The Disputable Discourse on Accuracy and Effectiveness of PGT-A in Light of Advancements in Genetic Tools 2 1 2 In the past three decades, the main concern regarding infertility treatment was whether checking embryos for chromosome aneuploidy before transfer to uterus has positive effect on results of in vitro fertilization (IVF), and correspondingly implantation, pregnancy, and live birth rates. During this long period of developments in the diagnosis and treatment of infertility, researchers made their best efforts to improve embryo quality by selecting the best ones for transfer and subsequently increasing outcomes of infertility treatment; yet, the substantial revolutions in the methods of genetic evaluation and performing preimplantation genetic testing for aneuploidies (PGT-A) assisted them for such purpose. Despite these developments, researchers have not yet been able to experimentally or clinically validate the effectiveness of these techniques. On the contrary, there is growing evidence that the effectiveness of these procedures is being questioned due to biological characteristics of early human embryos at pre-implantation stage (1). PGT-A, originally known as preimplantation genetic screening (PGS), was performed on a limited number of chromosomes using FISH on one blastomere of cleavage embryo. Today, the development of high throughput genomic methods such as next generation sequencing (NGS) and microarray-based comparative genomic hybridization (aCGH) and TE biopsy at blastocyst stage results in accurate diagnosis of aneuploidy; however, the case of mosaic embryos is a new challenge of IVF for which the application of PGT-A is matter of concern and debate (2). Regarding mosaicism detection in embryos, three conditions can be reported for trophectoderm biopsy and PGT-A results at blastocyst stage: (1) the reported euploid embryos may be 100% euploid or contain aneuploid cells elsewhere in the trophectoderm or inner cell mass. The actual explanation is that this embryo is not completely aneuploid, may be fully euploid or mosaic; (2) the reported aneuploidy embryos may actually be 100% aneuploid or may be mosaic due to presence of euploid cells elsewhere in the trophectoderm and inner cell mass. In fact, this embryo is not completely euploid; (3) the reported mosaic embryo is certainly mosaic, although it is not possible to reliably determine the true percentage of euploidy and aneuploidy through few cells collected from trophectoderm (1). Thus, the reported mosaicism is much lower than the actual rate, whereas the clinical significance of mosaicism in PGT-A has been overestimated. Still, many researchers falsely claim that mosaicism occurs in a small number of embryos at blastocyst stage, while in vivo and in vitro studies on mice and human embryonic stem cells have shown that the mosaicism at blastocyst stage is essentially a natural physiological phenomenon. On the other hand, mosaicism is one of the common findings of PGT-A following new generation of diagnostic tools. In addition, several studies have shown that ongoing pregnancies and live birth can be achieved even by transfer of mosaic embryos. For example, in one study, transfer of 102 mosaic embryos resulted in 46.6% live births, while the transfer of 268 euploid embryos resulted in 59.1% live births, respectively (3). It has been shown that mosaic embryos have different characteristics compared to euploid embryos. Based on this finding, this process has been proposed as a mechanism for aneuploidy correction in which aneuploid cells are selectively removed from the embryo. In mice, aneuploid embryonic cells are removed by autophagy and apoptosis and a similar mechanism has recently been identified in human embryos. In addition, it has been shown that the elimination of aneuploid cells from inner cell mass (ICM; embryonic cells) is greater than trophectoderm cells (TE; extraembryonic cells) (4). Application of spent culture media or blastocoelic fluid (BF) containing cell free DNA (cfDNA) is a new option for noninvasive PGT-A. The elimination of aneuploid cells through self-correction process of embryos leads to release of cfDNA in blastocoel and culture media. An interesting finding was that the cfDNA in BF of aneuploid embryos is more likely to be amplified, showing that more DNA is released form apoptosis of aneuploid cells. This could mean that the amount of cfDNA in BF could predict euploidy status of embryos. Therefore, the use cfDNA for PGT-A may lead to false positive results and subsequently elimination of euploid embryos from the total number of embryos of a couple (4, 5). In order to use any clinical test for diagnostic purposes, adequate levels of sensitivity and specificity must be achieved. Both invasive trophectoderm biopsy and the noninvasive cfDNA techniques have high levels of sensitivity and specificity for the diagnosis of euploid embryos, while their diagnostic ability for aneuploidy is very disappointing; therefore, high frequency of false positive results leads to elimination of numerous healthy euploid embryos. The evidence for this claim is based on various reports of the live birth of healthy neonates from the embryos which were tagged as aneuploid or mosaic through PGT-A (5). According to recent findings, the human early embryos have self-correction ability to remove the aneuploid blastomeres. Therefore, with the development of the embryo from the cleavage to the blastocyst stage, the aneuploidy of embryos decreases; also, lower rate of aneuploidy among day 5 embryos compared to day 3 confirms such decline. Currently, the maximum time for culturing and maintaining early embryos outside the uterus is 6 days, after which the embryos should be frozen or transferred into the uterus. Therefore, if the advancement of science and technology lays the ground for longer in vitro culture of human embryos without interfering with implantation rate, it will be possible to select more euploid embryos for transfer into the uterus and considerably enhance the success rate of infertility treatment (1, 4). Despite huge improvements in genetic testing and increasing the accuracy and efficiency of methods, the effectiveness of PGT-A in IVF has always been challenged. Simply put, based on the available evidence, the usefulness of these methods had not been proven in the last three decades, therefore, the rationale for recommending PGT-A routinely for all IVF cycles cannot be justified now since it may decrease the chance of a couple to have a child and waste their time, money, and energy for continuing treatment. 149 151 Mohammad Reza MR Sadeghi محمدرضا صادقی Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran sadeghi@ari.ir No Keyword 120128.pdf Gleicher N, Barad DH, Ben-Rafael Z, Glujovsky D, Mochizuki L, Modi D, et al. Commentary on two recently published formal guidelines on management of "mosaic" embryos after preimplantation genetic testing for aneuploidy (PGT-A). Reprod Biol Endocrinol. 2021;19(1):1-6.##Cornelisse S, Zagers M, Kostova E, Fleischer K, Wely MV, Mastenbroek S. Preimplantation genetic testing for aneuploidies (abnormal number of chromosomes) in in vitro fertilisation. Cochrane Database Syst Rev. 2020;9 (9):CD005291.##Zhang YX, Chen JJ, Nabu S, Yeung QS, Li Y, Tan JH, et al. The pregnancy outcome of mosaic embryo transfer: a prospective multicenter study and meta-analysis. Genes (Basel). 2020;11(9):973.##Bouba I, Hatzi E, Ladias P, Sakaloglou P, Kostoulas C, Georgiou I. Biological and clinical significance of mosaicism in human preimplantation embryos. J Dev Biol. 2021;9(2):18.##Brouillet S, Martinez G, Coutton C, Hamamah S. Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review. Reprod Biomed Online. 2020;40(6):779-96.##

Evaluating the Expression of Wnt Pathway Related Genes in Mouse Vitrified Preantral Follicles: An Experimental Study 2 1 2 Background: Wnt signaling pathway plays critical role in ovarian follicle development. Therefore, the aim of this study was to evaluate the effects of vitrification on the expression of Wnt pathway related genes in preantral follicles (PFs). Methods: Isolated PFs (n=982) of 14-16 day old female mice (n=45: 15 for each group) were divided into fresh (n=265), toxicity (n=272), and vitrified (n=265). The mRNA levels of Wnt2, Wnt4, Lrp5 and Fzd3 were evaluated by real-time PCR on the 2nd and 6th days of culture period. One-way ANOVA was conducted to analyze the data. Post hoc Tukey's HSD was used for multiple comparisons and p-value less than 0.05 was considered statistically significant. Results: The developmental parameters of fresh PFs were significantly higher than those of vitrified (p<0.001). There were no differences between fresh and vitrified PFs on the 2nd day of culture (p<0.001). Wnt4 expression levels decreased significantly in vitrified groups compared with fresh ones (p<0.001). Fzd3 and Lrp expression levels increased significantly in vitrified groups compared with those in the fresh group on the 2nd day (p<0.001). On the 6th day of culture period, the expression levels of Wnt2 and Fzd3 increased significantly in vitrified group compared to those of fresh group (p<0.001). Moreover, the expression levels of Wnt4 and Lrp increased significantly in toxicity groups compared to those of the control group (p<0.001). Conclusion: Vitrification increase the expression levels of Wnt2, Lrp and Fzd3 genes of PFs during in vitro culture. 151 159 Shahla Sh Babaki School of Biology, Damghan University School of Biology, Damghan University Iran Saeed S Zavareh Saeed Zavareh School of Biology, Damghan University School of Biology, Damghan University Iran zavareh.s@du.ac.ir Parisa P Farrok School of Biology, Damghan University School of Biology, Damghan University Iran Meysam M Nasiri School of Biology, Damghan University School of Biology, Damghan University Iran CryopreservationFolliculogenesisOvaryWnt/β-catenin pathway 120121.pdf Wood CE, Shaw JM, Trounson AO. Cryopreservation of ovarian tissue. Potential" reproductive insurance" for women at risk of early ovarian failure. Med J Aust. 1997;166(7):366-9.##Meirow D. Reproduction post-chemotherapy in young cancer patients. Mol Cell Endocrinol. 2000;169(1-2):123-31.##Varghese AC, Du Plessis SS, Falcone T, Agarwal A. Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: challenges for fertility preservation. Reprod Biol Endocrinol. 2008;6(1):47.##Hosseinzade E, Zavareh S, Lashkarboluki T. [The comparison of developed mouse vitrified preantral follicle with isolated preantral follicles from vitrified ovary]. Koomesh. 2016;17(4):981-9. Persian.##Valojerdi MR, Salehnia M. Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification. J Assist Reprod Genet. 2005;22(3):119-27.##Park S, Hong S, Lee S, Chung H, Cha K, Kim T. Chromosome and spindle configurations of human oocytes matured in vitro after vitrification at the germinal vesicle stage in stimulated cycle. Fertil Steril. 2004;82:S114.##Amidi F, Khodabandeh Z, Nori Mogahi MH. Comparison of the effects of vitrification on gene expression of mature mouse oocytes using cryotop and open pulled straw. Int J Fertil Steril. 2018;12(1):61-7.##Dalman A, Farahani NS, Totonchi M, Pirjani R, Ebrahimi B, Valojerdi MR. Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Cryobiology. 2017;79:29-36.##Fatehi R, Ebrahimi B, Shahhosseini M, Farrokhi A, Fathi R. Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression. Theriogenology. 2014;81(2):302-8.##Asadzadeh R, Khosravi S, Zavareh S, Ghorbanian MT, Paylakhi SH, Mohebbi SR. Vitrification affects the expression of matrix metalloproteinases and their tissue inhibitors of mouse ovarian tissue. Int J Reprod Biomed. 2016;14(3):173-80.##Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, et al. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction. 2009;138(2):319-27.##Lin C, Tsai S. The effect of cryopreservation on DNA damage, gene expression and protein abundance in vertebrate. Italian J Anim Sci. 2012;11(1):e21.##Mofarahe ZS, Salehnia M, Novin MG, Ghorbanmehr N, Fesharaki MG. Expression of folliculogenesis-related genes in vitrified human Ovarian tissue after two weeks in vitro culture. Cell J. 2017;19(1):18-26.##Gumus E, Kaloglu C, Sari I, Yilmaz M, Cetin A. Effects of vitrification and transplantation on follicular development and expression of EphrinB1 and PDGFA in mouse ovaries. Cryobiology. 2018;80:101-13.##Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781-810.##Rider V, Isuzugawa K, Twarog M, Jones S, Cameron B, Imakawa K, et al. Progesterone initiates Wnt-β-catenin signaling but estradiol is required for nuclear activation and synchronous proliferation of rat uterine stromal cells. J Endocrinol. 2006;191(3):537-48.##Hsieh M, Boerboom D, Shimada M, Lo Y, Parlow AF, Luhmann UF, et al. Mice null for Frizzled4 (Fzd4−/−) are infertile and exhibit impaired corpora lutea formation and function. Biol Reprod. 2005;73(6):1135-46.##Hsieh M, Johnson MA, Greenberg NM, Richards JS. Regulated expression of Wnts and Frizzleds at specific stages of follicular development in the rodent ovary. Endocrinology. 2002;143(3):898-908.##Ricken A, Lochhead P, Kontogiannea M, Farookhi R. Wnt signaling in the ovary: identification and compartmentalized expression of wnt-2, wnt-2b, and frizzled-4 mRNAs. Endocrinology. 2002;143(7):2741-9.##Boyer A, Goff AK, Boerboom D. WNT signaling in ovarian follicle biology and tumorigenesis. Trends Endocrinol Metab. 2010;21(1):25-32.##Boyer A, Lapointe É, Zheng X, Cowan RG, Li H, Quirk SM, et al. WNT4 is required for normal ovarian follicle development and female fertility. FASEB J. 2010;24(8):3010-25.##Wang HX, Li TY, Kidder GM. WNT2 regulates DNA synthesis in mouse granulosa cells through beta-catenin. Biol Reprod. 2010;82(5):865-75.##Wang HX, Tekpetey FR, Kidder GM. Identification of Wnt/β-catenin signaling pathway components in human cumulus cells. Mol Hum Reprod. 2008;15(1):11-7.##Staal FJ, Luis TC, Tiemessen MM. WNT signalling in the immune system: WNT is spreading its wings. Nat Rev Immunol. 2008;8(8):581-93.##Hatami S, Zavareh S, Salehnia M, Lashkarbolouki T, Karimi I. Comparison of oxidative status of mouse pre-antral follicles derived from vitrified whole ovarian tissue and vitrified pre-antral follicles in the presence of alpha lipoic acid. J Obstet Gynaecol Res. 2014;40(6):1680-8.##Castañon BI, Stapp A, Gifford C, Spicer L, Hallford D, Gifford JH. Follicle-stimulating hormone regulation of estradiol production: possible involvement of WNT2 and β-catenin in bovine granulosa cells. J Anim Sci. 2012;90(11):3789-97.##Wang HX, Gillio-Meina C, Chen S, Gong X-Q, Li TY, Bai D, et al. The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells. Biol Reprod. 2013;89(2):39.##Kashka RH, Zavareh S, Lashkarbolouki T. Augmenting effect of vitrification on lipid peroxidation in mouse preantral follicle during cultivation: modulation by coenzyme Q10. Syst Biol Reprod Med. 2016;62(6):404-14.##Segino M, Ikeda M, Aoki S, Tokieda Y, Hirahara F, Sato K. In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification. Hum Cell. 2003;16(3):109-16.##Hatami S, Zavareh S, Salehnia M, Lashkarbolouki T. Comparison of total oxidative status of mouse vitrified preantral follicles and drived from vitrified whole ovarian tissue. Iran J Reprod Med. 2014;12(6):36.##Zavareh S, Gholizadeh Z, Lashkarbolouki T. Evaluation of changes in the expression of Wnt/β-catenin target genes in mouse reproductive tissues during estrous cycle: an experimental study. Int J Reprod Biomed. 2018;16(2):69-76.##Li L, Ji SY, Yang JL, Li XX, Zhang J, Zhang Y, et al. Wnt/β-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components. Mol Cell Endocrinol. 2014;382(2):915-25.##Gifford JAH. The role of WNT signaling in adult ovarian folliculogenesis. Reproduction. 2015;150 (4):R137-48.##Choi J, Lee B, Lee E, Yoon BK, Bae D, Choi D. Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles. Cryobiology. 2008;56(1):36-42.##

The Association of Kinetic Variables with Blastocyst Development and Ploidy Status 2 1 2 Background: Despite a plethora of studies conducted so far, a debate is still unresolved as to whether TLM can identify predictive kinetic biomarkers or algorithms universally applicable. Therefore, this study aimed to elucidate if there is a relationship between kinetic variables and ploidy status of human embryos or blastocyst developmental potential. Methods: For conducting this retrospective cohort study, the normal distribution of data was verified using Kolmogorov-Smirnov test with the Lilliefors’ amendment and the Shapiro-Wilk test. Kinetic variables were expressed as median and quartiles (Q1, Q2, Q3, Q4). Mann-Whitney U-test was used to compare the median values of parameters. Univariate and multiple logistic regression models were used to assess relationship between blastocyst developmental potential or ploidy status and kinetics. Several confounding factors were also assessed. Results: Blastocyst developmental potential was positively correlated with the t4-t3 interval (s2) (OR=1.417, 95% CI of 1.288-1.560). s2 median value was significantly different between high- and low-quality blastocysts (0.50 and 1.33 hours post-insemination, hpi, respectively; p=0.003). In addition, timing of pronuclear appearance (tPNa) (OR=1.287; 95% CI of 1.131-1.463) had a significant relationship with ploidy changes. The median value of tPNa was statistically different (p=0.03) between euploid and aneuploid blastocysts (Euploid blastocysts=8.9 hpi; aneuploid blastocysts=10.3 hpi). Conclusion: The present findings are in line with the study hypothesis that kinetic analysis may reveal associations between cleavage patterns and embryo development to the blastocyst stage and ploidy status. 159 165 Francesca F Pennetta Baby Family and Fertility Center Baby Family and Fertility Center Italy Cristina C Lagalla Baby Family and Fertility Center Baby Family and Fertility Center Italy Raffaella R Sciajno Baby Family and Fertility Center Baby Family and Fertility Center Italy Nicoletta N Tarozzi Baby Family and Fertility Center Baby Family and Fertility Center Italy Marco M Nadalini Baby Family and Fertility Center Baby Family and Fertility Center Italy Carlotta C Zacà Baby Family and Fertility Center Baby Family and Fertility Center Italy Giovanni G Coticchio Baby Family and Fertility Center Baby Family and Fertility Center Italy giovanni.coticchio@9puntobaby.it Andrea A Borini Baby Family and Fertility Center Baby Family and Fertility Center Italy AneuploidyBlastocystEmbryoEmbryonic developmentMicroscopy 120115.pdf Munné S, Wells D. Detection of mosaicism at blastocyst stage with the use of high-resolution next-generation sequencing. Fertil Steril. 2017;107(5):1085-91.##Macklon NS, Geraedts JPM, Fauser BCJM. Conception to ongoing pregnancy: The “black box” of early pregnancy loss. Hum Reprod Update. 2002;8(4):333-43.##Munné S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online. 2003;791):91-7.##Scott KL, Hong KH, Scott RT. Selecting the optimal time to perform biopsy for preimplantation genetic testing. Fertil Steril. 2013;100(3):608-14.##Findikli N, Oral E. Time-lapse embryo imaging technology: does it improve the clinical results? Curr Opin Obstet Gynecol. 2014;26(3):138-44.##Lagalla C, Barberi M, Orlando G, Sciajno R, Bonu MA, Borini A. A quantitative approach to blastocyst quality evaluation: morphometric analysis and related IVF outcomes. J Assist Reprod Genet. 2015;32(5):705-12.##Coticchio G, Lagalla C, Sturmey R, Pennetta F, Borini A. The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART. Hum Reprod Update. 2019;25(4):422-38.##Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB, Remohi J. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod. 2011;26(10):2658-71.##Wong CC, Loewke KE, Bossert NL, Behr B, De Jonge CJ, Baer TM, et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat Biotechnol. 2010;28(10):1115-21.##Milewski R, Kuć P, Kuczyńska A, Stankiewicz B, Łukaszuk K, Kuczyński W. A predictive model for blastocyst formation based on morphokinetic parameters in time-lapse monitoring of embryo development. J Assist Reprod Genet. 2015;32(4):571-9.##Kirkegaard K, Ahlström A, Ingerslev HJ, Hardarson T. Choosing the best embryo by time lapse versus standard morphology. Fertil Steril. 2015;103(2):323-32.##Pennetta F, Lagalla C, Borini A. Embryo morphokinetic characteristics and euploidy. Curr Opin Obstet Gynecol. 2018;30(3):185-96.##Ciray HN, Campbell A, Agerholm IE, Aguilar J, Chamayou S, Esbert M, et al. Proposed guidelines on the nomenclature and annotation of dynamic human embryo monitoring by a time-lapse user group. Hum Reprod. 2014;29(12):2650-60.##Basile N, Nogales MDC, Bronet F, Florensa M, Riqueiros M, Rodrigo L, et al. Increasing the probability of selecting chromosomally normal embryos by time-lapse morphokinetics analysis. Fertil Steril. 2014;101(3):699-704.##Veeck LL, Zaninoviae N. An atlas of human blastocysts. 1st ed. New York: The Parthenon Publishing Group; 2003. 367 p.##Oda M, Kanoh Y, Watanabe Y, Masai H. Regulation of DNA replication timing on human chromosome by a cell-type specific DNA binding protein SATB1. PLoS One. 2012;7(8):e42375.##Balakier H, MacLusky NJ, Casper RF. Characterization of the first cell cycle in human zygotes: implications for cryopreservation. Fertil Steril. 1993;59(2):359-65.##Ahlstrm A, Westin C, Reismer E, Wikland M, Hardarson T. Trophectoderm morphology: an important parameter for predicting live birth after single blastocyst transfer. Hum Reprod. 2011;26(12):3289-96.##Hill MJ, Richter KS, Heitmann RJ, Graham JR, Tucker MJ, DeCherney AH, et al. Trophectoderm grade predicts outcomes of single-blastocyst transfers. Fertil Steril. 2013;99(5):1283-9.e1.##Pribenszky C, Losonczi E, Molnár M, Lang Z, Mátyás S, Rajczy K, et al. Prediction of in-vitro developmental competence of early cleavage-stage mouse embryos with compact time-lapse equipment. Reprod Biomed Online. 2010;20(3):371-9.##Conaghan J, Chen AA, Willman SP, Ivani K, Chenette PE, Boostanfar R, et al. Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial. Fertil Steril. 2013;100(2):412-9.e5.##Cruz M, Garrido N, Herrero J, Pérez-Cano I, Muñoz M, Meseguer M. Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality. Reprod Biomed Online. 2012;25(4):371-81.##Desai N, Ploskonka S, Goodman LR, Austin C, Goldberg J, Falcone T. Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles. Reprod Biol Endocrinol. 2014;12:54.##Coticchio G, Mignini Renzini M, Novara PV, Lain M, De Ponti E, Turchi D, et al. Focused time-lapse analysis reveals novel aspects of human fertilization and suggests new parameters of embryo viability. Hum Reprod. 2018;33(1):23-31.##

Correlation of Sperm Mitochondrial DNA 7345 bp and 7599 bp Deletions with Asthenozoospermia in Jordanian Population 2 1 2 Background: Alterations in sperm mitochondrial DNA (mtDNA) affect the functions of some OXPHOS proteins which will affect sperm motility and may be associated with asthenozoospermia. The purpose of this study was to investigate the correlation between 7599-bp and 7345-bp sperm mtDNA deletions and asthenozoospermia in Jordan. Methods: Semen specimens from 200 men including 121 infertile and 79 healthy individuals were collected at the Royal Jordanian Medical Services In-vitro fertilization (IVF) units. The mtDNA was extracted followed by mtDNA amplification. Polymerase chain reaction (PCR) was conducted for the target sequences, then DNA sequencing was performed for the PCR products. Chi-square, Fisher's and Spearman's tests were used to calculate the correlation. Results: The results showed a significant correlation between the presence of 7599-bp mtDNA deletion and infertility where the frequency of the 7599-bp deletion was 63.6 % in the infertile group compared to the fertile 34.2% (p<0.001, (OR=3.37, 95% CI=1.860 to 6.108)). Additionally, the sperm motility showed a significant association with the frequency of the 7599-bp deletion (p=0.001, r=-0.887). The 7345-bp mtDNA deletion showed no assoctiation with the infertility (p=0.65, (OR=0.837, 95% CI=0.464-1.51)) or asthenozoospermia (p=0.98, r=0.008). Conclusion: We demonstrated a significant correlation between asthenozoospermia and the 7599-bp mtDNA deletion but not the 7345-bp mtDNA deletion in the infertile men in Jordan. Screening for deletions in sperm mtDNA can be used as a pre-diagnostic molecular marker for male infertility. 165 173 Mazhar MS Al Zoubi Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University Jordan mszoubi@yu.edu.jo Ali M. AM Al-Talafha Department of Biological Sciences, Faculty of Science, Yarmouk University Department of Biological Sciences, Faculty of Science, Yarmouk University Jordan Emad E Al Sharu King Hussein Medical Centre, Royal Medical Services King Hussein Medical Centre, Royal Medical Services Jordan Bahaa B Al-Trad Department of Biological Sciences, Faculty of Science, Yarmouk University Department of Biological Sciences, Faculty of Science, Yarmouk University Jordan Ayman A AlZu'bi Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University Jordan Manal MI AbuAlarjah Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University Jordan Qasem Q Shehab Department of Clinical Sciences, Faculty of Medicine, Yarmouk University Department of Clinical Sciences, Faculty of Medicine, Yarmouk University Jordan Mohammad M Alsmadi Department of Gynecology and Reproductive Medicine, Faculty of Medicine, Saarland University Department of Gynecology and Reproductive Medicine, Faculty of Medicine, Saarland University Germany Khalid M. Kh Al-Batayneh Department of Biological Sciences, Faculty of Science, Yarmouk University Department of Biological Sciences, Faculty of Science, Yarmouk University Jordan AsthenozoospermiaGene deletionMale infertilityMitochondrial DNASperm motility 120113.pdf Brugo-Olmedo S, Chillik C, Kopelman S. Definition and causes of infertility. Reprod Biomed Online. 2001;2(1):41-53.##Demain LA, Conway GS, Newman WG. Genetics of mitochondrial dysfunction and infertility. Clin Genet. 2017;91(2):199-207.##Hull MG, Glazener CM, Kelly NJ, Conway DI, Foster PA, Hinton RA, et al. Population study of causes, treatment, and outcome of infertility. Br Med J (Clin Res Ed). 1985;291(6510):1693-7.##Kumar N, Singh AK. Trends of male factor infertility, an important cause of infertility: a review of literature. J Hum Reprod Sci. 2015;8(4):191-6.##Wang C, Swerdloff RS. Limitations of semen analysis as a test of male fertility and anticipated needs from newer tests. Fertil Steril. 2014;102(6):1502-7.##Ortega C, Verheyen G, Raick D, Camus M, Devroey P, Tournaye H. Absolute asthenozoospermia and ICSI: what are the options? Hum Reprod Update. 2011;17(5):684-92.##Baker HW, Clarke GN. Sperm morphology: consistency of assessment of the same sperm by different observers. Clin Reprod Fertil. 1987;5(1-2):37-43.##Amaral A, Lourenco B, Marques M, Ramalho-Santos J. Mitochondria functionality and sperm quality. Reproduction. 2013;146(5):R163-74.##Kao SH, Chao HT, Wei YH. Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa. Mol Hum Reprod. 1998;4(7):657-66.##Ambulkar PS, Waghmare JE, Chaudhari AR, Wankhede VR, Tarnekar AM, Shende MR, et al. Large scale 7436-bp deletions in human sperm mitochondrial DNA with spermatozoa dysfunction and male infertility. J Clin Diagn Res. 2016;10(11):GC09-GC12.##Pandey R, Mehrotra D, Catapano C, Choubey V, Sarin R, Mahdi AA, et al. Association of mitochondrial deoxyribonucleic acid mutation with polymorphism in CYP2E1 gene in oral carcinogenesis. J Oral Biol Craniofacial Res. 2012;2(1):4-9.##Kao S, Chao HT, Wei YH. Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Biol Reprod. 1995;52(4):729-36.##Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988;9(6):367-76.##Shamsi MB, Kumar R, Bhatt A, Bamezai RN, Kumar R, Gupta NP, et al. Mitochondrial DNA mutations in etiopathogenesis of male infertility. Indian J Urol. 2008;24(2):150-4.##St John JC, Jokhi RP, Barratt CL. Men with oligoasthenoteratozoospermia harbour higher numbers of multiple mitochondrial DNA deletions in their spermatozoa, but individual deletions are not indicative of overall aetiology. Mol Hum Reprod. 2001;7(1):103-11.##Talebi E, Karimian M, Nikzad H. 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Dietary Fatty Acid Intakes and the Outcomes of Assisted Reproductive Technique in Infertile Women 2 1 2 Background: The purpose of this study was evaluating the relationship between fatty acid (FA) intakes and the Assisted Reproductive Technique (ART) outcomes in infertile women. Methods: In this descriptive longitudinal study, a validated food frequency questionnaire (FFQ) was used to measure dietary intakes among 217 women with primary infertility seeking ART treatments at Isfahan Fertility and Infertility Center, Isfahan, Iran. The average number of total and metaphase II (MII) oocytes, the fertilization rate, the ratio of good and bad quality embryo and biochemical and clinical pregnancy were assessed. Analyses were performed using mean, standard deviation, Chi-square test, ANOVA, ANCOVA, logistic regression. Results: A total of 140 women were finally included in the study. There was a positive relationship between the average number of total and MII oocytes and the amount of total fatty acids (TFAs), saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), linoleic acids, linolenic acids, and oleic acids intakes, while eicosapentaenoic acids (EPAs) and docosahexaenoic acids (DHAs) intakes had an inverse relationship. Consuming more amounts of TFAs, SFAs, PUFAs, MUFAs, linoleic acids, and oleic acids was associated with the lower fertilization rate, whereas the consumption of linolenic acids and EPAs increased the fertilization rate. The ratio of good quality embryo was directly affected by the amount of PUFAs intakes. Additionally, there was a negative correlation between the amount of SFAs intakes and the number of pregnant women. Conclusion: TFAs, SFA, PUFA, and MUFA intakes could have both beneficial and adverse impacts on ART outcomes. 173 184 Maryam M Jahangirifar School of Nursing and Midwifery, Faculty of Medicine, Nursing and Health Sciences, Monash University School of Nursing and Midwifery, Faculty of Medicine, Nursing and Health Sciences, Monash University Australia Mahboubeh M Taebi Department of Midwifery and Reproductive Health, Isfahan University of Medical Sciences Department of Midwifery and Reproductive Health, Isfahan University of Medical Sciences Iran m_taebi@nm.mui.ac.ir Mohammad Hossein MH Nasr-Esfahani Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR Iran Motahar M Heidari-Beni Child Growth and Development Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences Child Growth and Development Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences Iran Gholam Hossein GhH Asgari Department of Community Nutrition, Isfahan University of Medical Sciences Department of Community Nutrition, Isfahan University of Medical Sciences Iran Assisted reproductive techniqueDietary fatsIn Vitro FertilizationInfertilityNutrition assessment 120114.pdf Rizk B, Olsen ME. 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Women’s Reports of Barriers to and Facilitators of Oral Medication Adherence During Ovarian Stimulation: A Mixed Methods Pilot Study 2 1 2 Background: Adherence to lifestyle modification recommendations remains problematic for women undergoing fertility treatment, raising concerns about the extent to which women adhere to prescribed medication regimens. Limited data have shown suboptimal oral medication adherence rates of 19% to 74%. The objective of this study was to explore what women perceive as barriers to and facilitators of oral medication adherence during fertility treatment cycles. Methods: An exploratory mixed methods pilot study was conducted among a sample of 30 women who were actively taking one to two cycles of letrozole or clomiphene citrate for ovarian stimulation in conjunction with intrauterine insemination cycles. Medication adherence barriers were measured using a 20-item survey. Medication adherence facilitators and personal experiences with fertility treatment were assessed with structured interviews. Medication adherence was assessed with electronic event monitoring. Results: The overall medication adherence median was 0.97 with a range of 0.75 to 1.00, and nine women (50%) demonstrated perfect adherence. The most commonly reported barriers were recently feeling sad, down, or blue (53%), and taking medication more than once per day (40%). Women with higher barrier scores had significantly lower medication adherence scores (p=0.02) compared to women with lower total barrier scores. Facilitators included using physical aides as reminders (60%) and establishing a daily routine (50%). No significant correlation was found between medication adherence scores and facilitators. Conclusion: The dynamic interplay between perceived barriers and facilitators and women’s medication-taking patterns could influence whether or not medication regimens are followed correctly. 184 201 Diane E DE Mahoney School of Nursing, University of Kansas Medical Center School of Nursing, University of Kansas Medical Center US dmahoney@kumc.edu Cynthia L CL Russell School of Nursing, University of Kansas Medical Center School of Nursing, University of Kansas Medical Center US Female infertilityOvarian stimulationPsychology 120112.pdf Chandra A, Copen CE, Stephen EH. Infertility and impaired fecundity in the United States, 1982-2010: data from the national survey of family growth. Natl Health Stat Report. 2013(67):1-18.##Thoma ME, McLain AC, Louis JF, King RB, Trumble AC, Sundaram R, et al. Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach. 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Int J Fertil Steril. 2014;8(3):303-14.##Gellad E, Grenard J, McGlynn EA. A review of barriers to medication adherence: a framework for driving policy options. Santa Monica, CA: RAND Corporation; 2009. 68 p.##Lakatos E, Szigeti JF, Ujma PP, Sexty R, Balog P. Anxiety and depression among infertile women: a cross-sectional survey from Hungary. BMC Womens Health. 2017;17(1):48.##Ogawa M, Takamatsu K, Horiguchi F. Evaluation of factors associated with the anxiety and depression of female infertility patients. Biopsychosoc Med. 2011;5(1):15.##Prasad S, Kumar Y, Nayar P, Prasad, S, Sharma G. A prospective study to assess the mental health and quality of life in women undergoing assisted reproduction. Fertil Sci Res. 2017;4(2):117-25.##Donarelli Z, Lo Coco G, Gullo S, Marino A, Volpes A, Salerno L, et al. Infertility-related stress, anxiety and ovarian stimulation: can couples be reassured about the effects of psychological factors on biological responses to assisted reproductive technology? Reprod Biomed Soc Online. 2016;3:16-23.##Nicoloro-SantaBarbara J, Busso C, Moyer A, Lobel M. Just relax and you'll get pregnant? Meta-analysis examining women's emotional distress and the outcome of assisted reproductive technology. Soc Sci Med. 2018;213:54-62.##Verkuijlen J, Verhaak C, Nelen WL, Wilkinson J, Farquhar C. Psychological and educational interventions for subfertile men and women. Cochrane Database Syst Rev. 2016;3(3):CD011034.##Chow KM, Cheung MC, Cheung IK. Psychosocial interventions for infertile couples: a critical review. J Clin Nurs. 2016;25(15-16):2101-13.##Frederiksen Y, O'Toole MS, Mehlsen MY, Hauge B, Elbaek HO, Zachariae R, et al. The effect of expressive writing intervention for infertile couples: a randomized controlled trial. Hum Reprod. 2017;32(2):391-402.##Boivin J, Griffiths E, Venetis CA. Emotional distress in infertile women and failure of assisted reproductive technologies: meta-analysis of prospective psychosocial studies. 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Predictors of medication adherence in the elderly: the role of mental health. Med Care Res Rev. 2018;75(6):746-61.##Thames AD, Moizel J, Panos SE, Patel SM, Byrd DA, Myers HF, et al. Differential predictors of medication adherence in HIV: findings from a sample of African American and Caucasian HIV-positive drug-using adults. AIDS Patient Care STDS. 2012;26(10):621-30.##Kalinderi K, Asimakopoulos B, Nikolettos N, Manolopoulos VG. Pharmacogenomics in IVF: a new era in the concept of personalized medicine. Reprod Sci. 2019;26(10):1313-25.##

Multiple Mutations in Exon-2 of Med-12 Identified in Uterine Leiomyomata 2 1 2 Background: Uterine leiomyomata (UL), commonly known as uterine fibroids, are benign smooth muscle tumors of the myometrium. They cause pelvic pain, abnormal uterine bleeding, and infertility in women of reproductive age. The ovarian hormone estrogen is the main stimulator for the fibroid growth. The etiology is not yet clearly understood; however, UL are believed to be monoclonal tumors arising from a common progenitor cell. Chromosomal cytogenetic abnormalities have been demonstrated in 40-50% of the fibroids. The most frequent tumor specific genetic alterations in UL were identified in exon-2 of Mediator Complex Subunit 12 (MED-12). Methods: In the present study, twenty-two multiple fibroids were evaluated both from the same uterus and from different uteri, of four women, for somatic mutations in hotspot region of MED-12. The tissue DNA of the UL's was isolated, amplified by PCR visualized on gel and sent for Sanger sequencing. Results: The results indicate several variants in exon-2 and flanking intronic regions, seven exonic variants and five intronic variants which provide evidence that multiple UL in the same uterus may not be clonal in origin. Conclusion: This study indicates genetic heterogeneity. UL may not have a clonal origin, these exon-2 variants of MED-12 gene could be involved in UL progression. 201 210 Ruqia R Firdaus Department of Genetics and Molecular Medicine, Vasavi Medical and Research Center, Lakdi-ka-pool Department of Genetics and Molecular Medicine, Vasavi Medical and Research Center, Lakdi-ka-pool India ruqiafirdaus@gmail.com Prabha P Agrawal Department of Gynaecology and Obstetrics, Medicover Hospitals, Hi–Tech City Department of Gynaecology and Obstetrics, Medicover Hospitals, Hi–Tech City India Manjula M Anagani Department of Gynaecology and Obstetrics, Medicover Hospitals, Hi–Tech City Department of Gynaecology and Obstetrics, Medicover Hospitals, Hi–Tech City India Kodati K Vijayalakshmi Department of Genetics and Molecular Medicine, Vasavi Medical and Research Center, Lakdi-ka-pool Department of Genetics and Molecular Medicine, Vasavi Medical and Research Center, Lakdi-ka-pool India Qurratulain Q Hasan Department of Biotechnology, Hyderabad Science Society Department of Biotechnology, Hyderabad Science Society India ClonalCodon 44Gene variantsMediator Complex Subunit 12Somatic mutationsUterine Leiomyoma 120120.pdf Flake GP, Andersen J, Dixon D. Etiology and pathogenesis of uterine leiomyomas: a review. Environ Health Perspect. 2003;111(8):1037-54. ##Stewart EA. Uterine fibroids. Lancet. 2001;357(9252):293-8.##Wallach EE, Vlahos NF. Uterine myomas: an overview of development, clinical features, and management. Obstet Gynecol. 2004:104(2):393-406.##Walker CL. Role of hormonal and reproductive factors in the etiology and treatment of uterine leiomyoma. Recent Prog Horm Res. 2002;57:277-94.##Shaik NA, Lone WG, Khan IA, Vaidya S, Rao KP, Kodati VL, et al. Detection of somatic mutations and germline polymorphisms in mitochondrial DNA of uterine fibroids patients. Genetic Test Mol Biomarkers. 2011;15(7-8):537-41.##Makinen N, Mehine M, Tolvanen J, Kaasinen E, Li YL, Lehtonen HJ, et al. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science. 2011;334(6053):252-5.##Croce S, Chibon F. MED12 and uterine smooth muscle oncogenesis: state of the art and perspectives. Eur J Cancer. 2015;51(12):1603-10.##Mehine M, Kaasinen E, Heinonen HR, Mäkinen N, Kämpjärvi K, Sarvilinna N, et al. Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers. Proc Natl Acad Sci USA. 2016;113(5):1315-20.##Darooei M, Khan F, Rehan M, Zubeda S, Jeyashanker E, Annapurna S, et al. MED12 somatic mutations encompassing exon 2 associated with benign breast fibroadenomas and not breast carcinoma in Indian women. J Cell Biochem. 2019;120(1):182-91.##Noor Ahmad S, Sujatha G, Vijaylakshmi K, Kaipa Prabhakar R, Qurratulain H. Polymorphic (CAG)n repeats in the androgen'receptor gene: a risk marker for endometriosis and uterine leiomyomas. Hematol Oncol Stem Cell Ther. 2009;2(1):289-93.##Lim WK, Ong CK, Tan J, Thike AA, Ng CC, Rajasegaran V, et al. Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma. Nat Genet. 2014;46(8):877-80.##Mäkinen N, Vahteristo P, Kämpjärvi K, Arola J, Bützow R, Aaltonen LA. MED12 exon 2 mutations in histopathological uterine leiomyoma variants. Eur J Hum Genet. 2013;21(11):1300-3.##Conaway RC, Conaway JW. Function and regulation of the mediator complex. Curr Opin Genet Dev. 2011;21(2):225-30.##Taatjes DJ. The human mediator complex: a versatile, genome-wide regulator of transcription. Trends Biochem Sci. 2010;35(6):315-22.##Näär AM, Taatjes DJ, Zhai W, Nogales E, Tjian R. Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev. 2002;16(11):1339-44.##Zhang X, Krutchinsky A, Fukuda A, Chen W, Yamamura S, Chait BT, et al. MED1/TRAP220 exists predominantly in a TRAP/Mediator subpopulation enriched in RNA polymerase II and is required for ER-mediated transcription. Mol Cell. 2005;19(1):89-100.##Malik S, Baek HJ, Wu W, Roeder RG. Structural and functional characterization of PC2 and RNA polymerase II-associated subpopulations of metazoan mediator. Mol Cll Biol. 2005;25(6):2117-29.##Baek HJ, Kang YK, Roeder RG. Human mediator enhances basal transcription by facilitating recruitment of transcription factor IIB during preinitiation complex assembly. J Biol Chem. 2006;281(22):15172-81.##Johnson KM, Wang J, Smallwood A, Arayata C, Carey M. TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev. 2002;16(14):1852-63.##Meyer KD, Lin SC, Bernecky C, Gao Y, Taatjes DJ. p53 activates transcription by directing structural shifts in mediator. Nat Struct Mol Biol. 2010;17(6):753-60.##Turunen M, Spaeth JM, Keskitalo S, Park MJ, Kivioja T, Clark AD, et al. Uterine leiomyoma-linked MED12 mutations disrupt mediator-associated CDK activity. Cell Rep. 2014;7(3):654-60.##Kämpjärvi K, Park MJ, Mehine M, Kim NH, Clark AD, Bützow R, et al. Mutations in Exon 1 highlight the role of MED 12 in uterine leiomyomas. Hum Mutat. 2014;35(9):1136-41.##Cani AK, Hovelson DH, McDaniel AS, Sadis S, Haller MJ, Yadati V, et al. Next-gen sequencing exposes frequent MED12 mutations and actionable therapeutic targets in phyllodes tumors. Mol Cancer Res. 2015;13(4):613-9.##Pfarr N, Kriegsmann M, Sinn P, Klauschen F, Endris V, Herpel E, et al. Distribution of MED12 mutations in fibroadenomas and phyllodes tumors of the breast—implications for tumor biology and pathological diagnosis. Genes Chromosomes Cancer. 2015;54(7):444-52.##McGuire MM, Yatsenko A, Hoffner L, Jones M, Surti U, Rajkovic A. Whole exome sequencing in a random sample of North American women with leiomyomas identifies MED12 mutations in majority of uterine leiomyomas. PloS One. 2012;7(3):e33251.##Mäkinen N, Heinonen HR, Moore S, Tomlinson IP, Van Der Spuy ZM, Aaltonen LA. MED12 exon 2 mutations are common in uterine leiomyomas from South African patients. Oncotarget. 2011;2(12):966-9.##Mäkinen N, Vahteristo P, Bützow R, Sjöberg J, Aaltonen LA. Exomic landscape of MED12 mutation‐negative and‐positive uterine leiomyomas. Int J Cancer. 2014;134(4):1008-12.##Ajabnoor G, Mohammed NA, Banaganapalli B, Abdullah LS, Bondagji ON, Mansouri N, et al. Expanded somatic mutation spectrum of MED12 gene in Uterine Leiomyomas of Saudi Arabian Women. Front Genet. 2018;9:552.##Je EM, Kim MR, Min KO, Yoo NJ, Lee SH. Mutational analysis of MED12 exon 2 in uterine leiomyoma and other common tumors. Int J Cancer. 2012;131(6):E1044-7.##Markowski DN, Bartnitzke S, Löning T, Drieschner N, Helmke BM, Bullerdiek J. MED12 mutations in uterine fibroids—their relationship to cytogenetic subgroups. Int J Cancer. 2012;131(7):1528-36.##Schwetye KE, Pfeifer JD, Duncavage EJ. MED12 exon 2 mutations in uterine and extrauterine smooth muscle tumors. Hum Pathol. 2014;45(1):65-70.##Halder SK, Laknaur A, Miller J, Layman LC, Diamond M, Al-Hendy A. Novel MED12 gene somatic mutations in women from the Southern United States with symptomatic uterine fibroids. Mol Genet Genomics. 2015;290(2):505-11.##Perot G, Croce S, Ribeiro A, Lagarde P, Velasco V, Neuville A, et al. MED12 alterations in both human benign and malignant uterine soft tissue tumors. PloS One. 2012;7(6):e40015.##Bertsch E, Qiang W, Zhang Q, Espona-Fiedler M, Druschitz S, Liu Y, et al. MED12 and HMGA2 mutations: two independent genetic events in uterine leiomyoma and leiomyosarcoma. Mod Pathol. 2014;27(8):1144-53.##Kim S, Xu X, Hecht A, Boyer TG. Mediator is a transducer of Wnt/β-catenin signaling. J Biol Chem. 2006;281(20):14066-75.##Al-Hendy A, Laknaur A, Diamond MP, Ismail N, Boyer TG, Halder SK. Silencing Med12 gene reduces proliferation of human leiomyoma cells mediated via Wnt/β-catenin signaling pathway. Endocrinology. 2017;158(3):592-603.##Piscuoglio S, Murray M, Fusco N, Marchiò C, Loo FL, Martelotto LG, et al. 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In Silico Analysis of Sperm From Ejaculates with Different Semen Characteristics 2 1 2 Background: Male infertility is associated with altered characteristics of the sperm within the ejaculate. It is possible to find molecular explanations for the observed phenotypes and their consequences. This study aimed to analyze, using a specialized software, a gene set of transcriptomic data from different types of ejaculates. Methods: Data from ejaculate samples categorized as normal, oligospermia, and teratozoospermia were obtained from Gene Expression Omnibus (GEO). After normalization, the data average for each sample category was calculated and analyzed independently using Ingenuity Pathway Analysis (IPA). Results: Five important canonical pathways are involved in normal and altered semen samples (Oligospermia and teratozoospermia) except sirtuin signaling and mitochondrial dysfunction pathways. The five most important biological processes are identified in all semen phenotypes, but the only difference is the genes connected with initiation of RNA transcription in oligospermic and asthenospermic samples. Conclusion: Surprisingly, different types of ejaculates share many pathways and biological processes; sperm proteomics as a new global approach gives clues for the development of strategies to explain the reason for observed phenotypes of ejaculated spermatozoa, their possible effect on fertility, and for implementing research strategies in the context of infertility diagnosis and treatment. 210 216 Jesús Alfredo JAB Gutiérrez Reproduction Group, Department of Microbiology and Parasitology, Medical School, University of Antioquia Reproduction Group, Department of Microbiology and Parasitology, Medical School, University of Antioquia Colombia Walter D WC Maya Reproduction Group, Department of Microbiology and Parasitology, Medical School, University of Antioquia Reproduction Group, Department of Microbiology and Parasitology, Medical School, University of Antioquia Colombia wdario.cardona@udea.edu.co FertilityMaleRNASpermatozoaTranscriptome 120122.pdf de los Rios J, Cardona WD, Berdugo JA, Correa C, Arenas A, Olivera-Angel M, et al. 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Available from: https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/##Jodar M, Sendler E, Krawetz SA. The protein and transcript profiles of human semen. Cell Tissue Res. 2016;363(1):85-96.##Selvaraju S, Parthipan S, Somashekar L, Kolte AP, Binsila BK, Arangasamy A, et al. Occurrence and functional significance of the transcriptome in bovine (Bos taurus) spermatozoa. Sci Rep. 2017;7:42392.##Fraser L, Brym P, Pareek CS, Mogielnicka-Brzozowska M, Jastrzębski JP, Wasilewska-Sakowska K, et al. Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq. Theriogenology. 2020;142:400-13.##Bai M, Sun L, Jia C, Li J, Han Y, Liu H, et al. Integrated analysis of miRNA and mRNA expression profiles reveals functional miRNA-targets in development testes of small tail han sheep. G3 (Bethesda). 2019;9(2):523-33.##Montjean D, De La Grange P, Gentien D, Rapinat A, Belloc S, Cohen-Bacrie P, et al. 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A RNA-Seq analysis to describe the boar sperm transcriptome and its seasonal changes. Front Genet. 2019;10:299.##Pelloni M, Paoli D, Majoli M, Pallotti F, Carlini T, Lenzi A, et al. Molecular study of human sperm RNA: Ropporin and CABYR in asthenozoospermia. J Endocrinol Invest. 2018;41(7):781-7.##KEGG. Kyoto Encyclopedia of Genes and Genomes [Internet]. Available from: https://www.ge-nome.jp/kegg/.##Fu H, Wada-Hiraike O, Hirano M, Kawamura Y, Sakurabashi A, Shirane A, et al. SIRT3 positively regulates the expression of folliculogenesis-and luteinization-related genes and progesterone secretion by manipulating oxidative stress in human luteinized granulosa cells. Endocrinology. 2014;155(8):3079-87.##Coussens M, Maresh JG, Yanagimachi R, Maeda G, Allsopp R. Sirt1 deficiency attenuates spermatogenesis and germ cell function. PloS One. 2008;3(2):e1571.##Liu C, Song Z, Wang L, Yu H, Liu W, Shang Y, et al. 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The presence, role and clinical use of spermatozoal RNAs. Hum Reprod Update. 2013;19(6):604-24.##Johnson GD, Sendler E, Lalancette C, Hauser R, Diamond MP, Krawetz SA. Cleavage of rRNA ensures translational cessation in sperm at fertilization. Mol Hum Reprod. 2011;17(12):721-6.##Lewis SE. Is sperm evaluation useful in predicting human fertility? Reproduction. 2007;134(1):31-40.##Ostermeier GC, Miller D, Huntriss JD, Diamond MP, Krawetz SA. Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature. 2004;429(6988):154.##Hermann BP, Cheng K, Singh A, Roa-De La Cruz L, Mutoji KN, Chen IC, et al. The mammalian spermatogenesis single-cell transcriptome, from spermatogonial stem cells to spermatids. Cell Rep. 2018;25(6):1650-67.e8.##Garcia-Herrero S, Meseguer M, Martinez-Conejero JA, Remohi J, Pellicer A, Garrido N. The transcriptome of spermatozoa used in homologous intrauterine insemination varies considerably between samples that achieve pregnancy and those that do not. 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Persistent Mullerian Duct Syndrome: A Rare Case of an Adult Infertile Male with Bilateral Cryptorchidism 2 1 2 Background: Persistent mullerian duct syndrome (PMDS) is a very rare form of internal male pseudohermaphroditism in individuals who are phenotypically males with 46 XY karyotypes harbouring internal female reproductive organs which are Mullerian derivatives. It occurs as a defect in the genes coding for the Mullerian inhibiting substance (MIS) or the anti Mullerian hormone (AMH) receptor, ultimately leading to failure of regression of Mullerian ducts. Case Presentation: A 29-year-old male with PMDS presented with complaints of primary infertility. Diagnosis was made with the help of high index of suspicion, radiological imaging, and karyotyping. Our patient underwent exploratory laparotomy with hysterectomy and bilateral orchidopexy. Conclusion: The purpose of this study was increasing awareness regarding rare entities and surgeons should have high clinical suspicion of PMDS when patient with bilateral undescended testis comes for the evaluation of primary infertility. 216 220 Prakash P Sankapal Department of Urology, Grant Government Medical College and Sir JJ Hospital Department of Urology, Grant Government Medical College and Sir JJ Hospital India Venkat VA Gite Department of Urology, Grant Government Medical College and Sir JJ Hospital Department of Urology, Grant Government Medical College and Sir JJ Hospital India balajigite@yahoo.com Agrawal A Mayank Department of Urology, Grant Government Medical College and Sir JJ Hospital Department of Urology, Grant Government Medical College and Sir JJ Hospital India Mahesh M Sane Department of Urology, Grant Government Medical College and Sir JJ Hospital Department of Urology, Grant Government Medical College and Sir JJ Hospital India Atul A Singal Department of Urology, Grant Government Medical College and Sir JJ Hospital Department of Urology, Grant Government Medical College and Sir JJ Hospital India Anti Mullerian hormone (AMH)CryptorchidismHysterectomyKaryotypingMullerian inhibiting substance (MIS)OrchidopexyPersistent mullerian duct syndrome (PMDS)Primary infertility 120119.pdf Nerune SM, Hippargi SP, Mestri NB, Mehhrotra NM. Persistant mullerian duct syndrome with ovarian endometriosis - a rare case report. J Chin Diagn Res. 2016:10(2):ED14-5.##Deepika AK. Persistent mullerian duct syndrome with transverse testicular ectopia: rare entity. J Clin Diagn Res. 2014;8(3):162-3.##Wuerstle M, Lesser T, Hurwitz R, Applebaum H, Lee SL. Persistent mullerian duct syndrome and transverse testicular ectopia: embryology, presentation, and management. J Pediatr Surg. 2007;42(12): 2116-9.##Guerrier D, Tran D, Vanderwiden JM, Hideux S, Van Outryve L, Legeal L, et al. The persistent mullerian duct syndrome: a molecular approach. J Clin Endocrinol Metab. 1989;68(1):46-52.##Loeff DS, Imbeaud S, Reyes HM, Meller JL, Rosenthal IM. Surgical and genetic aspects of persistence mullerian duct syndrome. J Paediatr Surg. 1994:29(1):61-5.## Manjunath BG, Shenoy VG, Raj P. Persistent müllerian duct syndrome: how to deal with the müllerian duct remnants-a review. Indian J Surg. 2010;72(1):16-9.##Wu HC, Chen JH, Lu HF, Shen WC. Persistent müllerian duct syndrome wtih seminoma: CT findings. AJR Am J Roentgenol. 2000;174(1):102-4.##Palanisamy S, Patel ND, Sabnis SC, Palanisamy N, Vijay A, Chinnusamy P. Laparoscopic hysterectomy with bilateral orchidectomy for persistent mullerian duct syndrome with seminoma testes: case report. J Minim Access Surg. 2015;11(4):273-5.##Gutte AA, Pendharkar PS, Sorte SZ. Transverse testicular ectopia associated with persistent Mullerian duct syndrome-the role of imaging. Br J Radiol. 2008;81(967):e176-8.##

Successful Management of Caesarean Scar Ectopic Pregnancies: A Report of Five Cases 2 1 2 Background: Cesarean section scar ectopic pregnancy (CSEP) is a rare and poten-tially life-threatening condition. A standardized management protocol is yet to be established owing to limited data available. Case Presentation: In this paper, five cases of CSEP over a period of 18 months at a tertiary referral hospital, managed medically with methotrexate administered both systemically and into the gestational sac at the time of feticide with potassium chloride (KCL) are presented. Surgical management was the second line therapy when medical treatment failed. Conclusion: With rising trends in cesarean deliveries, CSEP may be a challenge which requires close investigation regarding its diagnosis and treatment on the merits of case studies and available healthcare facilities. 220 225 Neena N Malhotra Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences India Nilofar N Noor Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences India Piyush P Bansal Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences India K. Aparna KA Sharma Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences India kaparnasharma@gmail.com Caesarean sectionEctopic pregnancyGestational sacMethotrexate 120127.pdf No authors listed. Diagnosis and management of ectopic pregnancy: Green-top Guideline No. 21. BJOG. 2016;123(13):e15-55.##Vial Y, Petignat P, Hohlfeld P. Pregnancy in a cesarean scar. Ultrasound. Obstet Gynecol. 2000;16(6):592-3.##Larsen JV, Solomon MH. Pregnancy in a uterine scar sacculus--an unusual cause of postabortal haemorrhage. A case report. South Afr Med J Suid-Afr Tydskr Vir Geneeskd. 1978;53(4):142-3.##Jayaram PM, Okunoye GO, Konje J. Caesarean scar ectopic pregnancy: diagnostic challenges and management options. Obstet Gynaecol. 2017;19(1):13-20.##Qian ZD, Guo QY, Huang LL. Identifying risk factors for recurrent cesarean scar pregnancy: a case-control study. Fertil Steril. 2014;102(1):129-34.e1.##Gonzalez N, Tulandi T. Cesarean scar pregnancy: a systematic review. J Minim Invasive Gynecol. 2017;24(5):731-8.##Jayaram P, Okunoye G, Al Ibrahim AA, Ghani R, Kalache K. Expectant management of caesarean scar ectopic pregnancy: a systematic review. J Perinat Med. 2018;46(4):365-72.##Seow KM, Wang PH, Huang LW, Hwang JL. Transvaginal sono-guided aspiration of gestational sac concurrent with a local methotrexate injection for the treatment of unruptured cesarean scar pregnancy. Arch Gynecol Obstet. 2013;288(2):361-6.##Sadeghi H, Rutherford T, Rackow BW, Campbell KH, Duzyj CM, Guess MK, et al. Cesarean scar ectopic pregnancy: case series and review of the literature. Am J Perinatol. 2010;27(2):111-20.##Cheung VYT. Local Methotrexate injection as the first-line treatment for cesarean scar pregnancy: review of the literature. J Minim Invasive Gynecol. 2015;22(5):753-8.##Gupta M, Kriplani A, Mahey R, Kriplani I. Successful management of caesarean scar live ectopic pregnancies with local KCL and systemic methotrexate. BMJ Case Rep. 2017;2017:bcr 2017221844.##Kaur R, Goel B, Sehgal A, Goyal P, Mehra R. Feticide with intracardiac potassium chloride to reduce risk of hemorrhage in medical termination of pregnancy. J Gynecol Women Healthc. 2018;1(1):1-4.##Roy MM, Radfar F. Management of a viable cesarean scar pregnancy: a case report. Oman Med J. 2017;32(2):161-6.##Deans R, Abbott J. Hysteroscopic management of cesarean scar ectopic pregnancy. Fertil Steril. 2010;93(6):1735-40.##Ben Nagi J, Helmy S, Ofili-Yebovi D, Yazbek J, Sawyer E, Jurkovic D. Reproductive outcomes of women with a previous history of caesarean scar ectopic pregnancies. Hum Reprod. 2007;22(7):2012-5.##Wang Q, Peng HL, He L, Zhao X. Reproductive outcomes after previous cesarean scar pregnancy: Follow up of 189 women. Taiwan J Obstet Gynecol. 2015;54(5):551-3.##