TESE8 (80)2738
Diagnostic MESA2 (40)3220
Therapeutic MESA4 (100)0434
Therapeutic PESA1 (100)001
Diagnostic TESE5 (38)8520
Total102 ( 50)100542547

Conclusions: Sperm retrieved by any surgical procedure can successfully be used for ICSI.   Choice of the procedure dependents on the nature of infertility.  Male infertility is still a challenge as sperm could not be retrieved by any of the available surgical procedure in 50 % of men.

Pamela L. Kurjanowicz1, 2; Sergey Moskovtsev1, 3; Clifford Librach1, 2, 3, 4
1CReATe Fertility Centre, Toronto, ON; 2Department of Physiology, University of Toronto, Toronto, ON; 3Department of Obstetrics & Gynaecology, University of Toronto, Toronto, ON; 4Department of Gynaecology, Women’s College Hospital, Toronto, ON

Introduction: Improvement in our understanding of idiopathic infertility is an important avenue of research for the advancement of diagnosis and treatment of infertility. Telomeres stabilize chromosome ends and regulate cell division, and have been of interest to reproductive scientists as a potential explanation for male semen disorders and idiopathic infertility. Numerous publications on sperm telomere length (spTL) and clinical tests of male fertility and pregnancy outcomes have been published, but with conflicting results. Here, we conduct a systematic review and comparative analyses to determine the cause of this variability.

Methods: We conducted a review of multiple major databases. Our search strategy utilized a combination of subject headings and text-words to identify articles related to ‘Telomeres’ and ‘Male Infertility’. Final conclusions of relevant articles were subject to comparative analysis to determine the level of concordance, followed by specific comparison of sample size, fertility status and age of participants, semen fraction analyzed, DNA isolation method (when applicable), and telomere length (TL) assay implemented.

Results: The search returned 188 records, from which 15 articles with direct comparisons between spTL and male fertility potential (i.e. clinical tests of sperm health, fertility outcomes, and/or offspring health) were included. Major findings were summarized into 9 categories, and each investigation characterized as ‘in agreement’ or ‘disagreement’. There was a consensus (100% concordance) that spTL distributions demonstrate inter-individual variability, does not correlate with morphology, and intra-individual spTL is greater than somatic cell TL. Discordant findings (66.7%-75% concordance) included: longer spTL is associated with improved semen analysis parameters, and more prevalent in fertile men. Studies that compared spTL and DNA fragmentation were strongly divergent (50% concordance). Our second analysis revealed three major differences in study design: evaluation of different semen fractions, evaluation of different male populations with variable fertility status and age; and utilization of TL assays that differ in upstream DNA processing.

Conclusions: We have shown that important differences in study design and assay selection dramatically alter final results of the investigation. Overall, our work demonstrates that targeted study design is crucial to accurately address scientific questions. This concept can be effectively translated to all research in the reproductive sciences, which deals with high heterogeneity in both the patient population and cell types of interest, particularly in spermatozoa.

Valeriy Kuznyetsov1, Ran Antes1, Siamak Bashar1, Zenon Ibarrientos1, Gelareh Motamedi1, Agata Sojecki1, Svetlana Madjunkova1, Clifford Librach1,2,3,4
1CReATe Fertility Centre, Toronto, ON; 2Department of Obstetrics and Gynecology and 3Department of Physiology, University of Toronto, Toronto, ON; 4Department of Gynecology, Women’s College Hospital, Toronto, ON

Introduction: Embryo aneuploidy is the most significant contributor to IVF outcome. Oocyte maturation and meiotic errors underlie the high frequency of aneuploidies with advanced maternal age. Selection of euploid embryos by preimplantation genetic screening (PGS) has been proven to increase implantation and pregnancy rates and reduce miscarriage rates.  Oocyte donation from young donors is commonly utilized as part of ART for patients of advanced age, premature ovarian failure, and for same sex male couples. However, the pregnancy rates reported per cycle for egg donor cycles are typically in the 50-70% range, lower than one might expect.  This is an important consideration from an emotional, physical and financial point of view, especially when combined with the need for surrogacy.   The aneuploidy rate among embryos originating from donated oocytes is not well characterized and may be underestimated by clinicians. Our aim was to analyze our PGS aneuploidy data for this population.

Material & Methods: Embryos (n=353 )included in the study were generated by fertilization of fresh oocytes with ICSI from egg donors aged 22-26 years, attending the CReATe Fertility Centre, Toronto, Canada. PGS to detect whole and segmental aneuploidies was performed using the 24Sure™ aCGH kit (BlueGnome) in CReATe’s PGS/PGD laboratory.  In all cases, DNA was obtained from a 3-5 cell blastocyst trophectoderm biopsy on Day 5 or 6 of development. This study was approved by institutional REB.

Results: The overall rate of aneuploidy in this young group of donors was 25.8% (91/353; range 0-100%). There was no difference in the euploidy rate between those embryos reaching blastocyst on day 5 (183/248; 73.8%) or day 6 (79/105; 75.2%). Of the 91 aneuploid (whole and segmental aneuploidies) embryos, 59 (64.8%) embryos had whole chromosome aneuploidy and 72.9% (43/59) of these were of a single chromosome. The rest (n=32) had double aneuploidies, complex aneuploidies and/or segmental aneuploidies.  Of the single chromosome aneuploid embryos, 41.9% (18/43) were trisomies and 58.1% were monosomies.

Conclusions: The aneuploidy rate in embryos derived from young egg donors was approximately 26%.  Therefore, aneuploidy screening in egg donor cases has the potential to increase per cycle implantation and reduce miscarriage rates.  As well, it would be expected to reduce the time to pregnancy, an important emotional, physical and financial consideration for ovum recipients.

Andrea Lanes1,2, Shelley Dougan1, Ann Sprague1, Moya Johnson1, Art Leader3, Mark Walker4
1BORN Ontario, Ottawa, ON; 2University of Ottawa,Ottawa, ON; 3Ottawa Fertility Centre, Ottawa, ON; The
Ottawa Hospital, Ottawa, ON

Introduction: Multiple marker prenatal screening uses maternal serum markers combined with nuchal translucency measurement (an ultrasound marker) to estimate risk of Down syndrome or a number of other aneuploidies. This algorithm incorporates corrections for ethnicity, smoking, diabetes and in vitro fertilization (IVF) for specific maternal serum screening markers. In Ontario, only PAPP-A, total hCG (2nd trimester), and µE3 have an IVF correction factor applied. The objective of this study was to investigate the accuracy of IVF identification on the prenatal screening record (sourced from the prenatal screening requisition) and to compare the screening markers in IVF and non-IVF pregnancies in the population of Ontario.

Material and methods: This was a population-based cohort analysis using BORN (Better Outcomes Registry & Network) Ontario and CARTR (Canadian Assisted Reproductive Technologies Register) Plus data from January 2013 through December 2014 in Ontario. We compared AFP, PAPP-A, free-βhCG, total hCG (1st trimester), total hCG (2nd trimester), DIA and µE3 among IVF and non-IVF pregnancies using linear regression.

Results: When IVF status identification from the prenatal screening record was compared to the gold standard for fertility treatment in Canada (the CARTR Plus database), the sensitivity was 92.5% and the specificity was 99.3%. When IVF status was identified using information on the prenatal screening record, maternal serum screening markers were significantly higher among IVF pregnancies for 1st trimester total hCG (MoM) (β=0.41; 95%CI: 0.18–0.64) and DIA (MoM) (β=0.26; 95%CI: 0.17–0.34). When CARTR Plus was used to identify IVF status, the most substantial differences were found for DIA (MoM) (β=0.18; 95%CI: 0.09–0.28) and AFP (MoM) (β=0.15; 95%CI: 0.13–0.18).

Conclusions: The maternal serum screening markers with the largest differences did not have correction factors for IVF treatment applied and would benefit from more accurate multiples of the median. Additionally, identification of conception method would be more appropriate for implementing correction factors if ascertained from CARTR Plus.

Maria Teresita Lao, David Peng, Essam Michael, Alex C Varghese
Astra Fertility Clinic, Mississauga, ON

Introduction: Approximately 15% of the couples worldwide have fertility problems with a male factor found in 45% of the cases. Multiple factors are known to impair spermatogenesis: exposure to environmental disruptors, genetics, testis pathologies, lifestyle etc. It has recently been shown that transcripts levels in spermatozoa are altered in defective spermatogenesis cases like oligozoospermia1 and teratozoospermia.  Another study shows significantly higher frequencies of chromosomes 13, 18, 21 disomies in the group of patients with moderate and severe oligozoospermia compared with the disomy frequencies of normozoospermic group2. The objective of the present study was to evaluate the effect of sperm concentration on embryo development, blastulation rate and pregnancy outcome.

Materials and methods: A total of 185 couples (female age < 36 years) undergoing ICSI for the first time between the years 2013 and 2015 at Astra Fertility Clinic were included in this retrospective study.  We did not include poor ovarian response patients and those who had less than 4 MII oocytes on egg retrieval.  Fertilization and cleavage rates, quality of embryos, blastulation rate, were analyzed in 2133 MII oocytes undergoing first ICSI treatment cycle. The couples were allocated in five groups, according to semen parameters: Group 1: patients with normal semen parameters (sperm concentration ≥16×106/mL, motility ≥50% [types a and b, according to the WHO criteria ), which served as the control group, Group 2: patients with mild oligozoospermia (sperm concentration 5-15×106 /mL), Group 3: patients with moderate oligozoospermia (sperm concentration 1-

Results:  The study did not find any significant difference in fertilization, cleavage rate and Day-2 and Day-3 embryo quality between the control (normozoospermic) and other sperm groups. However the blastocyst development rate on Day 5 was reduced in groups 2-5 compared to the normozoospermics.  Moreover Day- 6 blastulation rate were significantly more in group 2-5 compared to the control group. There were no significant difference in implantation rate between groups 1, 2, 3 and 5. However group 4 (severe oligozoospermia) presented with less implantation rate compared to other groups.

Conclusion: A negative relationship was observed between sperm concentration and the blastulation rate and this was more exemplified in the implantation rate of group with severe oligozoospermia. The study emphasizes the possible impact of molecular defects as the sperm concentration deteriorates and its impact on the blasulation rate as the zygote genome starts activation after the Day 3 of the embryo development in the mammalian systems. This preliminary analysis warrant more rigorous and well controlled prospective studies looking at sperm severity and pregnancy outcome.

1 Montjean D, De La Grange P, Gentien D, et al. Sperm transcriptome profiling in oligozoospermia. Journal of Assisted Reproduction and Genetics. 2012;29(1):3-10.

2 Durak ArasB et al,. Exploring the relationship between the severity of oligozoospermia and the frequencies of sperm chromosome aneuploidies. Andrologia. 2012 Dec;44(6):416-22

Cécile Le Saint, Laurie Vingataramin, Sabrina Alix, Simon Phillips, Stéphanie Belloc, Armand Zini, Isaac-Jacques Kadoch
Ovo Fertility, Montreal, QC

Introduction: Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa using a number of different assays. The two commonly used tests to measure sperm DNA damage are the sperm chromatin structure assay (SCSA) and the TUNEL assay. However, little is known about the correlation between assays and the inter-lab variability of these assays. We sought to validate the TUNEL assay, examine the correlation between sperm chromatin structure assay (SCSA) and the TUNEL assay and the inter-lab variability of the TUNEL assay.

Methods: Semen samples from 38 men referred to a male infertility clinic (OVO clinic) were tested for sperm DNA fragmentation by TUNEL assay using the Apo-Direct Kit with a bench top flow cytometer Accuri C6 (BD Pharmingen, CA, USA). These samples were freshly fixed before the TUNEL assay analysis. We determined the percentage of positive cells for the TUNEL assay. We proceeded to an analytical validation for clinical use. Samples were also analyzed by a French laboratory Eylau-Unilabs Laboratory which uses the TUNEL assay: In situ Cell Death Detection Kit-Fluorescein (Roche Diagnostic Gmbh, Penzberg, Germany) with a flow cytometer FC500 (Beckman Coulter, Villepinte, France), and by the andrology laboratory of the Royal Victoria Hospital which uses the SCSA test. Statistical analysis was performed using the GraphPad Prism software, version 5.04, La Jolla, California USA.

Results: Correlation between the three techniques was assessed, and good correlation was found between the TUNEL assay carried out at ovo Labo and Eylau Laboratory (R2 = 0.7325, p<0.0001). We also observed a good correlation between the TUNEL assay carried out at ovo labo and the SCSA assay (R2 = 0.7137, p<0.0001).

Conclusion: We observed a strong correlation between the results of the sperm chromatin structure assay (SCSA) and the TUNEL assay. Moreover, our data indicate that the TUNEL assay exhibits a low inter-laboratory variability.

Yiwen Liu1, Crystal Chan2,3
1University of Toronto Faculty of Medicine, Toronto, ON; 2Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON; 3Centre for Fertility and Reproductive Health, Mount Sinai Hospital, Toronto, ON

Introduction: Intrauterine insemination (IUI) is the first-line treatment for most couples with infertility, for which injectable gonadotropins are often chosen as adjunctive stimulation. Estrogen produced by growing follicles induces endometrial proliferation, leading to increased thickness of the endometrial stripe. The association between endometrial thickness and pregnancy outcome in gonadotropin stimulated IUI is not well studied. The only study involving patients using only gonadotrophins featured an uncharacteristically low pregnancy rate of 4.7%1. Using an arbitrary cut-off of 8mm, endometrial thickness was significantly associated with higher pregnancy rates using univariate analysis only (P=.020). The objective of the current study is to determine if endometrial thickness can predict pregnancy outcome in gonadotropin-stimulated IUI cycles.

Materials & Methods: A retrospective review of all gonadotropin-stimulated IUI cycles completed at Mount Sinai Hospital’s Centre for Fertility and Reproductive Health between in 2014 and 2015 (N= 658) was conducted. Data was collected on patient age, BMI, infertility diagnosis, number of antral follicles, anti-Mullerian hormone level, number of dominant follicles (≥1.5mm) and corresponding peak estrogen level, number of motile sperm, and peak endometrial thickness measured within 48 hours of hCG trigger. A positive beta-hCG 2 weeks after IUI indicated a conception, which included chemical pregnancies and early pregnancy loss. Negative outcomes included negative beta-hCG values as well as patient-reported bleeds. Mean endometrial thickness was compared in conception vs. non-conception cycles. Pregnancy rates were compared between cycles with endometrial thickness <6mm, 6-9mm, and >9mm.

Results: Cycles missing values for endometrial thickness (n=61) or pregnancy outcome (n=107) were excluded, resulting in a total of 490 cycles of which 100 resulted in a clinical pregnancy (20.4%). The mean endometrial thickness among conception cycles was 9.50mm (SD 7.64–11.3mm). No conception outcomes were observed below 5.2mm or above 14mm. Among 390 negative cycles, endometrial thickness ranged from 5mm to 17mm, with a mean of 9.15mm (SD 7.30–11.01mm). The mean endometrial thickness between these two groups was not significantly different (P=.100). Among endometrial thicknesses below 6mm, only 1 pregnancy was reported out of 5 completed cycles. For cycles between 6-9mm inclusive, 45 resulted in pregnancy (16.5%), while those above 9mm had 54 conceptions out of 213 cycles (24.5%).

Discussion: Chart review is ongoing, and retrieval of missing data will allow for further analysis. Threshold values of endometrial thickness for gonadotropin cycles and significantly associated factors will be determined and used to guide management of IUI, including recommendations for cancellation.

References:

 1Yavuz, A., Demirci, O., Sözen, H., & Uludoğan, M. (2013). Predictive factors influencing pregnancy rates after intrauterine insemination. Iran J Reprod Med, 11 (3), 227-234.

Carson K. Lo1,2, V. Logan Kennedy2, Mark H. Yudin3, Heather M. Shapiro4, Mona Loutfy1,2
1Department of Medicine, University of Toronto, Toronto, ON; 2Women’s College Research Institute, Women’s College Hospital, Toronto, ON; 3St. Michael’s Hospital, Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON; 4Mount Sinai Hospital, Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON

Introduction/Objective: Since the introduction of combination antiretroviral therapy, patients with HIV have successfully kept their viral loads to an undetectable level and reduced mortality and morbidity.  With this, pregnancy planning has become an important issue for HIV-positive individuals and couples.  In 2007, a study surveying Canadian fertility clinics found a lack of access to fertility services for HIV-positive patients.  Given the extensive efforts made to address this lack of services, a follow up assessment was warranted.  The purpose of this study is to compare access to Canadian fertility clinics and services for HIV-positive individuals and couples in 2014 to access in 2007.

Materials & Methods: Surveys were sent to medical or laboratory directors of assisted reproductive technology (ART) clinics registered under the Canadian Fertility and Andrology Society in 2014.  Main outcome measures include: the proportion of fertility clinics in Canada willing to provide ART to HIV-positive individuals and couples in 2014 was compared to 2007; the specific services offered and whether the Canadian HIV Pregnancy Planning Guidelines were implemented to inform practice were also assessed.

Results: Across Canadian provinces, 20/34 (59%) clinics completed the survey.  95% (19/20) of clinics accepted HIV-positive patients for consultation, but only 45% (9/20) offered a full range of ART. This was also isolated to four provinces.  Ten clinics (50%) in five provinces were aware that guidelines exist; half (n=5) having read them and four reported that they had implemented all of the guidelines in their practice.  Compared to 2007, more clinics had implemented separate facilities (P=0.028) to treat HIV-positive individuals, offered IVF (P=0.013) for HIV-positive female partners, and risk reduction techniques to couples with HIV-positive men and women (P=0.006).

Conclusions: Access to fertility clinics for people with HIV has improved over time but is still regionally dependent and access to full ART is still limited.  These findings suggest the need for further advocacy targeted towards certain geographical areas and optimizing access to comprehensive services.

Kathryn Lye1, Paula Mackie1, Farwah Iqbal1,2, Peter Szaraz1,2, Shlomit Kenigsberg1, Andrée Gauthier-Fisher1,
Clifford L. Librach
1, 2, 3, 4, 5
1CReATe Fertility Centre, Toronto, ON; 2Department of Obstetrics and Gynaecology, 3Department of Physiology; 4Institute of Medical Sciences, University of Toronto, Toronto, ON; 5Department 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.

Svetlana Madjunkova1, Ran Antes1, Valeriy Kuznyetsov1, Clifford Librach1, 2, 3, 4
1The CReATe Fertility Centre, Toronto, Canada; 2Department of Obstetrics and Gynecology and 3Department of Physiology, University of Toronto, Toronto, Canada; 4Department of Gynecology, Women’s College Hospital, Toronto, ON

Introduction: Next-generation sequencing (NGS) is an emerging new methodology for preimplantation genetic screening (PGS) of human embryos. Array comparative genomic hybridization (aCGH) is presently considered the gold standard.  However, chromosomal copy number assessment based on NGS may offer several advantages to aCGH including: enhanced detection of partial or segmental aneuploidies, increased dynamic range enabling enhanced detection of mosaicism in multi cellular samples, reduced cost per sample, and shorter turnaround time, coupled with the potential for automated DNA library preparation. We aimed to validate the NGS VeriSeq kit for PGS by comparing it to aCGH in a clinical setting.

Material & Methods: This study had institutional REB approval. 100 human trophectoderm (TE) biopsy samples were analyzed. These samples were selected from prior known aCGH results to represent a variety of findings: single aneuploidy (n=34), complex aneuploidy (≥2 chromosomes n=34), segmental aneuploidy (n=23), and euploidy (n=10). The same WGA performed on 2-5 cell day-5/6 TE biopsy was assessed with VeriSeq™ PGS kit for NGS on the MiSeq system (Illumina) and 24Sure™ aCGH kit (BlueGnome). Results from the NGS platform were analyzed blindly by two experienced geneticists and then compared to the previously reported aCGH results. The analysts were required to identify the chromosomal abnormality, mark the time to call each aberration, and number of repeated analyses of the affected region.

Results: NGS correctly detected all chromosomal abnormalities previously identified with aCGH, including numerical and structural (partial deletion and/or duplication) in all samples.  The reported results were identical from the two blinded analysts.  This represented a sensitivity and specificity of 100% for whole chromosome aneuploidy, del/dup ≥10Mb, and mosaicism ≥50%. However, unlike aCGH, the NGS platform was also able to identify segmental aberrations in the presence of ≤50% mosaicism. No embryos identified as euploid by aCGH were found to be abnormal by NGS.  NGS data was less time consuming to analyze than the aCGH (35.5±9.6min vs 40±10.8min, respectively), possibly due to higher dynamic range of the output data.

Conclusions: NGS appears to be a robust high-throughput methodology for human embryo PGS, with the potential advantages of reduced costs and greater precision. Given its reliability and high level of consistency with aCGH, NGS appears to be an accurate, reproducible and reliable PGS methodology for clinical use.

Leila Maghen1, German T. Videna1, Mahta R. Ghaffarzadeh1, Thomas G. Hannam2, William B. Schoolcraft3, Jason E. Swain4, Alexander Lagunov1
1CCRM Toronto, Toronto, ON; 2Hannam Fertility Centre, Toronto, ON; 3Colorado Centre for Reproductive Medicine, Lone Tree, CO, USA; 4CCRM IVF Network, Englewood, CO, USA

Introduction: Human sperm DNA integrity plays an important role in the paternal contribution to fertilization and subsequent embryo development.   The sperm chromatin structure assay (SCSA) and the Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) are the most common semi-quantitative DNA integrity tests. However, they can be prohibitive; as they require expensive equipment and are time-involved. Therefore, not all labs are able perform the assays “in house”. The sperm Hyal  uronan Binding Assay (HBA®) is a commercially available quantitative test to assess a percentage of bound or “mature” sperm. The binding score has been correlated with sperm euploidy and positive pregnancy outcomes.  The HBA® assay can be easily performed and has an adjunct method to select individual Hyaluronan (HA)-bound sperm for use during ICSI; something not feasible with current DNA fragmentation approaches.  Though a previous study has correlated DNA integrity with individual HA-bound sperm, the HBA index correlation with sperm DNA integrity remains unknown.

Method: A total of 20 semen samples were collected via masturbation. All samples were split and analyzed using the HBA® slides (Origio) and also assessed for DNA fragmentation using SCSA or TUNEL assay.  A minimum of 200 sperm were counted for both HBA® score and TUNEL assay and SCSA was performed on 5000 sperm. HBA® scores are presented as the percentage of sperm bound and DNA fragmentation assays are presented as percentage of sperm with DNA fragmentation. Regression analysis was performed to determine if any correlation was present between DNA fragmentation and HBA® binding score.

Results: Patients ranged from 19-49 years of age (37.2±6.7). SCSA values ranged from 7.8% to 37.3% while TUNEL values ranged from 5.8% to 50.6% (DFI ≥30%=abnormal).   HBA® scores ranged from 77% to 98% (<80% = abnormal). This study did not find a statistical correlation, between HBA® score and DNA fragmentation value (R2=0.057).

Conclusion: Despite a prior study showing a correlation between DNA damage and individual HA bound sperm, our results did not show any correlation between HBA® binding score and sperm DNA fragmentation. This may be due to alternate methods used to assess sperm DNA integrity between studies or the different patient population examined.  Use of sperm DNA fragmentation assessment may still provide useful information in conjunction with the HBA assay®.

Leila Maghen1, Mahta R. Ghaffarzadeh1, German T. Videna1, Thomas G. Hannam2, William B. Schoolcraft3,
Jason E. Swain
4, Alexander Lagunov1
1CCRM Toronto, Toronto, ON; 2Hannam Fertility Centre, Toronto, ON; 3Colorado Centre for Reproductive Medicine, Lone Tree, CO, USA; 4CCRM IVF Network, Englewood, CO,USA

Introduction: Intracytoplasmic sperm Injection (ICSI) is a common procedure used to address male-factor infertility. In conventional ICSI, sperm are visually selected according to their morphology and motility. However, sperm selected using this approach can still have damaged DNA and/or chromosomal aberrations and significantly impact fertilization and embryo development. Hyaluronan-binding offers another means of sperm selection for ICSI, with bound sperm reported to have higher DNA integrity and normal chromosomal complement compared to unbound sperm. The HBA® slide offers a means to assess the percentage of the sperm population that can bind hyaluronan, while the pICSI® procedure permits use of hyaluronan bound sperm for ICSI.  In this study we assessed HBA score in patients to direct type of sperm injection and compared outcomes of cycles using pICSI® bound sperm with conventional ICSI.

Method: Semen samples were obtained from 30 men undergoing fertility treatment.  Patients were separated into two groups (ICSI group=20 patients, 156 MII oocytes; pICSI group=10 patients, 42 MII oocytes) based on HBA results. The two groups were assessed for fertilization rate, usable blastocyst rates (>3BB) and total blastocyst rates, patients with HBA<80% received pICSI®. After swim up, sperm aliquots were placed into normal ICSI dishes per protocol or onto pICSI® dishes according to manufacturer’s instructions. Sperm were selected based on morphology/motility in the ICSI group under 400x and pICSI® bound sperm were obtained and then selected in a similar manner.   Data are presented as mean ± SEM. Statistical analysis was performed using Student’s t-test.

Results: Mean female age in ICSI and pICSI® groups were 35.5±3 and 33±4.3yrs, respectively. Average number of injected eggs was 8.2±5.5. There was no significant difference in fertilization rate, D5 usable blastocyst, D5/6 usable blastocyst and total blastocyst formation rate between the groups.

ICSI grouppICSI®P value
HBA score90.3±1.1857.2±10.7
Fertilization rate (%)82.6 ±0.0371.5 ± 0.07p<0.2
D5 usable blast rate (%)25.6 ±0.0737.8 ± 0.12P<0.4
D5/6 usable blast rate (%)53.3 ± 0.0747.8 ± 0.12P<0.7
Total blast (%)71.8 ± 0.0670 ± 0.03P<0.8

 

Conclusion: Use of pICSI® in cases with poor HBA sperm binding yields fertilization and blastocyst conversion rates comparable to patients with normal HBA scores undergoing conventional ICSI. pICSI® may be useful to improve laboratory outcomes in cases with low sperm HBA binding scores. Subsequent studies using oocyte splits with/without pICSI® will give further insight into the usefulness of this sperm selection method.

Lyne Massicotte, Mathieu Boilard
Nasci Biologie Médicale Inc., Longueuil, QC

Introduction: The standard method to evaluate the presence or absence of spermatozoa (spz) in an ejaculate is to observe a small fraction of the ejaculate under the microscope. Nonetheless, the fraction of the ejaculate that is evaluated remains very small (approximately 1µl). Therefore, in the best conditions the functional sensitivity of the test is 56 000 spz/ml according to the WHO manual for semen evaluation. A new flow cytometry methodology was developped to obtain better laboratory performance at detecting rare spz and characterizing them.

Materials and methods: Briefly, 100µl of sperm is diluted and incubated for 15 minutes at 37°C in 900ml of PBS already containing Hoechst (DNA detection) to help discriminate spz from white blood cells (wbc), other cell types and debris and DIOC6 (mitochonrial transmembrane potential) to assess their functional state. Samples are then analyzed by flow cytometry.

Results: Limit of quantification for spz concentration was 200 spz/ml and was 5000 wbc/ml for wbc.  Linearity for spz concentration ranges from 1×103 to 1×106 spz/ml (R2>0,99) and from 1×104 to 1×106 wbc/ml (R2>0,99) for wbc. Compared to motility, viability can be detected for 5h. Duplicates have produced less than 20% variation although linearity range. This variation was caused by the extreme rarity of the cells. A sample was obtained from a man diagnosed with azoospermia. Sperm concentration was measured by flow cytometry at 3400 spz/ml and the concentration of DIOC6-positive spermatozoa was 1500/ml. An extensive research of motile spermatozoa was then conducted under the microscope and 1 motile spz was finally visualized and filmed after 45 minutes. After being informed of the results, the fertility clinic attempted ICSI directly from an ejaculate which led to the birth of a healthy baby boy nine months later.

Conclusions: This case shows that no man should be diagnosed azoospermic unless the presently described protocol is performed. The present report also shows that PESA or TESA or the use of donor semen might not always be necessary for those patients ejaculating not enough spermatozoa to be detected during a routine microscopic semen evaluation.

Adel R. Moawad1,2, Burak Ozkosem1,2, Alex Yu1,2, Cristian O’Flaherty1,2,3
1Urology Research Laboratory, 2Research Institute of the McGill University Health Centre, Department of Surgery (Urology Division); 3McGill University, Department of Pharmacology and Therapeutics; McGill University, Montreal, QC

Introduction: Oxidative stress, the imbalance between the production of reactive oxygen species (ROS) and antioxidant activity is a major cause of male infertility. Peroxiredoxins (PRDXs) are major antioxidant enzymes of mammalian spermatozoa1. Their deficiency and/or inactivation have been linked with male infertility. It has been reported that infertile men have lower amounts of PRDXs in seminal plasma and spermatozoa than healthy donors2. Spermatozoa of Prdx6-/- male mice showed high degree of abnormality and subsequent low fertility3. Previous results suggested that inhibitors of PRDXs prevented human sperm capacitation4. Our objective was to evaluate whether the inhibition of PRDXs activity has an impact on sperm functions and subsequent fertilizing competence in mice.

Methods: CD1 mice spermatozoa were incubated in BWW medium with MJ33 (specific PRDX6 PLA2 activity inhibitor), or thiostrepton (TSP; 2-Cys PRDX peroxidase activity inhibitor) for 2h at 37°C. Total and rapid progressive motility and velocity parameters were measured by CASA. Capacitated spermatozoa were incubated in vitro with in vivo matured oocytes for 5h; the presumptive zygotes were then cultured in vitro for 4 days. Cleavage (24h post insemination (pi)) and blastocyst development (4 days pi) were evaluated. A series of experiments were also conducted to investigate the role of PRDX6 in sperm quality and fertilizing ability. Cholesterol efflux (using Amplex Red assay), zona binding (ZP), in vitro fertilization rates and embryo development were evaluated in Prdx6-/- and WT mice spermatozoa.

Results: TSP and MJ33 decreased sperm total motility and rapid progressive forward motility in a dose dependent manner and the lowest values were observed at 80 and 160 µM (p<0.05). Fertilization rates were lower when spermatozoa treated with MJ33 or TSP at 20 µM compared with control (p<0.05). Blastocyst development was dramatically decreased in TSP treated groups than controls (p<0.05). Percentages of blastocyst were also lower (p<0.05) in MJ33 treated groups than in controls. Sperm cholesterol efflux, ZP binding, preimplantation development rates were significantly lower in Prdx6-/- than in WT (P< 0.05).

Conclusions: Inhibition of PRDXs is associated with abnormal sperm function and compromised fertilizing ability. These studies suggest an essential role of sperm PRDXs to assure normal fertility in mice.  Funded by CIHR

1O’Flaherty C, de Souza A. 2011. Biol Reprod 84:238–247
2Gong S, San Gabriel M, Zini A, Chan P, O’Flaherty C. 2012. J Androl 33:1342–1351
3Ozkosem B, Feinstein S, Fisher A, O’Flaherty C. 2015. Biol Reprod 94:68:1–10
4O’Flaherty C. 2015. Asian Journal of Andrology 17: 583–590

Diane Myles Reid, Matthew Vlasschaert, Pat ChronisBrown, Ellen Greenblatt, Elena Kolomietz
Mount Sinai Hospital, Toronto, ON

Preimplantation genetic screening with comprehensive chromosomal screening (PGS-CCS) and subsequent selection and transfer of euploid embryos has been shown to improve clinical implantation rates and sustained implantation rates, particularly in patients with normal ovarian reserve. The ideal patient population who would most benefit from the application of PGS-CCS is still unclear. Minimal data is available for different patient populations, including those with a low ovarian reserve or poor prognosis for other reasons.

This is a retrospective observational study including the data from analysis of PGS-CCS results from February 2012-April 2016 for 993 blastocysts from 191 IVF cycles completed in 155 women . PGS-CCS was offered to infertile poor prognosis patients/couples defined as a history of multiple previous implantation failures in IVF, recurrent pregnancy loss, advanced maternal age (> 40 years) or an altered karyotype in one member of the couple .

Of all embryos in which DNA amplified and samples could be analyzed, 36.7.% were euploid and 63.3% were aneuploid, including segmental aneuploidies. Of the aneuploid embryos, 11.3% involved segmental aneuploidy, 26.9% were aneuploid for one chromosome and 12.2% were aneuploid for two chromosomes . In women >40 years of age, 82.0% of embryos were aneuploid. 24.4% of all IVF cycles did not have any euploid embryos for transfer and 29.5% of cycles had only one euploid embryo. Aneuploidy was detected for all 24 chromosomes and both monosomy and trisomy was detected for each of the 24 chromosomes.

This single center data, where all biopsies, procedures and PGS/PGD testing were performed on site, serves to further characterize aneuploidy in embryos of poor prognosis patients and allow for counselling that would include reasonable expectations.

Gary Nakhuda1, Rennie Chong-Kit2, Chris Willson2, Patti Freeman2
1Olive Fertility Centre, Vancouver, BC; 2Novavita Laboratory, Vancouver, BC

Introduction: AMH helps estimate ovarian reserve. Since initial commercial availability in 2002, the AMH assay has evolved from a plate-based ELISA to automated methods. Several factors determine the accuracy of results with any given method, and values produced by different techniques may not be directly comparable. We transitioned from an ELISA based method (Reprosource (RS)) to an automated, chemiluminescent system (Beckman Access2 (BA)). Retrospective comparison was performed to assess the clinical comparability of the 2 methods.

Methods: Regression analysis was performed to validate correlation between paired samples. Twenty-nine samples were collected in serum separator tubes, clotted for 45 minutes, aliquoted equally, then frozen. For each sample, 1 aliquot was shipped to Reprosource (Wodburn, MA) and analyzed with a laboratory developed ELISA validated with CLIA compliance; the other was thawed and processed on the Beckman Access2 at an accredited local laboratory (Novavita, Vancouver).

Subsequently, retrospective analysis was performed 8 months after switching methods, comparing cohorts tested by either technique. RS samples (n=1005) collected between December 2013- March 2015 were compared to BA samples (n=646) collected between April-December 2015.

Results: Paired samples were analyzed with Passing-Bablok regression and Bland-Altman plot, showing statistical correlation between methods (r=0.993, p<.001, 95%CI (0.9848 – 0.9967)), with the regression formula as follows:  BA= 0.0233447  +  1.322958 RS.

For retrospective cohort analysis, median AMH values were determined and age stratified for 2 sample populations. As predicted by the regression formula, BA levels were higher when stratified for age. Analyzed by Mann-Whitney-U, differences were statically significant for some age groups, but not all.

Conclusions: While excellent correlation was demonstrated between the 2 methods, the absolute values of BA results were consistently higher compared to results previously attained by the RS assay. When referenced to RS age-stratified values, respective BA results would appear higher, thus misinterpreted as suggestive of a greater ovarian reserve, potentially falsely reassuring patients. Therefore, when using different methods to analyze AMH, it is crucial to use reference values specific to the given assay, rather than historical reference ranges derived from a different method.

Gary Nakhuda, Elizabeth Taylor, Jason Hitkari, Albert Yuzpe, Gunu Warraich
Olive Fertility Centre, Vancouver, BC

Introduction: The endometrial receptivity array (ERA) was introduced as a diagnostic tool in cases of recurrent implantation failure (IF). By profiling the transcriptome of 238 genes, endometrial receptivity can be purportedly characterized. We began to offer ERA primarily to patients who had IF of euploid blastocysts. Here we report our initial experience with the ERA.

Methods: A retrospective review of ERA results was performed. The protocol used for endometrial preparation for the ERA mimicked that which was used in the previous failed frozen embryo transfer cycle. Generally, oral estradiol was escalated from 2-6mg in 5 day increments. If the endometrium appeared trilaminar and >8mm, vaginal progesterone (Endometrin) was administered TID for 5 full days (P+5) before Pipelle biopsy. Specimens were processed and shipped according to manufacturer’s instructions. Repeat biopsy cycles were performed as indicated by ERA results. A descriptive review of cases and outcomes is presented.

Results: From October 2014 – March 2016, 20 patients proceeded with ERA. Average patient age was 37.8 (range 28-48). Indications were as follows: Failed implantation of euploid blastocyst (1 failure, n=4; 2 failures, n=5; 3 failures, n= 2; 4 failures, n= 2); morphologically normal blastocysts (non-PGS) (1 failure, n=1; 2 failures, n=2, 6 failures, n=1); donor-blastocyst (2 failures, n=1); pre-FET in patients with only 1 euploid blastocyst (n=2).

Thirteen patients had initial ERA results that were receptive, including 2 patients who requested the ERA but had no history of implantation failure. Of the 7 biopsies that returned as non-receptive, all indicated a pre-receptivity. One indicated a displaced receptivity window by 12 hours, while 6 required a repeat ERA biopsy cycle for diagnosis. Five indicated receptivity on P+6; 1 indicated receptivity after P+7.

Six patients with pre-receptive results had subsequent transfers after protocol adjustment according to ERA. All 6 achieved clinical pregnancies, although 1 experienced a spontaneous miscarriage at 9 weeks.

Discussion: Our initial experience demonstrated that 65% (13/20) of patients were determined to have a receptive endometrium according to the ERA. Thirty-five percent (7/20) demonstrated a non-receptive endometrium, all of whom displayed a pre-receptive pattern. In patients who returned for transfer after adjusting progesterone exposure, all achieved pregnancy on the subsequent cycle.

While the data is limited in this case series, the ERA may be a promising technique to help characterize receptivity issues and provide actionable directives to improve implantation in instances of previous failure, perhaps most cogently in cases of failed euploid transfer.

Gabriela Pelinska1, Jeff Roberts2
1University of Alberta, Edmonton, AB; 2Pacifc Centre for Reproductive Medicine, Burnaby, BC

Introduction: Embryo/oocyte cryopreservation allows women undergoing gonadotoxic chemotherapy and radiation therapy, or surgical resection of reproductive organs the possibility of having a biologic child. Letrozole has been utilized during ovarian stimulation to lower estradiol levels, particularly in women with estrogen dependent cancers. We analyzed the fertility preservation program at a medium-sized IVF facility in Western Canada to assess a novel approach with high-dose letrozole for chemoprotection.

Methods: A retrospective analysis of female cancer patients who had undergone ovarian stimulation for embryo/oocyte cryopreservation from February 2007 to December 2014 was completed. Fifty-three patients (age range, 26 to 40 years, average age: 32 years) were analyzed. Patients received high dose letrozole (7.5mg) during gonadotropin stimulation. The efficacy of this regimen was assessed by duration of stimulation, gonadotropin dosage, peak estradiol levels and number of embryos/oocytes cryopreserved.

Results: Breast cancer was the most common indication for fertility preservation (81%), with lymphoma and cervical cancer being less common (6% each).  The average Day 3 FSH and Antral follicle count were 5.9 IU/L and 15.4, respectively.  Average total days of stimulation and gonadotropin dosage were 9.8 days and 2690 units. Peak estradiol levels were 2340 pmol/L. Mean time from consult to oocyte retrieval was 35.8 days. Average total and mature oocytes retrieved were 15.2 and 11.5 respectively. Thirty-eight women underwent IVF with half (19 patients) requiring ICSI. Average oocytes fertilized and embryos cryopreserved per patient were 9.5 and 8.5. Two women had embryo transfers, one patient having a surrogate carrier. Thirteen women cryopreserved oocytes, with average number oocytes cryopreserved being 15.8.

Conclusion: The use of high dose letrozole during ovarian stimulation for fertility preservation provides an additional margin of safety with low peak estradiol levels while maintaining favorable outcomes in embryo/oocyte cryopreservation.

Katherine Péloquin1, Audrey Brassard2
1Université de Montréal, Montreal, QC; 2Université de Sherbrooke, Sherbrooke, QC

Introduction: Most research examining the impact of infertility on psychological adjustment has generally reported no impact of diagnosis1,2. A few studies, however, have found gender differences in psychological or relationship adjustment based on gendered diagnosis (male versus female factor)1,3,4, suggesting that men and women experience infertility differently, partly based on which partner is infertile. Because infertility is closely tied to identity and self-esteem, and is sometimes stigmatised, we postulate that some individuals may be more affected by gendered diagnosis than others. Attachment theory5,6 may be a relevant framework from which to understand reactions to different diagnosis as well as gender differences in these reactions. We present preliminary results of a study examining the moderating effect of attachment insecurities (attachment anxiety and avoidance) and gender on the association between infertility diagnosis and adjustment.

Materials and Methods: Participants (25 women; 19 men) were seeking fertility treatment in Quebec. They completed questionnaires of attachment insecurities (Experiences in Close Relationships Scale7) and adjustment to infertility: Fertility Quality of Life Tool (FertiQoL)8 and Fertility Problem Inventory (FPI)9. A series of 2 (Sex) X 2 (Diagnosis) X 2 (Attachment insecurity) ANOVA were conducted to test research questions.

Results: Congruent with past research, we found no main effect of diagnosis for any of the adjustment variables. However, a significant Diagnosis X Attachment avoidance interaction was found for FertiQoL emotional (F(1,36)=4.48, p=.041, ηs2=.11), mind and body (F(1,36)=5.81, p=.021, ηs2=.4) and social (F(1,36)=10.25, p=.003, ηs2=.22) domains, and FPI relationship (F(1,36)=5.28, p=.027, ηs2=.13) and social concerns (F(1,36)=7.04, p=.012, ηs2=.16): individuals with low attachment avoidance were more negatively affected by male factor infertility, whereas individuals with high avoidance were more negatively affected by female factor infertility. Two three-way interactions were also marginally significant: Sex X Diagnosis X Attachment avoidance for FPI relationship concern (F(1,36)=2.71, p=.060, ηs2=.10); Sex X Diagnosis X Attachment anxiety for FertiQoL mind and body (F(1,36)=3.50, p=.069, ηs2=.09), suggesting possible gender differences in these associations.

Discussion: Findings suggest that individuals may be differentially impacted by diagnosis based on their interpersonal vulnerabilities (i.e., attachment) and gender. Clinically, these individual reactions may give rise to complex couple dynamics whereby each partner is uniquely impacted by infertility based on the diagnosis received and attachment, and this may have ramifications for the couple’s overall adjustment and treatment experience.

References:

  1. Greil AL, Slauson-Blevins K, McQuillan J. The experience of infertility: a review of recent literature. Sociology of Health & Illness 2010;32(1):140-162.
  2. Péloquin K, Lafontaine M-F. What are the correlates of infertility-related clinical anxiety? A literature review and the presentation of a conceptual model. Marriage & Family Review 2010;46(8):580-620.
  3. Vizheh M, Pakgohar M, Rouhi M, Veisy A. Impact of Gender Infertility Diagnosis on Marital Relationship in Infertile Couples: A Couple Based Study. Sexuality and Disability 2015;33(4):457-468.
  4. Wischmann T, Thorn P. (Male) infertility: what does it mean to men? New evidence from quantitative and qualitative studies. Reproductive BioMedicine Online 2013;27(3):236-243.
  5. Bowlby J. Attachment and loss: Vol 1. Attachment. New York, NY: Basic Books; 1982.
  6. Mikulincer M, Shaver PR. Attachment in adulthood: Structure, dynamics, and change. New York, NY: Guilford Press; US; 2007.
  7. Brennan KA, Clark CL, Shaver PR. Self-report measurement of adult attachment: An integrative overview. Attachment theory and close relationships. New York, NY: Guilford Press; US; 1998. p 46-76.
  8. Boivin J, Takefman J, Braverman A. The fertility quality of life (FertiQoL) tool: development and general psychometric properties. Human Reproduction 2011;26(8):2084-2091.
  9. Newton CR, Sherrard W, Glavac I. The Fertility Problem Inventory: measuring perceived infertility-related stress. Fertility & Sterility 1999;72(1):54-62.
  10. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatrica Scandinavica 1983;67(6):361-70.

Adam Rosen, Fang Zhou (Faith) Xu, Yaakov Bentov
University of Toronto, Toronto, ON

Introduction: Proper timing and embryo quality are two factors that influence IVF outcomes. Previous studies have shown better endometrial receptivity on day-5 fresh transfers compared to day-6, likely due to a forward-shifted window of implantation during ovarian hyperstimulation. Clinical outcomes, however, are improved when day-6 blastocysts are frozen for future use1. With blastocysts being used predominantly in practice, no work to-date has studied the outcomes of cavitating morula transferred on day-5. Thus, clinicians lack evidence to guide decision making when faced with only day-5 cavitating morulas.

Materials and Methods: This retrospective cohort study enrolled all patients that underwent IVF transfer with either a fresh day-5 cavitating morula or a frozen-thawed day-6 blastocyst at two IVF clinics in Toronto between January 2012 and December 2015. We reviewed outcomes from 170 cycles with 236 embryos transferred. 44 additional cycles met initial inclusion criteria, but outcomes could not be obtained from patient files. The study included 88 fresh day-5 cavitating morulas from 52 cycles (mean age=38.1) and 148 frozen-thawed day-6 blastocysts from 118 cycles (mean age=36.3). Measured outcomes were biochemical pregnancy (defined by serum B-hCG concentration >5.3 U/I 14-days post-transfer) and successful implantation (defined by the gestational sac detection on ultrasound 4-6 weeks post-transfer). Data was analyzed using a chi-squared test.

Results: The fresh day-5 cavitating morula and frozen-thawed day-6 blastocyst groups did not differ statistically in either biochemical or clinical pregnancy outcomes. 34.7% and 31.4% of cycles in day-5 cavitating morula and day-6 blastocyst groups, respectively, achieved a biochemical pregnancy (P>0.05). Successful implantation was achieved by 12.5% of day-5 cavitating morula group, compared to 18.9% of day-6 blastocyst group (P>0.05).

Conclusion: Study results suggest that, when faced with only day-5 cavitating morulas, physicians can base the decision to transfer, or wait a day and then freeze, on economic, scheduling, and patient-centered factors. Future research examining this question is required in a more controlled, better powered setting.

References
1) Shapiro, B. S., Daneshmand, S. T., Garner, F. C., Aguirre, M. & Ross, R. Contrasting patterns in in vitro fertilization pregnancy rates among fresh autologous, fresh oocyte donor, and cryopreserved cycles with the use of day 5 or day 6 blastocysts may reflect differences in embryo-endometrium synchrony. Fertil. Steril. 89, 20–6 (2008).

Talya Shaulov1 , Li Zhang1 , William Buckett1 , Asangla Ao1,2
1 Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University Health Centre (MUHC) Reproductive Centre, Montreal, QC; 2 Department of Human Genetics, McGill University, Montreal, QC

Introduction:
The effect of DM1 on ovarian reserve and outcomes of ovarian stimulation in IVF/PGD cycles is controversial in the literature. We therefore conducted a study to evaluate these characteristics among DM1 patients in Quebec seeking PGD.

Materials and Methods:
This is a retrospective cohort study. All couples in which the female was affected with DM1 and who underwent IVF/PGD cycles in a single university affiliated centre were included. Two control groups were used: the first comprised of patients treated with IVF/PGD in the same centre for X-linked recessive disorders, and the second of patients matched to DM1 patients in a 3:1 ratio for age and year of first IVF cycle, who underwent IVF or ICSI for infertility. Outcomes assessed in all three groups included markers for ovarian reserve (antral follicle count (AFC) and day 3 FSH), response to ovarian stimulation, embryo quality and clinical pregnancy rates (CPRs) per embryo transfer (ET). Comparisons were made between the 3 groups, and between the DM1 group and each control group separately.

Results:
Between September 1998 and January 2015, there were 36 IVF cycles performed in 10 patients in the DM1group; 25 IVF cycles performed in 15 patients in the X-linked recessive group; and 30 IVF cycles performed in 30 patients in the matched-control group. Median AFC was 14.5, 20 and 13 (p=0.41); and median day 3 FSH was 6.5, 5.3, and 6.6 IU/L (p=0.77) in 3 groups, respectively. Total days of stimulation (median: 10, 9, 9.5), total dose FSH (median: 2963, 2025, 2013 IU), number of oocytes collected (median: 10, 14, 11), and embryo quality did not differ significantly between cycles performed in the three groups. Number of embryos transferred at once and CPR per ET (27.3%, 30%, 43.5%) also did not differ significantly between groups. When comparisons were performed between the DM1 group and each control group separately, we noted that more days of stimulation and higher doses of FSH (+950IU) were necessary in the DM1 group than in the matched control group (p

Conclusion:
The results from this study show that female DM1 patients require significantly higher doses of gonadotropins to achieve similar clinical results from IVF/PGD than their age-matched controls. Ovarian reserve, however, does not seem to be affected in these patients.

Weon-Young Son, William Buckett
MUHC Reproductive Centre, McGill University Health Centre, Montreal, QC

Introduction: About 30% of malignant tumours occurring in women of childbearing age are breast cancer and approximately 10–15% of these breast cancers are diagnosed during reproductive years. Affected women are seeking options to preserve their fertility. The broadly established fertility preservation strategies include oocyte or/and embryo cryopreservation and these methods rely on ovarian stimulation. However, IVF procedures involve a delay in cancer treatment and produce relatively high estradiol levels, which may be potentially harmful in certain cancers such as estrogen receptor (ER)-positive breast cancer.  In those cases, immature oocyte collection followed by in vitro maturation (IVM) is a possible alternative. In this case report, we present a live birth resulting from the transfer of vitrified embryos produced from IVM oocytes of hCG-primed IVM cycles.

Methods: A 40 year old nulliparous woman diagnosed with an ER+ve and PR+ve infiltrating ductal carcinoma of the breast presented for fertility preservation. Because of concerns regarding ovarian stimulation from the couple and the oncological team, the couple chose to do IVM without ovarian stimulation rather than IVF cycles in order to avoid any possible risks. After local surgical excision, she had two hCG-primed IVM retrievals before starting chemotherapy.

Results: A total of 31 oocytes were obtained after performing two separate IVM cycles. After maturation, 24 MII oocytes were inseminated by ICSI and 18 embryos were vitrified at the cleavage stage. Four years after treatment of her breast cancer, the patient who was 44 years old returned in hopes of using the frozen embryos.  Three embryos derived from Germinal Vesicle-stage oocytes were warmed and replaced into her uterus.  Fourteen days later, the serum β-hCG was 271.0 IU/l and 6-7 weeks after embryo transfer an ongoing intrauterine single pregnancy with fetal heartbeat was confirmed. A healthy baby girl was delivered at 40+3 weeks gestation. The child is now a healthy 5 years old.

Conclusions: Although several modified IVF stimulation protocols have recently been offered, the possible risks associated with this hyperstimulation are still of concern, especially for ER(+) positive breast cancer patients.  In those cases, immature oocyte collection followed by IVM and embryo cryopreservation is a possible alternative.

KEYWORDS: Estrogen receptor-positive breast cancer, fertility preservation, in vitro maturation; live birth; vitrification.

Lih Yeen Tan1 , Liubov Krichevsky2 , Ellen Greenblatt1,3, Robert Casper1,3,4, Carl Laskin1,3,4, Sony Sierra1,4,9, Thomas Hannam5 , Mathias Gysler1,6, Megan Karnis7,8, Clifford Librach1,9,10, Prati Sharma1,9,
1 Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON; 2 Faculty of Medicine, University of Toronto, Toronto, ON 3 Department of Obstetrics and Gynaecology, Mount Sinai Hospital, Toronto, ON; 4 TRIO Fertility, Toronto, ON; 5 Hannam Fertility Centre, Toronto, ON; 6 Reproductive Care Centre, Mississauga, ON; 7 ONE Fertility, Burlington, ON; 8 Department of Obstetrics and Gynaecology, McMaster University, Hamilton, ON; 9 Department of Gynaecology, Women’s College Hospital, Toronto, ON; 10CReATe Fertility Centre; Toronto, ON

Introduction: Fragile X premutation (55-200 CGG repeats) is the most common genetic cause of 46,XX primary ovarian insufficiency (POI).  There is currently no published literature regarding the reproductive outcomes and efficacy of artificial reproductive technology (ART) in females with FXPOI. Much of the data regarding FXPOI patients are extrapolated from studies of women with other causes of POI. Trial and error in these women can be associated with loss of time to conception as well as substantial cost to the patient. This study aimed to determine whether ART treatment options and success rates differ between females with FXPOI and those with POI due to other causes.

Methods:  We performed a retrospective case control study of patients with POI from 7 fertility centres in Ontario from 2007 to 2015. The study group consisted of FXPOI patients, younger than 40 years old (n=19).  The control group consisted of females younger than 40 years old who were diagnosed with POI, with no Fragile X premutation (n=76). The FXPOI and control patients were grouped and matched by age (<30, 30-34, and 35-39) and by their ovarian reserve assessment utilising AMH testing (AMH

Results: 19 FXPOI patients who underwent a total of 70 treatment cycles and 76 control patients who underwent a total of 217 treatment cycles were identified. The overall pregnancy rate per cycle (stimulated and natural cycles) was significantly lower in the AMH

Conclusion: FXPOI females with very low ovarian reserve (AMH

Supported by CReATe Program Inc. and by a CREMS summer studentship.

Abu Rafea1,2
1 The Fertility Clinic, London Health Sciences Centre; 2 Western University, London, ON

Introduction: Culture media have played a major role in improving IVF cycle outcomes in the last decades.  These commercially available media fall into two culture systems: a) sequential (1 medium from zygote to day 3, and a second medium from day 3 to blastocyst); and b) single medium (culture from zygote to blastocyst stage). Studies suggest that the single medium system supports better embryo development to the blastocyst stage, and an equivalent or better pregnancy rate compared to the sequential system, driving a recent trend to move to single culture system. We report here the outcome of a project in our laboratory to transition from sequential to single medium culture system.

Methods: Study Part One – evaluated the development of frozen-thawed day 3 embryos from consenting patients to blastocysts in 3 commercial media. Embryos were cultured in Quinn’s Cleavage medium (sequential) and frozen by the slow method. Experiment 1 compared development of thawed embryos in Quinn’s Blastocyst (sequential) vs. Global (Single) media. Experiment 2 compared development of thawed embryos in 2 types of single culture media: Global + HSA vs. Global Total (containing protein). In each experiment allocation of frozen embryos were balanced between the 2 groups based on the quality at the time of freezing. Study Part Two compared blastocyst development in sequential culture to single medium culture. The outcomes compared were: a) fertilization rate; b) % blastocysts on day 5/6; c) % patients with day5/6 blastocysts; d) % patients with day5/6 vitrified blastocysts.

Results: In experiments 1 and 2, % embryo survival was similar between each of the two groups. The proportion of embryos reaching the blastocyst stage in experiment 1, tended to be slightly higher in the single medium compared to the sequential medium. In experiment 2, proportion of embryos reaching the blastocyst stage tended to be slightly higher in the Global Total vs. the Global + HSA medium. In study part 2, patient and cycle characteristics as well as % fertilization were comparable in cycles using sequential media vs. cycles using single medium. However, cycle outcomes were better in single vs. sequential medium culture with respect to: a) % blastocysts on day 5/6; b) % patients with day5/6 blastocysts; and c) % patients with day5/6 vitrified blastocysts.

Conclusions: Our results demonstrate an effective protocol for transitioning from a sequential to a single culture system. Embryo development to the blastocyst stage improved with the single medium culture compared to the sequential culture system.

Marcedes Vallance1 , Belen Herreo2 , Heather Brooks1 , William Buckett2
1 Outreach Health Services, Newmarket, ON; 2 McGill University Health Centre, Reproductive Centre, Montreal, QC

Introduction: One piece of information that parents of donor offspring often seek is information about half siblings to their child or children. These same recipients are often shocked to find that their donor has produced a number of offspring which they feel is higher than they anticipated. This can lead to patient’s anxiety and concerns. Reporting live births remains the only way to date for the sperm banks to curtail the activities of the donor, and retire him if the donor reaches the family unit limit. Moreover, it permits sperm banks to notify patients if any health problems arise with the donors. Live birth reporting from the use of donor sperm in Canada is not legally required, and thus remains to date, a voluntary process for patients and fertility clinic programs. The current guidelines from the American Society for Reproductive Medicine state that there should be no more than 25 live births per sperm donor in a population of 800,0001. Sperm banks follow these guidelines and often set their own, lower, voluntary limits relying on the accurate reporting by patients. Unfortunately, a vast number of sperm banks receive live birth information on only on a portion of the outcomes from the use of their donor samples, leading to less accurate information regarding number of offspring or family units per donor. The aim of this study was to evaluate whether a follow-up contact performed by the fertility clinic to patients that conceived using donor sperm would increase the number of live birth reports received by the sperm banks.

Methods: An intervention was implemented between a Canadian compliant donor sperm distributor and a reproductive centre in order to increase the number of live birth reports. Patients who conceived using donor sperm were contacted by phone or email to inform them about the importance of reporting their live birth(s) to the sperm bank, and instructions were sent directly by email if required. Data was collected with the cooperation of several sperm banks to confirm if a live birth report corresponding to the patient was submitted.

Results: A total of 322 patients were inseminated with donor sperm during a two year period. We compared live birth reports received by different sperm banks during 2014 to 2015. Only patients that conceived in 2015 were reached and informed about the importance of reporting their live birth using donor sperm. Overall, live birth rates/cycle (IVF/ICSI+IUI) for 2014 and 2015 were 18.1% and 16.8% respectively. In 2014, 41.9% of the patients reported their live birth to the sperm bank, whereas 60% of them reported it in 2015 (p<0.05). Patients who reported their live birth(s) were comprised of 29% heterosexual couples, %50 single women and 69.2% same sex couples. Moreover, 68% of patients that submitted a live birth report to the bank have chosen open ID sperm donors whereas 14.3% elected anonymous ID sperm donors.

Conclusions: A simple intervention at the level of the reproductive centre increased the number of live births reported by the patients to the sperm banks. The results also showed that same sex couples, single women, and patients who opted to use open ID donors are more likely to report live births using donor sperm. Thus, informing and reminding recipients of donor sperm about the importance of reporting live births leads to an overall improvement in regards to the quality of patient care.

1 http://www.asrm.org/error.aspx?aspxerrorpath=/membersonly/practice/2006gameteguide.pdf Retrieved April 1 2016.

Nabendu Murmu1 , Sreyashi Mitra1 , Alex C Varghese2
1 Department of Signal Transduction and Biogenic Amines, Chittaranjan National Cancer Institute, Kolkata, India, 2 Astra Fertility Clinic, Mississauga, ON

 Abstract:

Background: Emerging evidences across the world suggest that addiction to cannabis smoke has many deleterious health effects including the adverse effects on the fertility status. The aim of the present study was to determine the expression profile of the cell survival protein p-Akt and its downstream pro-apoptotic signaling in cannabis smoke addicted subfertile patients.

Method: Semen samples were collected from 80 male patients of reproductive age group in Southern Assam of North-East India. 46(57.5%), 25(31.25 %) and 9(11.325%) of the patients were found to be cigarette smokers, cannabis smokers and non-smokers respectively. ROS levels in seminal ejaculates were measured. Expression profiles of Bcl2, p-Akt and pro-apoptotic proteins Bax, Caspase 3, Caspase 9 were checked by flow cytometry and western blot analysis.

Results: The cannabis smoke addicted patients showed the highest level of seminal ROS production along with the highest percentage of sperm DNA damage, chromatin abnormalities and apoptotic cells among the three groups of patients. High expression of Bax, Caspase 3, Caspase 9 and low expression of p-Akt, Bcl2, has been observed in non-smoker and tobacco smoke addicted patients. Conversely, cannabis addicted patients showed highest expression of p-Akt, Bax and Caspase proteins and low expressions of Bcl2.

Conclusion: The present study indicates cannabis smoke addiction to be more detrimental for male reproductive health compared to the tobacco smoke addiction. The up-regulation of pro-survival protein Akt during sperm meiotic division could have triggered the oxidative apoptosis of sperm cells via pro-apoptotic proteins Bax, Caspase 3 and Caspase 9 in Cannabis addicted patients.

Brandon A. Wyse1*, Shlomit Kenigsberg1*, Hanna Balakier1 , Julieta Caballero1 , Itai Gat1 and Clifford L. Librach1,2,3 1 CReATe Fertility Centre, Toronto, ON; 2 Department of Obstetrics & Gynaecology, University of Toronto, ON; 3 Department of Gynaecology, Women’s College Hospital, Toronto, ON

Introduction
Cumulus cells (CCs) are a group of closely connected cells that surround the oocyte and participate in oocyte maturation and fertilization. It has been suggested that exchange of small molecules, like RNA, from CCs to the oocyte, potentially through gap-junctions and/or vesicular transport, controls meiotic arrest1. It is also known that long non-coding RNAs (lncRNAs), known regulators of cell signaling, are expressed in CCs2,3. To understand the contribution of CCs in oocyte maturation, our aim was to investigate the presence and differential expression of CC lncRNAs associated with different meiotic stages: germinal vesicle (GV), metaphase I (MI), and metaphase II (MII).

Materials and Methods
This study has U of T REB approval. CCs from 27 oocytes were obtained from three patients who underwent IVF. To enhance RNA yield, 3 samples of CCs were pooled together in 3 groupings according to the oocyte maturation: GV (n=3), MI (n=3), and MII (n=3). Total RNA was isolated using the NORGEN RNA micro kit. Comparative analysis of CCs-lncRNA was performed by a qPCR focused array (96 genes) for array analysis. A fold change of >2 and pP).

Results
In our analysis expression, 3 lncRNAs genes were found to be significantly different between the maturation stages. FOXN3-antisense and SENP3-EIF4A1 transcripts had 2-fold higher expression in GV oocytes as compared to MI and MII (P). RP-239B22.5 was overexpressed in GV compared to MI oocytes (P). FOXN3 is a member of the forkhead/winged helix transcription factor family and is part of the checkpoints for eukaryotic DNA damage-inducible cell cycle arrests at G1 and G2. The function of the SENP3-EIF4A1 and RP1-239B22.5 transcripts are unknown. We also discovered several lncRNAs that appear to be related to oocyte maturation (NEAT1, TUG1, and XIST), but a higher sample size is required to confirm their statistical significance.

Conclusion
Out of the 86 targeted lncRNA genes, we found 3 to be upregulated in CCs from immature GV oocytes in comparison to CCs from MI or MII oocytes. These interesting and novel findings have allowed us to target these and other lncRNA genes for further study towards understanding their role in oocyte maturation. In addition, we are currently examining CC-lncRNA genes in relation to various conditions that cause infertility.

1   Macaulay ADBiol Reprod. 2016 Dec;94(1

2   Huang et al (2016). Aberrant expression of long noncoding RNAs in cumulus cells isolated from PCOS patients.  J Assist Reprod Genet 33:111–121.

Grace Younes, Ashley Gilman, Samer Tannus, WeonYoung Son, Peter Chan, William Buckett
MUHC Reproductive Centre, Montreal, QC

Introduction
In the last two decades, the advent of ICSI and the application of various testicular sperm retrieval techniques have revolutionized treatment in men with OA and NOA.

Due to the assumed high spermatozoal sensitivity from exposure to reactive oxygen species and inflammatory proteins in the male excurrent ductal system, a higher rate of DNA damage has been noted in ejaculate compared to surgically retrieved testicular sperm.  There has been some debate in recent years as to whether surgically retrieved sperm is preferable to ejaculate in some couples with reproductive failure.

Materials and Methods
This is a descriptive retrospective study for all IVF cycles with surgically-retrieved sperm performed at MUHC Reproductive Centre from January 1 2009-December 31 2015.

During this period, 802 men underwent surgical sperm retrieval using TESE, microTESE, TESA, PESA and MESA due to various etiologies. All charts were reviewed and analyzed retrospectively.

Results
802 cycles with surgically retrieved sperm were found. 226 cycles (28.2%) with NOA, 362 (45.1%) with OA, 18 cycles with mixed cases of NOA and OA (2.2%) and the remaining 196 cycles (24.4%) were due to at least one previously failed IVF cycle, including cases with cryptospermia, oligoteratoasthenospermia, high DNA fragmentation, or repeated failures with normal spermograms.

Sperm retrieval rate (SRR) was highest in OA 99.17%, followed by 95.4% in repeated IVF failures.  In NOA, SRR was 76.55%. The implantation rate, clinical pregnancy rate (CPR) and live birth rate (LBR) per cycle were also higher in OA 28.1%, 31.8%, 19.6% respectively. These rates were 25.2%, 23.9% and 17.9% respectively in NOA and 24.9%, 25.5% and 14.79% respectively in repeated failure cases.

Interestingly, the highest no embryo transfer (ET) rate was found in the repeated failure group (44.76%) followed by NOA (31.41%) and OA (16.0%). Main reasons for no ET were due to lack of fertilization and poor embryo quality in the patients with repeated failures and OA while in the NOA this was because no sperm was found.

Discussion
This is so far the largest descriptive Canadian study about ART outcomes with surgically retrieved sperm.  Reassuringly, clinical outcomes are comparable to those reported by CARTR for all ART. However, more research is needed regarding the benefit surgically retrieved sperm in cases of repeated IVF failures.

Grace Younes, Samer Tannous, Weon.Young Son, Michael Haim Dahan
MUHC Reproductive Centre, Montreal, QC

Introduction: One in six Canadian couples suffer from infertility and in around half of these cases, male factors are to blame. Spermatozoa are very susceptible to oxidative stress, which is evidenced by a net increase of reactive oxygen species (ROS) due to an increase in their production and/or a decrease in antioxidant protection. The oxidative damage due to high levels of ROS has been associated with male infertility1.

The peroxiredoxins (PRDXs) are a major class of antioxidant enzymes in human spermatozoa2. PRDX6 is an enzyme with peroxidase and calcium-independent phospholipase A2 (PLA2) activities. We recently reported that male mice lacking PRDX6 are subfertile and their spermatozoa displayed high levels of oxidative damage resulting in low motility and abnormal chromatin structure3. Thus, the aim of the present study was to evaluate the role of PRDX6 PLA2 activity in the survival and protection of spermatozoa against oxidative stress.

Methods: Percoll-selected spermatozoa from healthy donors was incubated in Biggers, Whitten, and Whittingham medium (BWW) for 4 h at 37°C with or without MJ33, a specific PRDX6 PLA2 activity inhibitor. Oxidative stress was promoted by incubating aliquots of these spermatozoa with 2 mM hydrogen peroxide (H2O2). Sperm viability was assessed using the hypo-osmotic swelling test. Lipid peroxidation, a marker of oxidative damage, was determined by incubating spermatozoa with a fluorescence BODIPY C11 probe for 30 minutes at 37°C.  BODIPY C11 fluorescence was quantified by flow cytometry.

Results: MJ33 impaired sperm viability in a dose dependent manner, an effect that was exacerbated in H2O2-treated spermatozoa (two-way ANOVA and Bonferroni test; p<0.05). Lipid peroxidation levels increased in a dose-dependent manner after MJ33 treatment (Kruskal-Wallis one-way ANOVA and comparison of means ranks; p<0.05).

Conclusions: PRDX6 PLA2 activity is essential to protect human spermatozoa from oxidative stress and assure their survival. Future studies are ongoing to elucidate the regulation of pro-survival mechanisms by PRDX6. These studies will lead to novel therapeutic strategies for infertile patients.

Supported by CIHR

References

  1. Tremellen, K. “Oxidative stress and male infertility: a clinical perspective, Human Reproduction Update, 2008 (14):243–258.
  2. Gong S, San Gabriel M, Zini A, Chan P, O’Flaherty C. 2012. J Androl 33:1342–1351.
  3. Ozkosem B., et al., Advancing Age Increases Sperm Chromatin Damage and Impairs Fertility in Peroxiredoxin 6 Null Mice. Redox Biology, 2015. 5: p. 15-23.

Alex Yu1,2, Adel R. Moawad1,3, Cristian O’Flaherty1,2,3
1 The Research Institute-McGill University Health Centre, Montréal, QC; 2 Division of Experimental Medicine, McGill University, Montréal, QC; 3 Department of Surgery (Urology Division), McGill University, Montréal, QC

Introduction: One in six Canadian couples suffer from infertility and in around half of these cases, male factors are to blame. Spermatozoa are very susceptible to oxidative stress, which is evidenced by a net increase of reactive oxygen species (ROS) due to an increase in their production and/or a decrease in antioxidant protection. The oxidative damage due to high levels of ROS has been associated with male infertility1.

The peroxiredoxins (PRDXs) are a major class of antioxidant enzymes in human spermatozoa2. PRDX6 is an enzyme with peroxidase and calcium-independent phospholipase A2 (PLA2) activities. We recently reported that male mice lacking PRDX6 are subfertile and their spermatozoa displayed high levels of oxidative damage resulting in low motility and abnormal chromatin structure3. Thus, the aim of the present study was to evaluate the role of PRDX6 PLA2 activity in the survival and protection of spermatozoa against oxidative stress.

Methods: Percoll-selected spermatozoa from healthy donors was incubated in Biggers, Whitten, and Whittingham medium (BWW) for 4 h at 37°C with or without MJ33, a specific PRDX6 PLA2 activity inhibitor. Oxidative stress was promoted by incubating aliquots of these spermatozoa with 2 mM hydrogen peroxide (H2O2). Sperm viability was assessed using the hypo-osmotic swelling test. Lipid peroxidation, a marker of oxidative damage, was determined by incubating spermatozoa with a fluorescence BODIPY C11 probe for 30 minutes at 37°C.  BODIPY C11 fluorescence was quantified by flow cytometry.

Results: MJ33 impaired sperm viability in a dose dependent manner, an effect that was exacerbated in H2O2-treated spermatozoa (two-way ANOVA and Bonferroni test; p<0.05). Lipid peroxidation levels increased in a dose-dependent manner after MJ33 treatment (Kruskal-Wallis one-way ANOVA and comparison of means ranks; p<0.05).

Conclusions: PRDX6 PLA2 activity is essential to protect human spermatozoa from oxidative stress and assure their survival. Future studies are ongoing to elucidate the regulation of pro-survival mechanisms by PRDX6. These studies will lead to novel therapeutic strategies for infertile patients.

Supported by CIHR

References

  1. Tremellen, K. “Oxidative stress and male infertility: a clinical perspective, Human Reproduction Update, 2008 (14):243–258.
  2. Gong S, San Gabriel M, Zini A, Chan P, O’Flaherty C. 2012. J Androl 33:1342–1351.
  3. Ozkosem B., et al., Advancing Age Increases Sperm Chromatin Damage and Impairs Fertility in Peroxiredoxin 6 Null Mice. Redox Biology, 2015. 5: p. 15-23.

Xiao Yun Zhang1 , Weon-Yong Son1 , William Buckett1 , Asangla Ao1,2
1 McGill University Health Centre (MUHC) Reproductive Centre, Montreal, QC; 2 Departments of Human Genetics and obstetrics and Gynecology, McGill University, Montreal, QC

Introduction: Balanced translocation carriers are known to have a higher risk of facing infertility and repeated miscarriages. Preimplantation genetic diagnosis (PGD), by selecting chromosomally normal or balanced (N/B) embryos before implantation, is one of the options for them to have a healthy offspring. Studies showed differences in segregation pattern in embryos obtained from PGD cycles of male or female carriers. However, these differences did not affect the percentage of N/B embryos and clinical outcome. It is well known that with advanced female age, the risk of aneuploidies increase in the preimplantation embryos. The objective of this study was to analyze whether female age has an effect on clinical outcome of couples with balanced translocation who underwent PGD treatment.

Materials and Methods: We retrospectively analyzed data obtained in 84 reciprocal (51 cycles with young female age, <36, average age 30.7 vs 33 cycles with old female age, >36, average age 37.9) and 54 Robertsonian translocation PGD cycles (26 cycles with young female age, <36, average age 31.4 vs 28 cycles with old female age, >36, average age 38.6). The PGD test was performed by using chromosome specific FISH probes for reciprocal translocation and FISH probe panel (13,16,18,21,22 and 15,17,XY or 14,18) for Robertsonian translocation. Embryos were assigned as N/B when all the tested chromosome were euploid, and unbalanced/abnormal embryo when any of the tested chromosome were diagnosed as abnormal. Embryos diagnosed as N/B were transferred on day4 or day5.

Results: 705 embryos were tested for reciprocal translocation; there were no statistical differences in the proportion of N/B embryos (22% vs 19%), clinical pregnancy rate (26% vs 24%) and miscarriage rate (23% vs 37%) in young and old female age groups. Seventeen percent (9 out of 51 cycles) and 33% (11 out of 33 cycles) had no embryo transfer in both groups, respectively. All no embryo transfer cycles were due to absence of N/B embryos following PGD except for one cycle where N/B embryos had degenerated.

358 embryos were tested for Robertsonian translocation; and a significantly higher percentage of N/B embryos were obtained in young female age group compared to old female age group (32% vs 23%, P=0.04) with a lower trend of miscarriage rate (8% vs 25%) and a higher trend of clinical pregnancy rate (46% vs 28%) in the younger age group. The percentage of no embryo transfer cycles were similar (15% vs 18%).

 Conclusions: The chance of obtaining a N/B embryo from Robertsonian translocation carriers is significantly lower when the female age is 36 years old or older. This may be due to the inclusion of additional chromosome probes in the PGD test panel that are most common aneuploidies found in advanced maternal age. The rate of N/B embryos from reciprocal translocation carriers were similar, irrespective of the maternal age and in this group no additional chromosome probes were included.

Li Zhang1 , Sara Henderson1 , Shauna Reinblatt1 , Asangla Ao1,2 , MUHC-RC team1
1 Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University Health Centre (MUHC) Reproductive Centre, Montreal, QC; 2 Department of Human Genetics, McGill University, Montreal, QC

Introduction: Cree leukoencephalopathy (CLE), also called vanishing white matter leukodystrophy, is a rapidly fatal infantile autosomal recessive neurologic disorder, most commonly observed in the indigenous Cree and Chipewyan populations in northern Quebec and Manitoba.  This disorder is characterized by variable neurologic features, including progressive cerebellar ataxia, spasticity, and cognitive impairment associated with white matter lesions on brain imaging. The age at onset can range from early infancy to adulthood. To date, no preimplantation genetic diagnosis (PGD) for this disease has been reported. We present here our first clinical attempt at PGD of Cree leukoencephalopathy.

Materials and Method: In 2013, one couple (32-year-old female and 45-year-old male) was referred to our fertility center. Both of them are carrier of R195H in EIF2B5 gene. They have two unaffected children, one affected daughter, who died at 6 months, and one termination of pregnancy following prenatal diagnosis. For the IVF-PGD cycles, female patient underwent standard ovarian stimulation procedure in our clinic, and intracytoplasmic sperm injection (ICSI) was performed to avoid sperm contamination. 6 follicles were retrieved, of which 4 reached MII stage, 4 were fertilized, and 4 embryos were frozen on day 3 due to some technical reason. Embryo biopsy was performed on day 3 post-insemination. Fluorescent-based multiplex PCR was used for mutation analysis. Embryos diagnosed as unaffected were transferred on day 5 post fertilization. The study was approved by clinical ethics board of MUHC.

Results: A STR (short tandem repeat) based specific PGD test was developed for this couple. The gnomic DNA from their affected daughter and single sperm haplotype were used to identify the mutant allele.  A total of 7 informative STR markers and the specific mutation analysis were carried out for the PGD. The 4 frozen embryos were thawed and biopsied on day 3, in which 2 were diagnosed as affected, one as normal, and one as carrier. One normal embryo was transferred on day 5, unfortunately the patient did not become pregnant. The carrier embryo was not suitable for freezing.

Conclusion: Cree Leukoencephalopathy is a rare recessive disease, however, quite prevelent in Cree and Chipewyan populations in Canada. For those couple who do not want to terminate a pregnancy following prenatal diagnosis, PGD is an option to decrease the incident of affected child born in such small population.

Li Zhang1 , Asangla Ao1,2
1 Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University Health Centre (MUHC) Reproductive Centre, Montreal, QC; 2 Department of Human Genetics, McGill University, Montreal, QC

Background and objective: Sex selection, also known as gender selection, is the practice of choosing the gender (male or female) of a child before conception. In Preimplantation genetic diagnosis (PGD), sex selection is used for couples who wish to prevent X-linked recessive diseases, such as hemophilia, Duchenne muscular dystrophy (DMD), Becker’s muscular dystrophy (BMD), myotubular myopathy (MTM1), that are most often expressed only in male offspring.  By choosing to have a female child, a couple at risk for X-linked diseases may reduce their offspring’s chances of expressing the disease.

FISH (fluorescent in situ hybridization) is a simple method routinely used for sex selection, and it is now gradually replaced by array technology because FISH can be experience-dependent. However, array technology requires expensive equipment investment and may not be cost effective if it is utilized for sex selection only. Here, we present one universal method for sex selection based on multiple polymerase chain reactions (PCR).

Materials and Method: Human blood samples were taken, genomic DNA was extracted and single lymphocytes were collected. Four specific markers in Y chromosome and eleven markers in X chromosome with high heterozygosity for sex selection, and three markers with high heterozygosity in Chromosome 21 to detect trisomy 21were utilized. All the primers were designed by online software Primer3 and checked as unique by UCSC BLAT. Nested PCR based experiments were performed. In the first round PCR, all 17 pairs of primers were added, and four individual multiple PCR assay were performed in the second round PCR.

Results: Total 17 loci from Chromosome X, Y and 21 were tested. SRY, DYS390 and DYS391 are specific markers for Y chromosome. AMXY and AMEL are primers targeted in Amelogenin gene, which is located in both X and Y chromosomes with different fragment sizes.

The expected PCR amplification rate of gDNA was considered as 100%, and allele drop-out (ADO) rate of gDNA is considered as 0%. Our preliminary results show an amplification rate of 92.6% with ADO rate of 1.1% on single lymphocytes. The experiment were performed on 64 samples, the misdiagnosis rate is less than 0.1%.

Conclusion: Compared to DNA CHIP based technology, this multiplex PCR approach for sex selection can be cost-effective and less labor-intensive. In addition, this approach is accurate and universal, and can be simple alternate to FISH approach if FISH method is not available.

WanLi Zhou1 , Ekaterina Shlush2 , Clifford Librach2,3,4,5,6, Prati Sharma2
1 Faculty of Medicine; 2 Department of Obstetrics and Gynecology; 3 Department of Physiology; 4 Institute of Medical Sciences, University of Toronto, Toronto, ON; 5 Department of Gynecology, Women’s College Hospital, Toronto, ON; 6 CReATe Fertility Centre; Toronto, ON

Impact Statement: Performing SIS along with TVS as part of the routine infertility check-up is recommended to enhance the detection rate of uterine lesions and malformations.

Objective: It is estimated that 10-15% of women seeking treatments for subfertility have uterine cavity abnormalities. Therefore, evaluation of the uterine cavity before assisted reproductive techniques is of utmost importance. There have been various methods used for the evaluation of the uterine cavity, with the most common three being transvaginal sonography (TVS), saline infused sonohysterography (SIS), and hysteroscopy. TVS is preferred as the initial diagnostic procedure in evaluating the uterine cavity, due to its wide availability, cost-effectiveness, and well-tolerated nature. SIS involves infusion of saline into the endometrial cavity for distension and contrast during an ultrasound examination. Hysteroscopy is considered to be the gold standard investigative modality as it allows direct visualization of the endometrial cavity. However, due to its invasive nature, it often requires local or general anesthetics, or sedation, does not allow an assessment of the myometrium and adnexal structures, and is associated with more risks. TVS has traditionally been used as an initial diagnostic tool in the evaluation of the uterine cavity, but it has been shown to have moderate accuracy. We aimed to evaluate the diagnostic accuracy of TVS vs. SIS, and hypothesized that incorporating SIS in the initial evaluation of infertility will reduce the number of hysteroscopy procedures performed.

Methods: 696 sub-fertile patients underwent TVS, SIS, and hysteroscopy, between 2009 and 2015 at the CReATe Fertility Centre. The presence of focal abnormalities, including polyps, submucosal myomas, and intrauterine adhesions, as well as uterine malformations, including arcuate cavity, septae, and unicornuate uteri, were noted. Statistical analysis was performed, and sensitivity, specificity, positive predictive value, and negative predictive value for each testing modality was calculated.

Results: SIS showed significantly superior diagnostic accuracy compared to TVS, with an overall accuracy of 0.799 for uterine abnormalities, and 0.9184 for uterine malformations, compared to TVS’s diagnostic accuracy of 0.5585 for uterine abnormalities, and 0.8685 for uterine malformations.

Conclusion: Based on these results, we recommend performing SIS along with TVS as part of the routine infertility check-up to enhance the detection rate of uterine lesions and malformations. This way, hysteroscopy is reserved for those for which it would have therapeutic value to correct uterine abnormalities.

Funding:  This project was funded in part by CReATe Program Inc and by a CREMS summer studentship.