Freeze all Policy: An Expanded Strategy Before Its Clinical Approval 2 1 2 The introduction of vitriﬁcation method for cryopreservation of gametes/embryo in assissted reproductive technology (ART) increased the efficacy of freez-thawing method esspecislly its post-thawing survival rate. This efficient method provides an excellent solution to postpone embryo transfer in natural cycles with adequate number and timing for main challenges of ART such as polycystic ovarian syndrome (PCOS), pre-implantation genetics diagnosis/screening (PGD/PGS), advanced female age, low responders, repeated implantation failure (RIF), multiple pregnancy, third-party reproduction, etc (1). The first baby was born from frozen-thawed embryos in Australia in 1984 and subsequently in USA in1986. Regarding limitations of cryopreservation techniques, its low efficiency and low post-thawing survival rate of embryos, less than 1% of all cycles were frozen-thawed embryo transfer (FET) at that time. It increased up to 30% in 2004 and this rate is growing not only due to its better success rate compared to fresh embryo transfer but also because of worldwide advocacy of elective single embryo transfer (eSET) over multiple embryo transfer. At recent, it is estimated that more than 50% of the children born following ART are from frozen-thawing cycles (2). The success rate of IVF was less than 1% at the birth of Louise Brown, but its success rate has increased with the development of ART techniques up to 30% in 2000. The speed of growth has declined since then; however, the development of cryopreservation techniques leads to more success of frozen-thawing cycles than fresh ET, so that the overall success of frozen-thawing blastocyst transfer cycles is more than 50% and in some cases, the use of embryo transfer together with PGS techniques increase the ART success rate up to 80% (3, 1). At recent, an efficient cryopreservation technique for gametes, embryos and reproductive tissues has important functions that are critical and the main pratice of each ART program. It extends time for embryo evaluation, allows systematic application of elective single embryo transfer policy, enables egg banking for donation and/or for oocyte accumulation, permits fertility preservation for medical and non-medical indications, enhances cumulative live birth rate per oocyte retrieval cycle and also provides the opportunity to perform cycle segmentation. Following the successful birth of the first IVF baby and low success rate of fresh ET, Edwards and Steptoe discussed the negative effects of supper physiological levels of exogenous and endogenous hormones during ovarian stimulation on endometrium and its receptivity. Therefore, they suggested that freezing all embryos for subsequent transfers of thawed embryos is the best method (3). Unfortunately, the cryopreservation technology was very poor especially at early stages of its development, so the first IVF baby was born from a fresh ET and thus fresh ET became the default standard of ART. Over time, this approach has changed, so the rate of fresh ET reduced and in return the rate of frozen embryo transfer increased, so that the freeze all policy is going to be the dominant dialogue and practice in ART. Despite limited evidence, there is some concern on its effectiveness, increased cost-per-pregnancy and time-to-pregnancy (1). Similar to all medical procedures, in spite of several advantages and strengths of freeze all policy, it has some threats especially significantly increased rate of large-for-gestational age (LGA) babies in frozen cycles (4). There are few randomized clinical trials (RCT) in comparing freeze all protocols with fresh embryo transfer; however, most studies are not randomized and also there are four acceptable RCT works in different groups of infertile couples which are difficult to be compared. According to these results, benefits of freeze all strategy are likely for patients with good ovarian response and women with suboptimal/poor response had better results with fresh ET. Moreover, obstetric and perinatal outcomes regarding frozen ET are controversial (5). In addition, studies about cost and time concerns in freeze all policy do not seem to be valid since in some cases, the time spent and the cost per successful clinical pregnancy is less than that in fresh ET (6, 7). Since the health of ART born children during the whole length of life is important, considering above concerns, caution should be taken in using freeze all policy universally for all infertile couples. Therefore, more evidence is needed on the ramifications of freezing embryos and until then ART programs might adopt an individualized policy. Future studies may confirm the advantages of FET or refuse these findings and support fresh ET. Therefore, clinicians should carefully decide before adopting freeze all embryos as a beneficent policy for all patients. 341 343 Mohammad Reza MR Sadeghi محمدرضا صادقی Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran sadeghi@avicenna.ac.ir No Keyword 6.pdf Basile N, Garcia-Velasco JA. The state of "freeze-for-all" in human ARTs. J Assist Reprod Genet. 2016;33(12):1543-50.##Evans J, Hannan NJ, Edgell TA, Vollenhoven BJ, Lutjen PJ, Osianlis T, et al. Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence. Hum Reprod Update. 2014;20(6):808-21.##Shapiro BS, Garner FC. Recurrent implantation failure is another indication for the freeze-all strategy. Fertil Steril. 2017;108(1):44.##Blockeel C, Drakopoulos P, Santos-Ribeiro S, Polyzos NP, Tournaye H. A fresh look at the freeze-all protocol: a SWOT analysis. Hum Reprod. 2016;31(3):491-7.##Ata B, Seli E. A universal freeze all strategy: why it is not warranted. Curr Opin Obstet Gynecol. 2017;29(3):136-45.##Lattes K, Checa MA, Vassena R, Brassesco M, Vernaeve V. There is no evidence that the time from egg retrieval to embryo transfer affects live birth rates in a freeze-all strategy. Hum Reprod. 2017;32(2):368-74.##Roque M, Valle M, Guimarães F, Sampaio M, Geber S. Cost-effectiveness of the freeze-all policy. JBRA Assist Reprod. 2015;19(3):125-30.##

Vitrification of Mouse MII Oocyte Decreases the Mitochondrial DNA Copy Number, TFAM Gene Expression and Mitochondrial Enzyme Activity 2 1 2 Background: The objective of this study was determination of the changes in the reactive oxygen species (ROS) level, mitochondrial DNA (mtDNA) copy number and enzyme activity and transcription factor A (TFAM) gene expression in oocytes after vitrification. Methods: The oocytes at metaphase II (MII) stage (n=320) were collected from superovulated adult female mice (n=40). These oocytes were divided into vitrified and non-vitrified groups (n=160 in each group). After vitrification of oocytes, ROS level, mtDNA copy number; TFAM gene expression and mitochondrial enzymes activity (cytochrome C oxidase and succinate dehydrogenase) were assessed and compared with non-vitrified group. Visualization of the mitochondria was done using Mitotracker green staining under confocal microscope. Data were compared by independent T-test. Values of p<0.05 were considered as statistically significant. Results: The survival rate of oocytes after vitrification and warming was 96.05%. The intensity of cytochrome C oxidase activity, mtDNA copy number and TFAM gene expression in non-vitrified oocytes were significantly lower and the level of ROS was higher in vitrified oocytes in comparison with non-vitrified group (p<0.05). But the intensity of succinate dehydrogenase activity was not significantly different between the two groups. The pattern of mitochondrial distribution in two groups of study was similar but the intensity of Mitotracker green in non-vitrified oocytes was significantly higher than vitrified oocytes (p<0.05). Conclusion: This study showed that vitrification of mouse MII oocytes reduced the mtDNA copy number and mitochondrial cytochrome C oxidase activity by increasing ROS level, thus the subsequent embryo development may be affected. 343 352 Mahboobeh M Amoushahi Department of Anatomy, Tarbiat Modares University Department of Anatomy, Tarbiat Modares University Iran Mojdeh M Salehnia مژده صالح نیا Department of Anatomy, Tarbiat Modares University Department of Anatomy, Tarbiat Modares University Iran mogdeh@dr.com; salehnim@modares.ac.ir Seyed Javad SJ Mowla سید جواد مولی Department of Biotechnology, Faculty of Biological Sciences, Tarbiat Modares University Department of Biotechnology, Faculty of Biological Sciences, Tarbiat Modares University Iran Cytochrome c oxidasemtDNA copy numberReactive oxygen speciesSuccinate dehydrogenase 724.pdf Roque M, Valle M, Kostolias A, Sampaio M, Geber S. Freeze-all cycle in reproductive medicine: current perspectives. 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The Effect of Curcumin on TNF-α, IL-6 and CRP Expression in a Model of Polycystic Ovary Syndrome as an Inflammation State 2 1 2 Background: Having low-grade chronic inflammation such as elevated C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) plays a crucial role in polycystic ovary syndrome (PCOS). This study aimed at investigating the therapeutic effects of curcumin on IL-6, CRP and TNF-α and symptoms of polycystic ovary syndrome. Methods: In this research, 72 female adult Wistar rats were divided into control (n=12), PCOS (n=12) and curcumin-treated PCOS groups (n=48). PCOS was induced by injection of estradiol valerate (2 mg/kg- one-step). PCOS rats were divided into control and experimental groups which received daily intraperitoneal injection of curcumin. After 60 days of syndrome induction, ovaries were collected for histological and immunohistochemical evaluations. Serum IL-6 and CRP was detected by the ELISA kit. Data were analyzed using In-Stat 3 via one-way analysis of variance (ANOVA) and p<0.05 was considered statistically significant. Results: Histological studies showed a significant reduction in thickness of theca layer and increase in the number of corpus luteum (CL) diameter in the curcumin-treated group compared with the PCOS group; also inflammatory markers such as IL-6 and CRP significantly decreased in groups treated with curcumin compared with PCOS groups. Regarding immunohistochemical analysis, the expression of TNF-α in granulosa layer and follicular fluid of follicles and ovarian cysts in PCOS group was more than the control group's expression. However, expression of this factor in the ovaries treated with curcumin was decreased. Conclusion: This study showed that the anti-inflammatory and antioxidant effects of curcumin on PCOS may be due to its inhibitory effect on expression and levels of TNF-α, serum IL-6 and CRP. 352 361 Shima Sh Mohammadi Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Iran Parvin P Kayedpoor Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Iran Latifeh L Karimzadeh-Bardei Laboratory’s Animal Center & Cellular and Molecular Research Laboratory, Faculty of Biological Sciences, Kharazmi University Laboratory’s Animal Center & Cellular and Molecular Research Laboratory, Faculty of Biological Sciences, Kharazmi University Iran Mohammad M Nabiuni Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University Iran CRPCurcuminExperimental modelIL-6ImmunohistochemistryInflammationPCOSTNF-α 717.pdf Amer SAK. 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Endocr Abstracts. 2006;11:705.##Heutling D, Schulz H, Nickel I, Kleinstein J, Kaltwasser P, Krzyzanowska K, et al. Endothelial, inflammatory and endocrine markers in women with PCOS before and after metformin treatment. Exp Clin Endocrinol Diabetes. 2006;114(S 1):P15-195.##Ortega I, Stener-Victorin E, Villanueva JA, Sokalska A, Stanley SD, et al. Letrozole increases growth of rat theca-interstitial cells and Cyp17a1 gene expression in the rat ovary. Fertil Steril. 2011;96(7):447-59.##Marino JS, Iler J, Dowling AR, Chua S, Bruning JC, Coppari R, et al. Adipocyte dysfunction in a mouse model of polycystic ovary syndrome (PCOS): evidence of adipocyte hypertrophy and tissue-specific inflammation. PLoS One. 2012;7(10):e48643.##Schaaf C, Shan B, Buchfelder M, Losa M, Kreutzer J, Rachinger W, et al. Curcumin acts as anti-tumorigenic and hormone-suppressive agent in murine and human pituitary tumour cells in vitro and in vivo. 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Occult Form of Premature Ovarian Insufficiency in Women with Infertility and Oligomenorrhea as Assessed by Poor Ovarian Response Criteria 2 1 718 2 Background: The purpose of this study was to evaluate the ability of poor ovarian response criteria to classify women presenting with infertility and oligomenorrhea as having "occult" premature ovarian insufficiency. Methods: This was a cross sectional study conducted at Aziz Medical Center, Karachi, Pakistan from 1st August 2015 to 31st July 2016. Women with infertility and oligomenorrhea were included. All eligible women underwent day 2 FSH level and an early follicular phase transvaginal ultrasound to assess the antral follicular count (AFC). All women then underwent the confirmatory test, of Anti- Mullerian Hormone (AMH) level. The main outcome measure was assignment to occult premature ovarian insufficiency (POI) after screening that used the criteria set out in fertility guideline for predicting the likely ovarian response to gonadotrophin stimulation. Another measure was to compare the sensitivity and specificity of the two index criteria, of FSH and AFC, relative to the emerging reference standard, of the AMH criterion. Results: The three criteria together classified 59 (34.91%) women as occult POI in those with oligomenorrhea. The sensitivity, specificity, negative predictive value and positive predictive value of FSH relative to AMH for these women were 77.8%, 95.7%, 90.2% and 89.4%, respectively whereas the same values of AFC relative to AMH were 92.6%, 99.1 %, 96.6% and 98%, respectively. Conclusion: Women with menstrual irregularity and infertility are at a higher risk for satisfying criteria of poor ovarian response irrespective of age. A policy incorporating these surrogate markers can be used to screen these women for occult premature ovarian insufficiency. 361 368 Rubina R Izhar Department of Gynaecology and Obstetrics, Abbasi Shaheed Hospital and Karachi Medical and Dental College Department of Gynaecology and Obstetrics, Abbasi Shaheed Hospital and Karachi Medical and Dental College Pakistan Samia S Husain Department of Gynaecology and Obstetrics, Abbasi Shaheed Hospital and Karachi Medical and Dental College Department of Gynaecology and Obstetrics, Abbasi Shaheed Hospital and Karachi Medical and Dental College Pakistan samiahusain_scorpio@ hotmail.com Suhaima S Tahir Aziz Medical Center Aziz Medical Center Pakistan Sonia S Husain Department of Gynaecology and Obstetrics, Abbasi Shaheed Hospital and Karachi Medical and Dental College Department of Gynaecology and Obstetrics, Abbasi Shaheed Hospital and Karachi Medical and Dental College Pakistan Female infertilityOccult premature ovarian insufficiencyOligomenorrheaOvarian reservePremature ovarian ageingPremature ovarian failure 718.pdf Nelson LM. Primary ovarian insufficiency. N Engl J Med. 2009;360:606-14.##Tucker EJ, Grover SR, Bachelot A, Touraine P, Sinclair AH. Premature ovarian insufficiency: new perspectives on ge-netic cause and phenotypic spectrum. Endocr Rev. 2016;37(6):609-35.##European Society for Human Reproduction and Em-bryology (ESHRE) Guideline Group on POI, Webber L, Davies M, Anderson R, Bartlett J, Braat D, et al. ESHRE Guideline: management of women with premature ovarian insufficiency. Hum Reprod. 2016;31(5):926-37.##Gleicher N, Weghofer A, Barad DH. Defining ovarian reserve to better understand ovarian aging. Reprod Biol Endocrinol. 2011;9:23.##Gleicher N, Kushnir VA, Barad DH. Prospectively assessing risk for premature ovarian senescence in young females: a new paradigm. Reprod Biol Endo-crinol. 2015;13:34.##Cameron IT, O'Shea FC, Rolland JM, Hughes EG, de Kretser DM, Healy DL. Occult ovarian failure: a syndrome of infertility, regular menses, and elevated follicle-stimulating hormone concentrations. J Clin Endocrinol Metab. 1988;67(6):1190-4.##Bedoschi G, Turan V, Oktay K. Fertility preservation options in women with endometriosis. Minerva Ginecol. 2013;65(2):99-103.##Cohen J, Chabbert-Buffet N, Darai E. Diminished ovarian reserve, premature ovarian failure, poor ovarian responder--a plea for universal definitions. J Assist Reprod Genet. 2015;32(12):1709-12.##Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L; ESHRE working group on poor ovarian response definition. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertiliza-tion: the Bologna criteria. Hum Reprod. 2011;26(7):1616-24.##National Institute for Health and Care Excellence. Fertility problems: assessment and treatment. London: NICE; 2013 p.##The Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004;19:41-7.##Iliodromiti S, Kelsey TW, Wu O, Anderson RA, Nelson SM. The predictive accuracy of anti-Mül-lerian hormone for live birth after assisted conception: a systematic review and meta-analysis of the literature. Hum Reprod Update. 2014;20(4):560-70.##Laven JS. Primary Ovarian Insufficiency. Semin Reprod Med. 2016;34(4):230-4.##Baker VL. Primary ovarian insufficiency in the adolescent. Curr Opin Obstet Gynecol. 2013;25(5):375-81.##Gleicher N, Kushnir VA, Barad DH. Prospectively assessing risk for premature ovarian senescence in young females: a new paradigm. Reprod Biol Endocrinol. 2015;13:34.##Shestakova IG, Radzinsky VE, Khamoshina MB. Occult form of premature ovarian insufficiency. Gynecol Endocrinol. 2016;32(sup2):30-2.##Gizzo S, Andrisani A, Noventa M, Quaranta M, Esposito F, Armanini D, et al. Menstrual cycle length: a surrogate measure of reproductive health capable of improving the accuracy of biochemical/sonographical ovarian reserve test in estimating the reproductive chances of women referred to ART. Reprod Biol Endocrinol. 2015;13:28.##Devine K, Mumford SL, Wu M, DeCherney AH, Hill MJ, Propst A. Diminished ovarian reserve in the United States assisted reproductive technology population: diagnostic trends among 181,536 cycles from the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System. Fertil Steril. 2015;104(3):612-19.e3.##Toner JP, Seifer DB. Why we may abandon basal follicle-stimulating hormone testing: a sea change in determining ovarian reserve using antimüllerian hormone. Fertil Steril. 2013;99(7):1825-30.##Jamil Z, Fatima SS, Ahmed K, Malik R. Anti-Mul-lerian Hormone: Above and Beyond Conventional Ovarian Reserve Markers. Dis Markers. 2016;2016:5246217.##Fleming R, Seifer DB, Frattarelli JL, Ruman J. As-sessing ovarian response: antral follicle count versus anti-Müllerian hormone. Reprod Biomed Online. 2015;31(4):486-96.##Pearson K, Long M, Prasad J, Wu YY, Bonifacio M. Assessment of the Access AMH assay as an automated, high-performance replacement for the AMH Generation II manual ELISA. Reprod Biol Endocrinol. 2016;14:8.##van Helden J, Weiskirchen R. Performance of the two new fully automated anti-Müllerian hormone immunoassays compared with the clinical standard assay. Hum Reprod. 2015;30(8):1918-26.##Majumder K, Gelbaya TA, Laing I, Nardo LG. The use of anti-Müllerian hormone and antral follicle count to predict the potential of oocytes and embryos. Eur J Obstet Gynecol Reprod Biol. 2010;150(2):166-70.##Iliodromiti S, Anderson RA, Nelson SM. Technical and performance characteristics of anti-Müllerian hormone and antral follicle count as biomarkers of ovarian response. Hum Reprod Update. 2015;21(6):698-710.##Mutlu MF, Erdem M, Erdem A, Yildiz S, Mutlu I, Arisoy O, et al. Antral follicle count determines poor ovarian response better than anti-Müllerian hormone but age is the only predictor for live birth in in vitro fertilization cycles. J Assist Reprod Genet. 2013;30(5):657-65.##Oner G, Ulug P, Elmali F. Ovarian reserve markers in unexplained infertility patients treated with clo-miphene citrate during intrauterine insemination. Arch Med Sci. 2015;11(6):1250-4.##Nelson SM, Fleming R, Gaudoin M, Choi B, Santo-Domingo K, Yao M. Antimüllerian hormone levels and antral follicle count as prognostic indicators in a personalized prediction model of live birth. Fertil Steril. 2015;104(2):325-32.##Park HJ, Lee GH, Gong du S, Yoon TK, Lee WS. The meaning of anti-Müllerian hormone levels in patients at a high risk of poor ovarian response. Clin Exp Reprod Med. 2016;43(3):139-45.##

The Effects of Adjuvant Therapies on Embryo Transfer Success 2 1 2 Background: Many adjuvant therapies are employed during IVF treatment in an attempt to improve outcomes. The objective of our study was to evaluate the impact of thirteen adjuvants (Intralipid, steroids, melatonin, coenzyme Q10, Filgrastim, testosterone, DHEA, growth hormone, antibiotics, hCG infusion, aspirin, enoxaparin/heparin and dopamine agonists) on the success of embryo transfers. Methods: This is a retrospective cohort study of all embryo transfers between January 2010 and April 2015 from a multi-site IVF clinic. To ensure data independence, random number was applied to each included transfer and used to pick an individual transfer for each patient (n=13,372). Outcomes were clinical pregnancy, live birth and pregnancy loss. Univariate comparison with Chi square testing and logistic regression analysis were used. The level of significance was p<0.05. Results: Steroid use was significantly associated with both reduced clinical pregnancy loss (aOR 0.39, CI 0.19-0.76) and improved live birth rates (aOR 1.40, CI 1.11-1.77). While aspirin was associated with improved live birth rates (aOR 1.48, CI 1.08-2.02), melatonin was linked with reduced rates (aOR 0.66, CI 0.45-0.96). Analyses for all other adjuvant therapies did not reach statistical significance after logistic regression. Conclusion: Many of the interventions investigated in this study fail to significantly demonstrate any effects on the success of embryo transfers. Our analysis results show negative effects with the use of melatonin; however, use of aspirin or steroids demonstrated promising, potentially beneficial outcomes. Additional exploration is needed to guide evidence-based practice. 368 379 Rachael R Shirlow Monash University Monash University Australia rachael.shirlow@gmail.com Martin M Healey University of Melbourne University of Melbourne Australia Michelle M Volovsky Monash University Monash University Australia Vivien V Maclachlan Monash IVF Monash IVF Australia Beverley B Vollenhoven Monash University Monash University Australia Adjuvant drug therapyAspirinMelatoninSteroids 3.pdf Farquhar C, Rishworth JR, Brown J, Nelen WL, Marjoribanks J. Assisted reproductive technology: an overview of Cochrane Reviews. Cochrane Database Syst Rev. 2015;(7):CD010537.##Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Blastocyst quality affects the success of blastocyst-stage embryo transfer. Fertil Steril. 2000;74(2):282-7.##Fatemi HM, Popovic-Todorovic B. Implantation in assisted reproduction: a look at endometrial receptivity. Reprod Biomed Online. 2013;27(5):530-8.##Lessey BA. Embryo quality and endometrial receptivity: lessons learned from the ART experience. J Assist Reprod Genet. 1998;15(4):173-6.##Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Hum Reprod Update. 2006;12(6):731-46.##Kwak-Kim J, Gilman-Sachs A. Clinical implication of natural killer cells and reproduction. Am J Reprod Immunol. 2008;59(5):388-400.##Miko E, Manfai Z, Meggyes M, Barakonyi A, Wilhelm F, Varnagy A, et al. Possible role of natural killer and natural killer T-like cells in implantation failure after IVF. Reprod Biomed Online. 2010;21(6):750-6.##Roussev RG, Ng SC, Coulam CB. Natural killer cell functional activity suppression by intravenous immunoglobulin, intralipid and soluble human leukocyte antigen-G. Am J Reprod Immunol. 2007;57(4):262-9.##Roussev RG, Acacio B, Ng SC, Coulam CB. Duration of intralipid's suppressive effect on NK cell's functional activity. Am J Reprod Immunol. 2008;60(3):258-63.##Boomsma CM, Keay SD, Macklon NS. Peri-implantation glucocorticoid administration for assisted reproductive technology cycles. Cochrane Database Syst Rev. 2007;(1):CD005996.##Tamura H, Nakamura Y, Korkmaz A, Manchester LC, Tan DX, Sugino N, et al. Melatonin and the ovary: physiological and pathophysiological implications. Fertil Steril. 2009;92(1):328-43.##Turi A, Giannubilo SR, Brugè F, Principi F, Battistoni S, Santoni F, et al. Coenzyme Q10 content in follicular fluid and its relationship with oocyte fertilization and embryo grading. Arch Gynecol Obstet. 2012;285(4):1173-6.##Würfel W. Treatment with granulocyte colony-stimulating factor in patients with repetitive implantation failures and/or recurrent spontaneous abortions. J Reprod Immunol. 2015;108:123-35.##Ferrario M, Secomandi R, Cappato M, Galbignani E, Frigerio L, Arnoldi M, et al. Ovarian and adrenal androgens may be useful markers to predict oocyte competence and embryo development in older women. Gynecol Endocrinol. 2015;31(2):125-30.##Zeyneloglu HB, Onalan G. Remedies for recurrent implantation failure. Semin Reprod Med. 2014;32(4):297-305.##Kroon B, Hart RJ, Wong BM, Ford E, Yazdani A. Antibiotics prior to embryo transfer in ART. Cochrane Database Syst Rev. 2012;(3):CD008995.##Ye H, Hu J, He W, Zhang Y, Li C. The efficacy of intrauterine injection of human chorionic gonadotropin before embryo transfer in assisted reproductive cycles: Meta-analysis. J Int Med Res. 2015;43(6):738-46.##Dirckx K, Cabri P, Merien A, Galajdova L, Gerris J, Dhont M, et al. Does low-dose aspirin improve pregnancy rate in IVF/ICSI? A randomized double-blind placebo controlled trial. Hum Reprod. 2009;24(4):856-60.##Groeneveld E, Broeze KA, Lambers MJ, Haapsamo M, Dirckx K, Schoot BC, et al. Is aspirin effective in women undergoing in vitro fertilization (IVF)? Results from an individual patient data meta-analysis (IPD MA). Hum Reprod Update. 2011;17(4):501-9.##Fiedler K, Würfel W. Effectivity of heparin in assisted reproduction. Eur J Med Res. 2004;9(4):207-14.##Tang H, Hunter T, Hu Y, Zhai SD, Sheng X, Hart RJ. Cabergoline for preventing ovarian hyperstimulation syndrome. Cochrane Database Syst Rev. 2012;(2):CD008605.##Fernando S, Rombauts L. Melatonin: shedding light on infertility?--A review of the recent literature. J Ovarian Res. 2014;7:98.##Batıoğlu AS, Sahin U, Gürlek B, Oztürk N, Unsal E. The efficacy of melatonin administration on oocyte quality. Gynecol Endocrinol. 2012;28(2):91-3.##Showell MG, Brown J, Clarke J, Hart RJ. Antioxidants for female subfertility. Cochrane Database Syst Rev. 2013;(8):CD007807.##Nagels HE, Rishworth JR, Siristatidis CS, Kroon B. Androgens (dehydroepiandrosterone or testosterone) for women undergoing assisted reproduction. Cochrane Database Syst Rev. 2015;(11):CD009749.##Lu Q, Shen H, Li Y, Zhang C, Wang C, Chen X, et al. Low testosterone levels in women with diminished ovarian reserve impair embryo implantation rate: a retrospective case-control study. J Assist Reprod Genet. 2014;31(4):485-91.##Duffy JM, Ahmad G, Mohiyiddeen L, Nardo LG, Watson A. Growth hormone for in vitro fertilization. Cochrane Database Syst Rev. 2010;(1):CD000099.##Volovsky M, Healey M, MacLachlan VB, Vollenhoven BJ. Intrauterine human chorionic gonadotropin (HCG) infusion prior to embryo transfer (ET) may be detrimental to pregnancy rate. Fertil Steril. 2016;106(3):e52.##Craciunas L, Tsampras N, Coomarasamy A, Raine-Fenning N. Intrauterine administration of human chorionic gonadotropin (hCG) for subfertile women undergoing assisted reproduction. Cochrane Database Syst Rev. 2016;(5):CD011537.##Bentov Y, Esfandiari N, Burstein E, Casper RF. The use of mitochondrial nutrients to improve the outcome of infertility treatment in older patients. Fertil Steril. 2010;93(1):272-5.##Meldrum DR, Casper RF, Diez-Juan A, Simon C, Domar AD, Frydman R. Aging and the environment affect gamete and embryo potential: can we intervene? Fertil Steril. 2016;105(3):548-59.##Ben-Meir A, Burstein E, Borrego-Alvarez A, Chong J, Wong E, Yavorska T, et al. Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging. Aging Cell. 2015;14(5):887-95.##Aleyasin A, Abediasl Z, Nazari A, Sheikh M. Granulocyte colony-stimulating factor in repeated IVF failure, a randomized trial. Reproduction. 2016;151(6):637-42.##Barad DH, Yu Y, Kushnir VA, Shohat-Tal A, Lazzaroni E, Lee HJ, et al. A randomized clinical trial of endometrial perfusion with granulocyte colony-stimulating factor in in vitro fertilization cycles: impact on endometrial thickness and clinical pregnancy rates. Fertil Steril. 2014;101(3):710-5.##Kahyaoglu I, Yılmaz N, Timur H, Inal HA, Erkaya S. Granulocyte colony-stimulating factor: A relation between serum and follicular fluid levels and in-vitro fertilization outcome in patients with polycystic ovary syndrome. Cytokine. 2015;74(1):113-6.##Seshadri S, Sunkara SK, Khalaf Y, El-Toukhy T, Hamoda H. Effect of heparin on the outcome of IVF treatment: a systematic review and meta-analysis. Reprod Biomed Online. 2012;25(6):572-84.##Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, et al. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med. 2006;12(9):1065-74.##Brook N, Khalaf Y, Coomarasamy A, Edgeworth J, Braude P. A randomized controlled trial of prophylactic antibiotics (co-amoxiclav) prior to embryo transfer. Hum Reprod. 2006;21(11):2911-5.##Yeung TW, Chai J, Li RH, Lee VC, Ho PC, Ng EH. A randomized, controlled, pilot trial on the effect of dehydroepiandrosterone on ovarian response markers, ovarian response, and in vitro fertilization outcomes in poor responders. Fertil Steril. 2014;102(1):108-115.e1.##Dentali F, Ageno W, Rezoagli E, Rancan E, Squizzato A, Middeldorp S, et al. Low-dose aspirin for in vitro fertilization or intracytoplasmic sperm injection: a systematic review and a meta-analysis of the literature. J Thromb Haemost. 2012;10(10):2075-85.##Siristatidis CS, Basios G, Pergialiotis V, Vogiatzi P. Aspirin for in vitro fertilisation. Cochrane Database Syst Rev. 2016;11:CD004832.##Ruopp MD, Collins TC, Whitcomb BW, Schisterman EF. Evidence of absence or absence of evidence? A reanalysis of the effects of low-dose aspirin in in vitro fertilization. Fertil Steril. 2008;90(1):71-6.##Gelbaya TA, Kyrgiou M, Li TC, Stern C, Nardo LG. Low-dose aspirin for in vitro fertilization: a systematic review and meta-analysis. Hum Reprod Update. 2007;13(4):357-64.##

Efficacy of Intrauterine Injection of Granulocyte Colony Stimulating Factor (G-CSF) on Treatment of Unexplained Recurrent Miscarriage: A Pilot RCT Study 2 1 2 Background: Endometrium undergoes several changes in structure and cellular composition during pregnancy. Granulocyte Colony-stimulating Factor (GCS-F) is an important cytokine with critical role in embryo implantation and pregnancy. The aim of the present study was to evaluate the impact of intrauterine injection of G-CSF in patients who suffer from unexplained recurrent miscarriage (RM). Methods: In the present randomized clinical trial, a total of 68 patients were randomly allocated into two study groups including intrauterine G-CSF (n=23, 300 μg) injection and control group (n=27, no G-CSF injection). Eighteen out of 68 patients were excluded from the final analysis due to different reasons. All patients were in Ovulation Induction (I/O) cycle. In G-CSF group, intrauterine injection of G-CSF was done twice in the cycle. All enrolled patients were under 40 years old and had at least two unexplained pregnancy losses. Pregnancy was evaluated by titer of βhCG, presence of gestational sac (implantation) and fetal heart rate (clinical pregnancy) was assessed by vaginal ultrasonography. Student’s T test and Mann-Whitney U were used for analysis. The p≤0.05 was determined as statistically significant. Results: No significant differences were observed between the two study groups when the rates of chemical pregnancy (26.1% vs. 29.6%, p=0.781), implantation (26.1% vs. 22.2%, p=0.750), clinical pregnancy (17.4% vs. 11.1%, p=0.689) and abortion (33% vs. 37.5%, p=0.296) were compared. Conclusion: In our study, no significant difference was observed between the two study groups when the rates of chemical pregnancy, implantation, clinical pregnancy and abortion were compared. 379 386 Simin S Zafardoust Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi MM Akhondi محمدمهدی آخوندی Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Reza MR Sadeghi محمدرضا صادقی Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Afsaneh A Mohammadzadeh افسانه محمدزاده Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Atousa A Karimi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Sheyda Sh Jouhari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Soheila S Ansaripour Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran soh.ansaripour@gmail. com Granulocyte colony-stimulating factorIntrauterine injectionRecurrent miscarriage 721.pdf Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril. 1996;66(1):24-9.##Ford HB, Schust DJ. Recurrent pregnancy loss: etiology, diagnosis, and therapy. Rev Obstet Gynecol. 2009;2(2):76-83.##Warren JE, Silver RM. Genetics of pregnancy loss. Clin Obstet Gynecol. 2008;51(1):84-95.##Devi Wold AS, Pham N, Arici A. Anatomic factors in recurrent pregnancy loss. Semin Reprod Med. 2006;24(1):25-32.##Summers PR. Microbiology relevant to recurrent miscarriage. Clin Obstet Gynecol. 1994;37(3):722-9.##Sallerfors B, Olofsson T. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) secretion by adherent monocytes measured by quantitative immunoassays. Eur J Haematol. 1992;49(4):199-207.##Nagata S, Tsuchiya M, Asano S, Kaziro Y, Yamazaki T, Yamamoto O, et al. Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature. 1986;319(6052):415-8.##Brizzi MF, Avanzi GC, Pegoraro L. Hematopoietic growth factor receptors. Int J Cell Cloning. 1991;9(4):274-300.##Tsai KJ, Tsai YC, Shen CK. G-CSF rescues the memory impairment of animal models of Alzheimer's disease. J Exp Med. 2007;204(6):1273-80.##Franzke A, Piao W, Lauber J, Gatzlaff P, Könecke C, Hansen W, et al. G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases. Blood. 2003;102(2):734-9.##Guidi L, Mocci G, Marzo M, Rutella S. Treatment of Crohn's disease with colony-stimulating factors: An overview. Ther Clin Risk Manag. 2008;4(5):927-34.##Mannon PJ, Leon F, Fuss IJ, Walter BA, Begnami M, Quezado M, et al. Successful granulocyte-colony stimulating factor treatment of Crohn's disease is associated with the appearance of circulating interleukin-10-producing T cells and increased lamina propria plasmacytoid dendritic cells. Clin Exp Immunol. 2009;155(3):447-56.##Takano H, Ueda K, Hasegawa H, Komuro I. G-CSF therapy for acute myocardial infarction. Trends Pharmacol Sci. 2007;28(10):512-7.##Ohtsuka M, Takano H, Zou Y, Toko H, Akazawa H, Qin Y, et al. Cytokine therapy prevents left ventricular remodeling and dysfunction after myocardial infarction through neovascularization. FASEB J. 2004;18(7):851-3.##Santjohanser C, Knieper C, Franz C, Hirv K, Meri O, Schleyer M, et al. Granulocyte-colony stimulating factor as treatment option in patients with recurrent miscarriage. Arch Immunol Ther Exp (Warsz). 2013;61(2):159-64.##Würfel W, Santjohanser C, Hirv K, Bühl M, Meri O, Laubert I, et al. High pregnancy rates with administration of granulocyte colony-stimulating factor in ART-patients with repetitive implantation failure and lacking killer-cell immunglobulin-like receptors. Hum Reprod. 2010;25(8):2151-2; author reply 2152.##Hannan NJ, Evans J, Salamonsen LA. Alternate roles for immune regulators: establishing endometrial receptivity for implantation. Expert Rev Clin Immunol. 2011;7(6):789-802.##Suresh K. An overview of randomization techniques: An unbiased assessment of outcome in clinical research. J Hum Reprod Sci. 2011;4(1):8-11.##Bansal AS. Joining the immunological dots in recurrent miscarriage. Am J Reprod Immunol. 2010;64(5):307-15.##Clark DA, Chaouat G, Arck PC, Mittruecker HW, Levy GA. Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase [correction of prothombinase]. J Immunol. 1998;160(2):545-9.##Hiby SE, Regan L, Lo W, Farrell L, Carrington M, Moffett A. Association of maternal killer-cell immunoglobulin-like receptors and parental HLA-C genotypes with recurrent miscarriage. Hum Reprod. 2008;23(4):972-6.##Takasaki A, Ohba T, Okamura Y, Honda R, Seki M, Tanaka N, et al. Clinical use of colony-stimulating factor-1 in ovulation induction for poor responders. Fertil Steril. 2008;90(6):2287-90.##Sills ES, Walsh DJ, Shkrobot LV, Palermo GD, Walsh AP. Clinical experience with intravenous immunoglobulin and tnf-a inhibitor therapies for recurrent pregnancy loss. Ulster Med J. 2009;78(1):57-8.##Scarpellini F, Sbracia M. Use of granulocyte colony-stimulating factor for the treatment of unexplained recurrent miscarriage: a randomised controlled trial. Hum Reprod. 2009;24(11):2703-8.##Salmassi A, Schmutzler AG, Schaefer S, Koch K, Hedderich J, Jonat W, et al. Is granulocyte colony-stimulating factor level predictive for human IVF outcome? Hum Reprod. 2005;20(9):2434-40.##Lédée N, Gridelet V, Ravet S, Jouan C, Gaspard O, Wenders F, et al. Impact of follicular G-CSF quantification on subsequent embryo transfer decisions: a proof of concept study. Hum Reprod. 2013;28(2):406-13.##Zeyneloglu HB, Onalan G, Durak T, Alyazici I, Unal E. Granulocyte macrophage colony stimulating factor (G-CSF) administration for art patients with repeated implantation failure (RIF): which route is best? Fertil Steril.100(3):S291-S2.##Wong LF, Porter TF, Scott JR. Immunotherapy for recurrent miscarriage. Cochrane Database Syst Rev. 2014;(10):CD000112.##Christiansen OB, Pedersen B, Rosgaard A, Husth M. A randomized, double-blind, placebo-controlled trial of intravenous immunoglobulin in the prevention of recurrent miscarriage: evidence for a therapeutic effect in women with secondary recurrent miscarriage. Hum Reprod. 2002;17(3):809-16.##Tang AW, Alfirevic Z, Turner MA, Drury J, Quenby S. Prednisolone Trial: Study protocol for a randomised controlled trial of prednisolone for women with idiopathic recurrent miscarriage and raised levels of uterine natural killer (uNK) cells in the endometrium. Trials. 2009;10:102.##

Comparison of Outcomes of IVF Cycles Between Transferred Frozen Thawed Embryos and Fresh Embryos by a 2 Year Survey 2 1 2 Background: Infertility as one of most concerning topics in childbearing age mothers needs better managements with less complications and IVF can be assumed as an efficient method. This study aimed to compare pregnancy outcomes in fresh and frozen embryos transferred in IVF cycles. Methods: In a retrospective study, 11201 patients underwent IVF cycles from 21st March 2013 to 20th March 2014 and they were categorized into two groups according to age, previous tubal disease and surgery, tubal ligation, and previous ectopic pregnancy variables. Clinical pregnancy, ectopic pregnancy, multiple pregnancy, spontaneous abortion and preterm labor rates were compared in both groups. Results: 11201 patients were categorized in two groups. Results of 4149 frozen-thawed embryo transfer cycles showed 1281 clinical pregnancies (30.9%) and 7052 fresh embryo transfer cycles which led to 2085 clinical pregnancies (29.6%) without significant differences between groups (p=0.14). Ectopic pregnancy rates in frozen and fresh groups were 38 (3%) and 52 (2.5%), respectively (p=0.409). Spontaneous abortion rates in frozen and fresh groups were 8.4% and 9.4%, respectively (p=0.32). Preterm labor was seen in both frozen and fresh groups as 28.2% and 23.4%, respectively (p=0.002) and finally, multigestational pregnancy rate was 25.7% and 22.8% in frozen and fresh groups (p=0.06). Conclusion: According to this study, frozen and fresh embryo transfer cycles were not significantly different in clinical pregnancy, ectopic pregnancy, multigestational pregnancy rates, but preterm labor was dominant in frozen group, which should not be overlooked. 386 390 Ensieh E Shahrokh Tehraninejad Department of Endocrinology & Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR Department of Endocrinology & Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR Iran Zeynab Z Vosoog Department of Endocrinology & Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR Department of Endocrinology & Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR Iran Fatemeh F Farajzadeh-Vajari Department of Gynecology and Obstetrics, Babak Hospital, Tehran University of Medical Sciences Department of Gynecology and Obstetrics, Babak Hospital, Tehran University of Medical Sciences Iran Ectopic pregnancyFresh embryo transferFrozen-thawed embryo transferIn Vitro Fertilization 723.pdf Centers for Disease Control and Prevention (CDC). Ectopic pregnancy mortality - Florida, 2009-2010. MMWR Morb Mortal Wkly Rep. 2012;61(6):106-9.##Shaw JL, Dey SK, Critchley HO, Horne AW. Current knowledge of the aetiology of human tubal ectopic pregnancy. Hum Reprod Update. 2010;16(4):432-44.##Lesny P, Killick SR, Robinson J, Maguiness SD. Transcervical embryo transfer as a risk factor for ectopic pregnancy. Fertil Steril. 1999;72(2):305-9.##Anderson FW, Hogan JG, Ansbacher R. Sudden death: ectopic pregnancy mortality. Obstet Gynecol. 2004;103(6):1218-23.##Steptoe PC, Edwards RG. Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet. 1976;1(7965):880-2.##Huang B, Hu D, Qian K, Ai J, Li Y, Jin L, et al. Is frozen embryo transfer cycle associated with a significantly lower incidence of ectopic pregnancy? An analysis of more than 30,000 cycles. Fertil Steril. 2014;102(5):1345-9.##Londra L, Moreau C, Strobino D, Garcia J, Zacur H, Zhao Y. Ectopic pregnancy after in vitro fertilization: differences between fresh and frozen-thawed cycles. Fertil Steril. 2015;104(1):110-8.##Jun SH, Milki AA. Ectopic pregnancy rates with frozen compared with fresh blastocyst transfer. Fertil Steril. 2007;88(3):629-31.##Cheng LY, Lin PY, Huang FJ, Kung FT, Chiang HJ, Lin YJ, et al. Ectopic pregnancy following in vitro fertilization with embryo transfer: A single-center experience during 15 years. Taiwan J Obstet Gynecol. 2015;54(5):541-5.##Shapiro BS, Daneshmand ST, De Leon L, Garner FC, Aguirre M, Hudson C. Frozen-thawed embryo transfer is associated with a significantly reduced incidence of ectopic pregnancy. Fertil Steril. 2012;98(6):1490-4.##Shen C, Shu D, Zhao X, Gao Y. Comparison of clinical outcomes between fresh embryo transfers and frozen-thawed embryo transfers. Iran J Reprod Med. 2014;12(6):409-14.##Ishihara O, Kuwahara A, Saitoh H. Frozen-thawed blastocyst transfer reduces ectopic pregnancy risk: an analysis of single embryo transfer cycles in Japan. Fertil Steril. 2011;95(6):1966-9.##Aflatoonian A, Karimzadeh Maybodi MA, Aflatoonian N, Tabibnejad N, Amir-Arjmand MH, Soleimani M, et al. Perinatal outcome in fresh versus frozen embryo transfer in ART cycles. Int J Reprod Biomed (Yazd). 2016;14(3):167-72.##Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet. 1978;2(8085):366.##

Coexistent Circumvallate Placenta and Battledore Insertion of Umbilical Cord Resulting in Grave Obstetric Outcome: A Case Report 2 1 2 Background: Various placental and cord abnormalities have been known to adversely affect the obstetric outcome. Circumvallate placenta and Battledore insertion of the umbilical cord are both rare and known to be associated with poor obstetric outcome individually. Case Presentation: In this case report, the woman presented at 8 months of gestation with preeclampsia with IUFD to North Easter Indira Gandhi Regional Institute of Health and Medical Science Shillong on 22/7/16 and delivered a macerated fetus vaginally. After delivery, examination revealed both a circumvallate placenta and Battledore insertion of umbilical cord. This might have attributed to preeclampsia and ultimately IUFD in this case as she had no other identifiable cause for IUFD. Conclusion: If such placental and cord abnormalities are suspected or diagnosed antenatally by ultrasonography, the pregnancy should be regarded as high risk. Such woman would require more stringent follow up in the antenatal period and continuous intrapartum monitoring to avoid any catastrophe and to achieve a favorable maternal and fetal outcome. 390 393 Nalini N Sharma Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences India Nalinisharma100@rediffmail.com Rituparna R Das Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences India Sushila S Salam Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences India Roma R Jethani Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences India Ahanthem A Singh Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences Department of Obstetrics and Gynaecology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences India Battledore insertion of umbilical cordCircumvallate placentaPreeclampsiaPregnancy outcome 5.pdf Cunningham FG, Leveno KJ, Bloom SL, Hauth JC, Rouse DJ, Spong CY. Williams Obstetrics. 22nd ed. New York: McGraw Hill Professional; 2009. 1600 p.##Benirschke K, Kaufmann P. Pathology of the human placenta. 3rd ed. New York: Springer; 2006. 870 p.##Paalman RJ, Vander Veer CG, Rapids G. Circumvallate placenta. Am J Obstet Gynecol. 1953;65(3):491-7.##Rolschau J. Circumvallate placenta and intrauterine growth retardation. Acta Obstet Gynecol Scand Suppl. 1978;72:11-4.##Suzuki S. Clinical significance of pregnancies with circumvallate placenta. J Obstet Gynaecol Res. 2008;34(1):51-4.##Taniguchi H, Aoki S, Sakamaki K, Kurasawa K, Okuda M, Takahashi T, et al. Circumvallate placenta: associated clinical manifestations and complications-a retrospective study. Obstet Gynecol Int. 2014;2014:986230.##Liu CC, Pretorius DH, Scioscia AL, Hull AD. Sonographic prenatal diagnosis of marginal placental cord insertion: clinical importance. J Ultrasound Med. 2002;21(6):627-32.##Ebbing C, Kiserud T, Johnsen SL, Albrechtsen S, Rasmussen S. Prevalence, risk factors and outcomes of velamentous and marginal cord insertions: a population-based study of 634,741 pregnancies. PLoS One. 2013;8(7):e70380.##Tufail S, Nawaz S, Sadaf M, Sail SS. Association between battledore placenta and perinatal complications. J Rawal Med Coll. 2012;16(2):159-61.##Reddy VM, Kumar SS, Reddy NS. Prevalence and pattern of abnormalities occurring in placenta and umbilical cord. Int J Med Res Health Sci. 2013;2(4):935-40.##Holzman J, Zalud I, Bartholomew ML. Ultrasound of the placenta. Donald Sch J Ultrasound Obstet Gynaecol. 2007;1(4):47-60.##Hasegawa J, Matsuoka R, Ichizuka K, Sekizawa A, Okai T. Ultrasound diagnosis and management of umbilical cord abnormalities. Taiwan J Obstet Gynecol. 2009;48(1):23-7.##

Early Fertilization Abnormalities in the Human: An Exhausted Area of Research? 2 1 722 2 Introduction The techniques of assisted reproduction have admittedly unveiled the process of human oocyte fertilization and subsequent embryonic development. In specialized laboratories, male and female gametes are routinely handled and their immediate fusion product, the pronuclear stage, can nowadays be cultured up to the hatching blastocyst under in vitro conditions. Comprehensive overviews of the observed variable features of oocytes, zygotes, embryos and blastocysts have been provided elsewhere (1-4). An important side effect of monitoring early events of reproduction is the detection of various factors that may affect regular fertilization and/or cause developmental abnormalities. Quite recently, time-lapse systems (TLS) have been propagated as an ideal tool of continuous monitoring without disturbing the culture conditions and it appears as an immense advantage over the common assessments of pronuclear formation and cleavage at a given time. However, the focus of morphokinetic parameters obtained by TLS has mostly been on embryo development in order to select the "best" embryo and thus improve implantation rates (5). In the present comment, some issues that delt with the chromosomal constitution of zygotes and embryos produced in vitro were focused, evaluating whether TLS could play a role in the early detection of abnormal events. This subjective commentary was inspired for the most part by the knowledge obtained from cytogenetic analyses of abnormally fertilized oocytes in our laboratory. It amplifies the thoughts presented in a recent brief letter (6) and advocates further basic research using and preferably combining so-called morphokinetic aspects and (cyto) genetics during assisted reproduction. Such an approach appears indispensable if the purpose is to shed light on some still ambiguous mechanisms of fertilization disorders. Examples for abnormal pronuclear stages: In one of our case reports, a tripronuclear oocyte resulting from ICSI was reported that turned out to have a tetraploid chromosome constitution (7). Our conclusion was that diploid sperm must exist and being able to fertilize an oocyte through this event had been considered rather improbable by some investigators. Without appearance of an additional female pronucleus (PN) caused by non-extrusion of the second polar body (PB), this phenomenon would have escaped detection. In other words, a diploid spermatozoon that regularly fertilizes an oocyte should result in a single diploid male PN accompanying the haploid female PN. The corresponding bipronuclear stage would be classified as normal and could develop into a diandric triploid embryo. This assumption has recently been confirmed by Savage et al. (8) who applied genotyping to a partial molar pregnancy after ICSI. In the female, there are also diploid gametes that predominantly appear as "giant" oocytes. Two laboratories (9, 10) have independently shown that these oocytes can be fertilized and therefore cause digynic triploidy in the embryo. The problem is that giant oocytes can develop either two or three pronuclei (PNi) after penetration of a spermatozoon, indicating that the abnormality might be overlooked if only the number of PNi is assessed and oocyte size is not taken into account. In a review on triploidy-causing mechanisms (11), endoreduplication in one of the two PNi has been listed as a possibility because the evidence for this phenomenon was found in the maternal PN of two abnormally fertilized oocytes (12, 13). It is conceivable that endoreduplication can also occur in the male PN. Independent of its parental origin, however, endoreduplication is another good example for the fact that only the number of pronuclei could be assessed and not their genetic content during the routine laboratory procedure. The formation of a single PN is a peculiar abnormality that should be mentioned here. It is known that some of these monopronuclear oocytes can develop into diploid embryos (14) and result in the birth of normal healthy babies (15). The probability of obtaining a biparental diploid embryo appears to be higher after conventional in vitro fertilization (IVF) than after intracytoplasmic sperm injection (ICSI) but births have almost exclusively been reported after transfer of embryos that developed from monopronuclear IVF oocytes. Currently, there is no general consent on how to proceed with monopronuclear oocytes. Besides deviations from the regular number of PNi and variations in their genetic constitution, there is a rare morphologic abnormality at the pronuclear stage; an early division of the ooplasm that resembles embryonic cleavage. Such a case of premature cytokinesis in an abnormally fertilized oocyte with three PNi was observed before (16). Division of the ooplasm separated one PN from the other two PNi. This event could restore a biparental diploid cell line provided that the detrimental extra PN is excluded and even produce a normal embryo if the haploid cell does not develop further. Suggestions for further research: 1) Among the points that still need clarification is the origin of regularly fertilized but monopronuclear oocytes that result in diploid embryos carrying the genomes of both parents. Besides a delayed appearance of the second PN that has been found in 25% of the cases (17), some other mechanisms are under discussion. First, maternal and paternal chromatin are immediately enclosed in a common pronuclear envelope. This process has been shown to occur in mouse oocytes that were injected with spermatozoa close to the metaphase spindle (18). The resulting diploid PN was characterized by a larger size and a greater number of "nucleolus-like bodies". Second, two individual PNi are formed comparable to the normal fertilization pattern but then undergo a very early membrane fusion. Third, two individual PNi are initially present but one PN experiences a premature breakdown of its envelope (19). It has been proposed that TLS might be useful to elucidate events during the zygote stage (2). In fact, a continuous observation of oocytes from sperm penetration up to zygote formation should provide information on the delayed appearance of PNi, on pronuclear fusion and premature breakdown of the pronuclear membrane whereas immediate enclosure of both parental genomes in a common envelope will possibly escape detection. Moreover, if the purpose is to evaluate the predictive power of pronuclear size, i.e. determining whether diploid single PNi are larger than those with a haploid constitution, exact measurements of the pronuclear diameter followed by genetic analyses will be necessary. In cases of presumed premature pronuclear breakdown, it would be interesting to know whether the chromatin lacking a membrane participates in ensuing mitotic divisions. Therefore, the chromosomal constitution of the resulting embryos should be investigated. 2) Fertilization disorders occurring in vitro must be carefully screened to reconcile the observed mechanisms with the variety of existing theoretical models for the formation of genetic abnormalities. Let us look for example at the report of Sunde et al. (20) who examined hydatidiform moles showing one biparental cell population and one androgenetic cell population. To explain some of these mosaic cases, the authors suggested "fertilization of one oocyte by one spermatozoon, followed by duplication of the paternal pronucleus, creating a ‘temporary tripronuclear zygote’ with two identical paternal pronuclei ... ". If this statement has to be interpreted as a change from the originally bipronuclear to the tripronuclear state, it should become visible by continuous monitoring of the oocyte. Indeed, a comparable delayed formation of PNi has been described: following the observation of two PNi at about 18 hr after injection of a single spermatozoon into an oocyte, some hours later there were four and then even five PNi (21). Though time-lapse imaging has allowed the detection of this unexpected behaviour, it did not provide information on the origin of the additional three PNi. A simple cytogenetic examination of the abnormal pronuclear stage would have been helpful to decide at least whether the number of whole chromosome sets has increased (the chromosomal constitution should then be in the pentaploid range) or whether an approximately diploid constitution is present. In the latter case, splitting of one or both chromatid sets according to our previously published concept (22) has to be considered. The mechanisms for the development of mosaics proposed by Sunde et al. (20) would further require an "asymmetric cytokinesis", leading to the distribution of different PNi to different halves of the oocyte (compare figure 4, page 1029 of the corresponding paper). Therefore, more information on the frequency of early division of the oocyte is needed. In the mouse, it was shown that the gentle compression of oocytes during meiotic maturation can lead to the formation of two cells of similar size within the zona pellucida instead of extrusion of a small first PB (23). The authors considered one of these halves to be an extruded large first polar body. Furthermore, both cells could be fertilized by two independent spermatozoa, resulting in the development of twin embryos that may even amalgamate to form a "chimeric hermaphrodite". A similar concept has been put forward by Winberg et al. (24) who suggested "dispermic fertilization of a parthenogenetically activated oocyte, that is, fertilization by two spermatozoa of two identical haploid ova". The "presence of both maternal half-genomes and two paternal PNi in the same ‘tetragametic’ zygote" proposed by Boklage (25) may refer to the same phenomenon though an illustration has not been provided here. As a final example for hypothetical mechanisms that need clarification, I should like to cite the publication by Wegner et al. (26). Here, it was assumed that one of the paternal PNi of a dispermic tripronuclear zygote may remain unreplicated and become distributed to one of the daughter cells of the two cell embryo. At this point, it would fuse with the nucleus of the blastomere and create a triploid cell line whereas the other blastomere would be diploid. Observation of such an abnormal, long-term existence of a PN and its distribution to an embryonic cell would be another challenging task for TLS but of course, ensuing genetic analyses are required to confirm the suspected development of triploidy. Conclusion The examples mentioned at the beginning clearly show that there is a variety of abnormalities at the pronuclear stage that affect the chromosomal constitution of the embryo. Some of the adverse events can be identified by counting the number of PNi whereas others happen within a PN and escape detection. In the latter cases, TLS will obviously not be helpful. On the other hand, it is generally acknowledged that PNi are dynamic structures so that the reliability of their traditional scoring at a fixed and single point in time has to be questioned. TLS may improve our understanding of these early dynamic processes. The continuous observation of fertilized oocytes and developing embryos may also enable us to address some of the unresolved problems discussed above. In this respect, annotating the appearance of individual PNi (tPN1a; tPN2a; tPN3a etc.) and their disappearance or fading (tPN1f; tPN2f etc.) as proposed by Ciray et al. (27) will be of utmost importance. Observations during this period will help to reveal abnormalities such as the above-mentioned late appearance of extra PNi (21), the phenomenon of three PNi that turn into two (5), or to assess the frequency of premature cytokinesis. However, possible obstacles and limitations of TLS must be considered. Existing abnormalities of the oocyte, extrusion of the second PB and formation of PNi could be obscured by cumulus cells in case of routine in vitro fertilization. The position of the oocyte in the well, the type of microscopy and image acquisition, the available device or software are variables that may restrict the annotation of a desired parameter (27). Furthermore, it cannot be expected that every laboratory will be equipped with time-lapse systems in the near future and those who use it must be willing to spend time for scientific topics including the willingness to communicate their data. Most important, however, abnormal developmental stages should be examined (cyto) genetically. Due to these numerous prerequisites, relevant data will emerge only slowly. Nevertheless, a more profound monitoring of early fertilization events in conjunction with appropriate genetic methods may open a new and promising chapter in research. Conflict of Interest The author declares no conflict of interest. 393 397 Bernd B Rosenbusch Department of Gynaecology and Obstetrics, University of Ulm Department of Gynaecology and Obstetrics, University of Ulm Germany bernd.rosenbusch@uniklinik-ulm.de No Keyword 722.pdf Rienzi L, Balaban B, Ebner T, Mandelbaum J. The oocyte. Hum Reprod. 2012;27 Suppl 1:i2-21.##Papale L, Fiorentino A, Montag M, Tomasi G. The zygote. Hum Reprod. 2012;27 Suppl 1:i22-49.##Prados FJ, Debrock S, Lemmen JG, Agerholm I. The cleavage stage embryo. Hum Reprod. 2012;27 Suppl 1:i50-71.##Hardarson T, Van Landuyt L, Jones G. The blastocyst. Hum Reprod. 2012;27 Suppl 1:i72-91.##Basile N, Caiazzo M, Meseguer M. What does morphokinetics add to embryo selection and in-vitro fertilization outcomes? Curr Opin Obstet Gynecol. 2015;27(3):193-200.##Rosenbusch B. Time-lapse systems in assisted reproduction: the importance of the earliest steps of fertilization. JBRA Assist Reprod. 2015;19(3):155.##Rosenbusch BE, Schneider M, Hanf V. Tetraploidy and partial endoreduplication in a tripronuclear zygote obtained after intracytoplasmic sperm injection. Fertil Steril. 1998;69(2):344-6.##Savage P, Sebire N, Dalton T, Carby A, Seckl MJ, Fisher RA. Partial molar pregnancy after intracytoplasmic sperm injection occurring as a result of diploid sperm usage. J Assist Reprod Genet. 2013;30(6):761-4.##Balakier H, Bouman D, Sojecki A, Librach C, Squire JA. Morphological and cytogenetic analysis of human giant oocytes and giant embryos. Hum Reprod. 2002;17(9):2394-401.##Rosenbusch B, Schneider M, Gläser B, Brucker C. Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy. Hum Reprod. 2002;17(9):2388-93.##Rosenbusch BE. Mechanisms giving rise to triploid zygotes during assisted reproduction. Fertil Steril. 2008;90(1):49-55.##Rosenbusch B, Schneider M, Sterzik K. Triploidy caused by endoreduplication in a human zygote obtained after in-vitro fertilization. Hum Reprod. 1997;12(5):1059-61.##Rosenbusch B, Glaeser B, Brucker C, Schneider M. Endoreduplication of the hyperhaploid maternal complement and abnormal pronuclear formation in a human zygote obtained after intracytoplasmic sperm injection. Ann Genet. 2002;45(3):157-9.##Rosenbusch B. The chromosomal constitution of embryos arising from monopronuclear oocytes in programmes of assisted reproduction. Int J Reprod Med. 2014;2014:418198.##Itoi F, Asano Y, Shimizu M, Honnma H, Murata Y. Birth of nine normal healthy babies following transfer of blastocysts derived from human single-pronucleate zygotes. J Assist Reprod Genet. 2015;32(9):1401-7.##Rosenbusch BE, Schneider M. Separation of a pronucleus by premature cytokinesis: a mechanism for immediate diploidization of tripronuclear oocytes? Fertil Steril. 2009;92(1):394.e5-8.##Staessen C, Janssenswillen C, Devroey P, Van Steirteghem AC. Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization. Hum Reprod. 1993;8(2):221-3.##Krukowska A, Tarkowski AK. Mouse zygotes with one diploid pronucleus formed as a result of ICSI can develop normally beyond birth. Mol Reprod Dev. 2005;72(3):346-53.##Azevedo AR, Pinho MJ, Silva J, Sá R, Thorsteinsdóttir S, Barros A, et al. Molecular cytogenetics of human single pronucleated zygotes. Reprod Sci. 2014;21(12):1472-82.##Sunde L, Niemann I, Hansen ES, Hindkjaer J, Degn B, Jensen UB, et al. Mosaics and moles. Eur J Hum Genet. 2011;19(10):1026-31.##Burchill AA. Number of pronuclei and ploidy in IVF/ICSI-derived embryos. In: Campbell A, Fishel S, editors. Atlas of time lapse embryology. Boca Raton FL: CRC Press, Taylor and Francis Group; 2015. p. 39-42.##Rosenbusch BE. A preliminary concept, deduced from cytogenetic analyses, for explaining different types of multipronuclear oocytes obtained after intracytoplasmic sperm injection. Fertil Steril. 2010;94(6):2479-81.##Otsuki J, Nagai Y, Lopata A, Chiba K, Yasmin L, Sankai T. Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite. Hum Reprod. 2012 ;27(2):380-7.##Winberg J, Gustavsson P, Lagerstedt-Robinson K, Blennow E, Lundin J, Iwarsson E, et al. Chimerism resulting from parthenogenetic activation and dispermic fertilization. Am J Med Genet A. 2010;152A(9):2277-86.##Boklage CE. Traces of embryogenesis are the same in monozygotic and dizygotic twins: not compatible with double ovulation. Hum Reprod. 2009;24(6):1255-66.##Wegner RD, Kistner G, Becker R, Stumm M, Knoll U, Entezami M, et al. Fetal 46,XX/69,XXY mixoploidy: origin and confirmation by analysis of fetal urine cells. Prenat Diagn. 2009;29(3):287-9.##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.##