Human Papillomavirus Infection Is an Unresolved Challenge in Assisted Reproductive Techniques 2 1 2 Human papillomavirus (HPV) is the most common type of sexually transmitted infection in men and women at reproductive age with devastating socio-economic impacts across nations. The total cost for prevention and treatment of HPV-related diseases was more than 8 billion dollars in 2010 (1). This DNA virus can infect both cutaneous and mucosal squamous epithelium; its high-risk variant strains frequently lead to formation of benign lesions (warts or papillomas) and intraepithelial neoplasms such as cervical cancer and, to a lesser extent, other malignancies including vulva, vagina, anus, oropharynx, and penile cancer (2). Most HPV infections are subclinical, and few of them have mild to moderate symptoms. A lot of studies on pathogenesis of HPV have been conducted among women, while our data on its infection and pathogenesis among men is limited. The overall prevalence of high-risk and non-pathogenic strains of HPV in different communities was estimated to be about 12% in 2012 which varies in different age groups, depending on their sexual activity. The incubation interval of HPV ranges between 1-8 months. Compared to the complications of HPV infection in women, men experience transient types without clinical consequences, which is a neglected area of research in HPV treatment programs. However, research on men has recently received increasing attention due to association of HPV with genital warts, penile, anorectal, and oropharyngeal cancers (1). HPV-6 and HPV-11 are the most common low-risk types of the infection often leading to anogenital warts, while HPV-16 and-18 are the most common oncogenic or high-risk types and the cause of more than 70% of cervical cancers (2). The infected men might transmit HPV to their sexual partners. Therefore, the study of HPV pathogenesis in men and its possible role in male and female infertility and early development or miscarriage of fetus has particular importance. Recent studies suggest the possible roles of HPV in male infertility, yet it is not clear whether HPV infections can silently damage the reproductive system or not. Therefore, the detection of HPV is important in the diagnosis of infertility in order to opt for different assisted reproductive techniques (ART) among HPV-positive patients (3). Literature review over the past two decades shows that the prevalence of HPV infection in infertile men with leukocytospermia is higher than the fertile ones. It has been shown that HPV infection in men is the cause of unexplained infertility; based on previous research, the rate of HPV infection of semen is approximately 2-31% in overall community, while its prevalence in couples with unexplained infertility is nearly 10-35.7%. Similarly, there is a lot of evidence on a possible link between HPV sperm infection and male infertility, indicating that HPV infection is associated with decreased sperm motility, increased sperm DNA fragmentation and idiopathic asthenozoospermia; in fact, active transcription of well-defined HPV-specific genes in men can result in infertility via damaging sperm motility. In addition, higher levels of anti-sperm antibodies are detected in men infected with HPV (4). Infection with HPV-16 has been reported to affect oocyte fertilization, fetal development, and increased blastomeres apoptosis which consequently influences embryo implantation in the endometrium. In animal studies, HPV-16 and -18 via E6/E7 transcripts block embryonic development at two-cell stage. In addition, HPV infection in women is usually associated with tubal factor infertility through stimulation of the immune response or production of high levels of proinflammatory cytokines (1, 2, 5). HPV is deemed similar to other STIs viruses in binding to two separate regions along the equatorial segment of spermatozoa. The presence of glycosaminoglycans or other molecules on sperm surface appears to mediate the interaction and binding of HPV to sperm. In addition, the equatorial segment is the region that sperm attaches to the plasma membrane of oocyte through fertilization process. Binding of HPV to sperm does not affect its ability to fertilize the oocyte; however, HPV-infected sperm can transmit viral genome to oocyte, which may be expressed in the developing blastocysts. Nevertheless, it is still unclear whether HPV infection can affect the subsequent zygote, embryo, and fetus development(1, 2). Association of HPV infection with preterm birth, miscarriage, or premature rupture of membranes is another scenario for its effect on reproduction. In vitro transfection of embryonic trophectoderm by plasmids containing HPV genome increases the rate of apoptosis 3-6 times in comparison to control embryos. Therefore, trophoblast cell death is the cause of placental insufficiency, failure of invasion to endometrium, and ultimately premature rupture of membrane and early miscarriage(1-3). In about 10 to 30% of women, HPV virus will be cleared by the affected individuals' immune system within 3 months, and in 90 % of cases, it will go away within two years. HPV infection remains in 10% of infected women whose cellular immune response is unable to remove the viral load. Therefore, these women are always at risk for HPV-related diseases and possibly cervical cancer. Accordingly, vaccination of high-risk individuals especially young women is necessary since they are vulnerable to cervical cancer as a result of high HPV viral load (1). Currently, the research for effective protocols addressing washing and selecting the sperm for ART is a hot topic. In fact, these guidelines should provide instructions on how to remove HPV from the surface of sperm. The routine method of semen processing applied in ART has been found to be ineffective in eliminating HPV bound to sperm (6). It seems that vaccination of men can be an effective strategy to prevent HPV-related sperm disorders and infertility until researchers discover and introduce new methods for removal of HPV from semen and sperm surface. Though HPV vaccine can prevent genital warts, cervical dysplasia, and cervical cancer following HPV infection, vaccination of men and women against HPV can also reduce its harmful consequences on human fertility. In vitro studies have shown that vaccination can prevent HPV-related sperm damages, trophoblast apoptosis, and spontaneous abortion. Moreover, it can alleviate the serious concern of sperm banks about HPV infection of donated sperm (1, 3). Therefore, the effects of HPV on early embryo development are not conclusive and the research in the field is still in its infancy. However, the available experimental findings confirmed the possible negative effects of HPV on increased blastocyst apoptosis and decreased trophoblast cell attachment to the endometrium. Accordingly, new scientific approaches should be used to better understand the exact role of HPV infection in male and female infertility and early pregnancy development. Regarding current evidence on the relationship between HPV gametes infection and abnormalities at early stages of human reproduction, vaccination of young men, women, and couples is a reliable option to protect their fertility and improve ART outcomes in idiopathic infertility that might be linked to HPV infection. 071 73 Mohammad Reza MR Sadeghi محمدرضا صادقی Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran No Keyword 130160.pdf Pereira N, Kucharczyk KM, Estes JL, Gerber RS, Lekovich JP, Elias RT, et al. Human papillomavirus infection, infertility, and assisted reproductive outcomes. J Pathog. 2015;2015:578423.##Zacharis K, Messini CI, Anifandis G, Koukoulis G, Satra M, Daponte A. Human papilloma virus (HPV) and fertilization: a mini review. Medicina. 2018;54(4):50. ##Muscianisi F, De Toni L, Giorato G, Carosso A, Foresta C, Garolla A. Is HPV the novel target in male idiopathic infertility? a systematic review of the literature. Front Endocrinol (Lausanne). 2021;12:643539.##Souho T, Benlemlih M, Bennani B. Human papillomavirus infection and fertility alteration: a systematic review. PLoS One. 2015;10(5):e0126936.##Siristatidis C, Vaidakis D, Sertedaki E, Martins WP. Effect of human papilloma virus infection on in‐vitro fertilization outcome: systematic review and meta‐analysis. Ultrasound Obstet Gynecol. 2018;51(1):87-93. ##De Toni L, Cosci I, Carosso A, Barzon L, Engl B, Foresta C, et al. Hyaluronidase-based swim-up for semen selection in patients with human papillomavirus semen infection. Biol Reprod. 2021;104(1):211-22.##

Therapeutic Effects of Edaravone on Azoospermia: Free Radical Scavenging and Autophagy Modulation in Testicular Tissue of Mice 2 1 2 Background: Chemotherapeutic agents such as cyclophosphamide and busulfan have been shown to have a negative impact on the spermatogenesis process. Based on this fact, the objective of this study was to investigate the effects of edaravone on spermatogenesis in busulfan-induced mice. Methods: Forty adult male mice were equally divided into the four groups: 1) control, 2) edaravone, 3) busulfan, and 4) busulfan + edaravone. Then, the sperm parameters, histopathological examinations, and serum levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also assessed. Caspase-3, Beclin-1, and ATG-7 mRNA levels were also determined using real-time PCR. Results: Our results revealed that treatment of mice with edaravone in busulfan-induced azoospermia significantly improves sperm parameters, including total count, morphology, and viability (p<0.05). Furthermore, edaravone administration led to a significant increase in serum testosterone (p<0.0001) and FSH (p<0.001) levels, as well as testis weight (p<0.05) and volume (p<0.01). Edaravone also prevented a decrease in the number of testicular cells including spermatogonia (p<0.0001), primary spermatocytes (p< 0.001), round spermatids (p<0.0001), Sertoli (p<0.01), and Leydig cells (p<0.0001) in busulfan-treated mice. Additionally, in busulfan-induced azoospermia, edaravone significantly reduced the percentage of sperm with immature chromatin (p<0.0001). Following treatment with edaravone, a decrease in reactive oxygen species (ROS) and an increase in glutathione (GSH) production were noted compared to busulfan-treated mice. Further-more, caspase-3 (p<0.05), Beclin-1, and ATG-7 (p<0.001) genes expression decreased significantly in treatment groups compared to busulfan-induced azoospermia. Conclusion: According to our findings, edaravone can improve spermatogenesis in busulfan-induced azoospermia through free radical scavenging and autophagy modulation in testicular tissue. 073 84 Mahsa M Ghafari Novi Mens Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences Mens Health and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences Iran Mohammadamin M Sabbagh Alvani Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Mohammadreza MR Mafi Balani Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Abbas A Aliaghaei Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Azar A Afshar Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Fakhroddin F Aghajanpour Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Reza R Soltani Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Maryam M Salimi Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Ahad Hasan AH Syed Hasani Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Shabnam Sh Abdi Department of Anatomical Sciences & Cognitive Neuroscience, Faculty of Medicine, Tehran Medical Sciences, Islamic Azad University Department of Anatomical Sciences & Cognitive Neuroscience, Faculty of Medicine, Tehran Medical Sciences, Islamic Azad University Iran Mohammad-Amin MA Abdollahifar Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran m_amin58@yahoo.com, abdollahima@sbmu.ac.ir Pourya P Raee Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences Iran pourayarayi3@gmail.com Autophagy modulationAzoospermiaBusulfanEdaravoneFree radical scavenger 120143.pdf Ziaeipour S, Rezaei F, Piryaei A, Abdi S, Moradi A, Ghasemi A, et al. Hyperthermia versus busulfan: Finding the effective method in animal model of azoospermia induction. Andrologia. 2019;51(11): e13438.##Panahi S, Karamian A, Sajadi E, Aliaghaei A, Na-zarian H, Abdi S, et al. Sertoli cell–conditioned medium restores spermatogenesis in azoospermic mouse testis. Cell Tissue Res. 2020;379(3):577-87.##Caroppo E, Colpi GM. Hormonal treatment of men with nonobstructive azoospermia: what does the evidence suggest? J Clin Med. 2021;10(3):387.##Schlegel PN, Sigman M, Collura B, De Jonge CJ, Eisenberg ML, Lamb DJ, et al. Diagnosis and treat-ment of infertility in men: AUA/ASRM guideline part II. Fertil Steril. 2021;115(1):62-9.##Zhang HL, Zhao LM, Mao JM, Liu DF, Tang WH, Lin HC, et al. Sperm retrieval rates and clinical out-comes for patients with different causes of azoosper-mia who undergo microdissection testicular sperm extraction-intracytoplasmic sperm injection. Asian J Androl. 2021;23(1):59-63.##Zhankina R, Baghban N, Askarov M, Saipiyeva D, Ibragimov A, Kadirova B, et al. Mesenchymal stro-mal/stem cells and their exosomes for restoration of spermatogenesis in non-obstructive azoospermia: a systemic review. Stem Cell Res Ther. 2021;12(1): 229.##Barati E, Nikzad H, Karimian M. Oxidative stress and male infertility: current knowledge of patho-physiology and role of antioxidant therapy in dis-ease management. Cell Mol Life Sci. 2020;77(1): 93-113.##Dutta S, Henkel R, Sengupta P, Agarwal A. Physiological role of ROS in sperm function. In: Parekattil SJ, Esteves SC, Agarwal A, editors. Male Infertility. Switzerland: Springer, Cham; 2020. p. 337-45.##Sanocka D, Kurpisz M. Reactive oxygen species and sperm cells. Reprod Biol Endocrinol. 2004;2:12.##Tanaka M. [Pharmacological and clinical profile of the free radical scavenger edaravone as a neuro-protective agent]. Nihon Yakurigaku Zasshi. 2002; 119(5):301-8. Japanese.##Ismail H, Shakkour Z, Tabet M, Abdelhady S, Kobaisi A, Abedi R, et al. Traumatic brain injury: Oxidative stress and novel anti-oxidants such as Mitoquinone and Edaravone. Antioxidants (Basel). 2020;9(10):943.##Chen H, Chen Y, Wang X, Yang J, Huang C. Edaravone attenuates myocyte apoptosis through the JAK2/STAT3 pathway in acute myocardial in-farction. Free Radic Res. 2020;54(5):351-9.##Kassab AA, Aboregela AM, Shalaby AM. Edara-vone attenuates lung injury in a hind limb ische-mia-reperfusion rat model: a histological, immuno-histochemical and biochemical study. Ann Anat. 2020;228:151433.##Ommati MM, Attari H, Siavashpour A, Shafaghat M, Azarpira N, Ghaffari H, et al. Mitigation of cholestasis-associated hepatic and renal injury by edaravone treatment: evaluation of its effects on oxidative stress and mitochondrial function. Liver Res. 2021;5(3):181-93.##Tsounapi P, Saito M, Dimitriadis F, Koukos S, Shimizu S, Satoh K, et al. Antioxidant treatment with edaravone or taurine ameliorates diabetes-in-duced testicular dysfunction in the rat. Mol Cell Biochem. 2012;369(1-2):195-204.##Zhao X, Zhang E, Ren X, Bai X, Wang D, Bai L, et al. Edaravone alleviates cell apoptosis and mito-chondrial injury in ischemia–reperfusion-induced kidney injury via the JAK/STAT pathway. Biol Res. 2020;53(1):28.##Wang B, Lin W. Edaravone protects against pan-creatic and intestinal injury after acute pancreatitis via nuclear factor-κB signaling in mice. Biol Pharm Bull. 2020;43(3):509-15.##Yalcin Comert HS, Imamoglu M, Ercin ME, Eksi E, Kandaz M, Alver A, et al. Effect of edaravone on lungs and small intestine in rats with induced radiotherapy. Indian J Exp Biol. 2021;59:162-7.##Zarei L, Sadrkhanlou R, Shahrooz R, Malekinejad H, Eilkhanizadeh B, Ahmadi A. Protective effects of vitamin E and Cornus mas fruit extract on methotrexate-induced cytotoxicity in sperms of adult mice. Vet Res Forum. 2014;5(1):21-7.##Fernández JL, Muriel L, Goyanes V, Segrelles E, Gosálvez J, Enciso M, et al. Simple determination of human sperm DNA fragmentation with an im-proved sperm chromatin dispersion test. Fertil Steril. 2005;84(4):833-42.##Ziaeipour S, Ahrabi B, Naserzadeh P, Aliaghaei A, Sajadi E, Abbaszadeh H-A, et al. Effects of sertoli cell transplantation on spermatogenesis in azoo-spermic mice. Cell Physiol Biochem. 2019;52(3): 421-34.##Ayoubi M, Naserzadeh P, Hashemi MT, Rostami MR, Tamjid E, Tavakoli MM, et al. Biochemical mechanisms of dose-dependent cytotoxicity and ROS-mediated apoptosis induced by lead sulfide/ graphene oxide quantum dots for potential bio-imaging applications. Sci Rep. 2017;7(1):12896.##Tamamura M, Saito M, Kinoshita Y, Shimizu S, Satoh I, Shomori K, et al. Protective effect of edaravone, a free‐radical scavenger, on ischa-emia‐reperfusion injury in the rat testis. BJU Int. 2010;105(6):870-6.##Homma T, Kobayashi S, Sato H, Fujii J. Edara-vone, a free radical scavenger, protects against fer-roptotic cell death in vitro. Exp Cell Res. 2019; 384(1):111592.##Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, et al. ROS and ROS-mediated cellular signaling. Oxid Med Cell Longev. 2016;2016:4350965.##Sukmawan R, Yada T, Toyota E, Neishi Y, Kume T, Shinozaki Y, et al. Edaravone preserves coro-nary microvascular endothelial function after is-chemia/reperfusion on the beating canine heart in vivo. J Pharm Sci. 2007;104(4):341-8.##Ito K, Ozasa H, Horikawa S. Edaravone protects against lung injury induced by intestinal ische-mia/reperfusion in rat. Free Radic Biol Med. 2005; 38(3):369-74.##Hayashi C, Ito M, Ito R, Murakumo A, Yamamoto N, Hiramatsu N, et al. Effects of edaravone, a radical scavenger, on hepatocyte transplantation. J Hepatobiliary Pancreat Sci. 2014;21(12):919-24.##Shichinohe H, Kuroda S, Yasuda H, Ishikawa T, Iwai M, Horiuchi M, et al. Neuroprotective effects of the free radical scavenger Edaravone (MCI-186) in mice permanent focal brain ischemia. Brain Res. 2004;1029(2):200-6.##Zhao ZY, Luan P, Huang SX, Xiao SH, Zhao J, Zhang B, et al. Edaravone Protects HT22 Neurons from H2O2-induced Apoptosis by Inhibiting the MAPK Signaling Pathway. CNS Neurosci Ther. 2013;19(3):163-9.##Li Y, Liu H, Zeng W, Wei J. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells. PLoS One. 2017;12(3):e0174437.##Shimazaki H, Watanabe K, Veeraveedu PT, Ha-rima M, Thandavarayan RA, Arozal W, et al. The antioxidant edaravone attenuates ER-stress-medi-ated cardiac apoptosis and dysfunction in rats with autoimmune myocarditis. Free Radical Res. 2010; 44(9):1082-90.##Qiao J, Wu Y, Liu Y, Li X, Wu X, Liu N, et al. Busulfan triggers intrinsic mitochondrial-depend-ent platelet apoptosis independent of platelet ac-tivation. Biol Blood Marrow Transplant. 2016;22 (9):1565-72.##Cao Y, Klionsky DJ. Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res. 2007;17(10):839-49.##Wirawan E, Lippens S, Vanden Berghe T, Roma-gnoli A, Fimia GM, Piacentini M, et al. Beclin1: a role in membrane dynamics and beyond. Auto-phagy. 2012;8(1):6-17.##Yin J, Zhou Z, Chen J, Wang Q, Tang P, Ding Q, et al. Edaravone inhibits autophagy after neuronal oxygen-glucose deprivation/recovery injury. Int J Neurosci. 2019;129(5):501-10.##

Relationship of Clinical and Ultrasonographic Grades of Varicocele with Semen Analysis Profile and Testicular Volume 2 1 2 Background: Varicoceles are a major cause of infertility. The purpose of this study was to determine the relationship of the clinical and ultrasonographic grades of varicocele with the semen analysis profile and testicular volume among men undergoing scrotal ultrasonography. Methods: This cross-sectional analytical study involved 109 males undergoing scrotal ultrasonography for various indications in Shiraz, Iran, between January 2019 and January 2020. Varicoceles were graded with color Doppler ultrasonography (CDU) by an expert radiologist (Sarteschi's criteria) before an experienced urologist determined the clinical grade (Dubin and Amelar criteria) and requested further investigations. Next, the demographics, reasons for referral, testicular volumes, and semen analysis profiles across the different clinical/ultrasonographic grades were compared. Key statistical measures included Cohen's kappa coefficient, the Mann–Whitney and Kruskal-Wallis tests, and Spearman correlation. Data were analyzed using SPSS v. 21 with p-values <0.05 indicating statistical significance. Results: Ultrasonographic grades 1 and 2 provided the highest correlation with subclinical cases, while ultrasonographic grades 3, 4, and 5 corresponded with clinical grades 1, 2, and 3, respectively. Further comparisons were made between subclinical and clinical cases, which were similar in terms of reason for referral, total testicular volume, testicular volume differential, and semen analysis profile. Notably, total testicular volumes below 30 ml were associated with oligoasthenoteratospermia. Conclusion: The present study showed a relatively high correlation between varicocele grading based on clinical evaluation and CDU. However, the grades were similar in testicular volume parameters and semen analysis indices. Hence, decision-making should be guided by the infertility history, testicular atrophy, and abnormal semen analysis. 084 93 Amin A Abolhasani Foroughi Department of Radiology, Medical Imaging Research Center, Shiraz University of Medical Sciences Department of Radiology, Medical Imaging Research Center, Shiraz University of Medical Sciences Iran Manoochehr M Dallaki Department of Radiology, Medical Imaging Research Center, Shiraz University of Medical Sciences Department of Radiology, Medical Imaging Research Center, Shiraz University of Medical Sciences Iran Seyed Ali SA Hosseini Department of Biochemistry, Student Research Committee, Shiraz University of Medical Sciences Department of Biochemistry, Student Research Committee, Shiraz University of Medical Sciences Iran Ali A Ariafar Department of Urology, School of Medicine, Shiraz University of Medical Sciences Department of Urology, School of Medicine, Shiraz University of Medical Sciences Iran ariafara@sums.ac.ir InfertilityMale urogenital diseasesUltrasonographyVaricocele 120138.pdf Fretz PC, Sandlow JI. Varicocele: current concepts in pathophysiology, diagnosis, and treatment. Urol Clin North Am. 2002;29(4):921-37.##Lundy SD, Sabanegh Jr ES. Varicocele management for infertility and pain: a systematic review. Arab J Urol. 2017;16(1):157-70.##Cimador M, Castagnetti M, Gattuccio I, Pensabene M, Sergio M, De Grazia E. The hemodynamic ap-proach to evaluating adolescent varicocele. Nat Rev Urol. 2012;9(5):247-57.##Pilatz A, Altinkilic B, Köhler E, Marconi M, Weid-ner W. Color Doppler ultrasound imaging in vari-coceles: is the venous diameter sufficient for pre-dicting clinical and subclinical varicocele? World J Urol. 2011;29(5):645-50.##Sarteschi M. Lo studio del varicocele con eco-color-Doppler. G It Ultrason. 1993;4:43-9.##Sigman M, Jarow JP. Ipsilateral testicular hypotro-phy is associated with decreased sperm counts in infertile men with varicoceles. J Urol. 1997;158(2): 605-7.##Sharlip I, Jarow J, Belker A, Damewood M, Howards S, Lipshultz L, et al. Report on varicocele and infertility. Fertil Steril. 2004;82:S142-S5.##Belay RE, Huang GO, Shen JKC, Ko EYK. Diag-nosis of clinical and subclinical varicocele: how has it evolved? Asian J Androl. 2016;18(2):182-5.##Bhatt S, Jafri S, Wasserman N, Dogra VS. Imaging of non-neoplastic intratesticular masses. Diagn In-terv Radiol. 2011;17(1):52-63.##Arai T, Kitahara S, Horiuchi S, Sumi S, Yoshida K. Relationship of testicular volume to semen profiles and serum hormone concentrations in in-fertile Japanese males. Int J Fertil Womens Med. 1998;43(1):40-7.##Kurtz MP, Zurakowski D, Rosoklija I, Bauer SB, Borer JG, Johnson KL, et al. Semen parameters in adolescents with varicocele: association with testis volume differential and total testis volume. J Urol. 2015;193(5 Suppl):1843-7.##Dubin L, Amelar RD. Varicocele size and results of varicocelectomy in selected subfertile men with varicocele. Fertil Steril. 1970;21(8):606-9.##Foroughi AA, Yazdanpanah E, Nazeri M, Eghbali T, Arasteh P, Ariafar A. Clinical grading and color Doppler ultrasonography-based grading of varico-cele: how compatible are the two grading systems? World J Urol. 2019;37(7):1461-5.##Jedrzejewski G, Osemlak P, Wieczorek AP, Nach-ulewicz P. Testicular sonographic color doppler dynamic tissue perfusion measurements in adole-scents with varicocele. Urol Int. 2019;103(1):55-61.##Sigman M. There is more than meets the eye with varicoceles: current and emerging concepts in pathophysiology, management, and study design. Fertil Steril. 2011;96(6):1281-2.##Agarwal A, Sharma R, Harlev A, Esteves SC. Effect of varicocele on semen characteristics ac-cording to the new 2010 World Health Organiza-tion criteria: a systematic review and meta-ana-lysis. Asian J Androl. 2016;18(2):163-70.##Jensen CFS, Østergren P, Dupree JM, Ohl DA, Sønksen J, Fode M. Varicocele and male in-fertility. Nat Rev Urol. 2017;14(9):523-33.##Ghaed MA, Makian SA, Moradi A, Maghsoudi R, Gandomi-Mohammadabadi A. Best time to wait for the improvement of the sperm parameter after varicocelectomy: 3 or 6 months? Arch Ital Urol Androl. 2020;92(3).##Zheng YQ, Gao X, Li ZJ, Yu YL, Zhang ZG, Li W. Efficacy of bilateral and left varicocelectomy in infertile men with left clinical and right subclinical varicoceles: a comparative study. Urology. 2009; 73(6):1236-40.##Kim HJ, Seo JT, Kim KJ, Ahn H, Jeong JY, Kim JH, et al. Clinical significance of subclinical vari-cocelectomy in male infertility: Systematic review and meta‐analysis. Andrologia. 2016;48(6):654-61.##Owen RC, McCormick BJ, Figler BD, Coward RM. A review of varicocele repair for pain. Transl Androl Urol. 2017;6(Suppl 1):S20-S9.##Damsgaard J, Joensen UN, Carlsen E, Erenpreiss J, Jensen MB, Matulevicius V, et al. Varicocele is associated with impaired semen quality and re-productive hormone levels: a study of 7035 healthy young men from six European countries. Eur Urol. 2016;70(6):1019-29.##Semiz I, Tokgöz Ö, Tokgoz H, Voyvoda N, Seri-foglu I, Erdem Z. The investigation of correlation between semen analysis parameters and intra-parenchymal testicular spectral Doppler indices in patients with clinical varicocele. Ultrasound Q. 2014;30(1):33-40.##Akbay E, Cayan S, Doruk E, Duce M, Bozlu M. The prevalence of varicocele and varicocele‐ related testicular atrophy in Turkish children and adolescents. BJU Int. 2000;86(4):490-3.##Oliva A, Multigner L. Chronic epididymitis and Grade III varicocele and their associations with semen characteristics in men consulting for couple infertility. Asian J Androl. 2018;20(4):360-5.##Sakamoto H, Saito K, Ogawa Y, Yoshida H. Effects of varicocele repair in adults on ultraso-nographically determined testicular volume and on semen profile. Urology. 2008;71(3):485-9.##Krishna Reddy S, Basha Shaik A, Sailaja S, Ven-kataramanaiah M. Outcome of varicocelectomy with different degrees of clinical varicocele in infertile male. Advanc Androl. 2015;2015:1-9.##

Comparison of Pregnancy Outcomes Between Fresh Embryo Transfer in a Natural IVF Cycle and IUI Cycle Among Infertile Young Women 2 1 2 Background: The purpose of the current study was comparing pregnancy outcomes for natural cycle in vitro fertilization (IVF) per fresh embryo transfer (ET) and oocyte pick-up (OPU) in intrauterine insemination (IUI). Methods: This was a retrospective cohort study of women who underwent either IUI (n=246) or OPU with fresh ET for natural cycle IVF (n=291), conducted between April 2017 and February 2018 at the Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku, Tokyo, Japan. Patients in both groups did not receive ovarian stimulation and luteal support; gonadotropin-releasing agonist spray was administered 35 hr before OPU or IUI. The clinical pregnancy rate was compared between the IUI and IVF groups. Data analysis was based on the number of cycles. The p≦0.05 was considered significant. Results: The clinical pregnancy rate per OPU in the IVF group was higher than the one in IUI group (20.6% vs. 10.1%), and the difference was significant (p<0.01). The pregnancy rate for natural cycle IVF calculated per fresh ET was 36.8%. The miscarriage rate did not significantly differ between the IVF (4.1%) and IUI (8.0%) groups. Conclusion: Fresh ET in natural cycle IVF provides a higher implantation rate than IUI. 093 100 Yoko Y Gekka Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan 405fmv@gmail.com Koji K Nakagawa Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan Hideaki H Watanabe Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan Keiji K Kuroda Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan Takashi T Horikawa Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan Satoru S Takamizawa Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan Rikikazu R Sugiyama Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Center for Reproductive Medicine and Implantation Research, Sugiyama Clinic Shinjuku Japan Fresh embryo transferIUINatural cycle IVFPregnancy rate 120129.pdf von Wolff M. The role of Natural Cycle IVF in assisted reproduction. Best Pract Res Clin Endo-crinol Metab. 2019;33(1):35-45.##van der Gaast MH, Eijkemans MJC, van der Net JB, de Boer EJ, Burger CW, van Leeuwen FE, et al. Optimum number of oocytes for a successful first IVF treatment cycle. Reprod Biomed Online. 2006; 13(4):476-80.##Takeshima K, Jwa SC, Saito H, Nakaza A, Kuwa-hara A, Ishihara O, et al. Impact of single embryo transfer policy on perinatal outcomes in fresh and frozen cycles-analysis of the Japanese assisted re-production technology registry between 2007 and 2012. Fertil Steril. 2016;105(2):337-46.e3.##Basirat Z, Adib Rad H, Esmailzadeh S, Jorsaraei SG, Hajian-Tilaki K, Pasha H, et al. Comparison of pregnancy rate between fresh embryo transfers and frozen-thawed embryo transfers following ICSI treatment. Int J Reprod Biomed. 2016;14(1):39-46.##Feraretti AP, Goossens V, Kupka M, Bhattacharya S, de Mouzon J, Castilla JA, et al. Assisted re-productive technology in Europe, 2009: resulting generated from European register by ESHRE. Hum Reprod. 2013;28(9):2318-31.##Nakagawa K, Oba M, Ehara K, Ishigaki N, Ino N, Itakura A, et al. Clinical outcomes of assisted re-productive technology treatment by using a self-injection of recombinant human chorionic gonado-tropin as the final maturation trigger. Reprod Med Biol. 2018;17(2):203-8.##Nakagawa K, Nishi Y, Sugiyama R, Juen H, Taka-hashi C, Ojiro Y, et al. A programmed schedule of oocyte retrieval using mild ovarian stimulation (clo-miphene citrate and recombinant follicle-stimulating hormone). Reprod Med Biol. 2011;11(2):85-9.##Nakagawa K, Shirai A, Nishi Y, Sugiyama R, Kuri-bayashi Y, Sugiyama R, et al. A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted repro-ductive technology. Reprod Med Biol. 2010;9(3): 163-8.##Nakagawa K, Ojiro Y, Nishi Y, Sugiyama R, Moto-yama H, Sugiyama R. Perinatal outcomes of patients who achieved pregnancy with a morphologically poor embryo via assisted reproductive technology. Arc Gynecol Obstet. 2016;293(1):183-8.##Nakagawa K, Takahashi C, Nishi Y, Juen H, Sugi-yama R, Kuribayashi Y, et al. Hyaluronan-enriched transfer medium improves outcome in patients with multiple embryo transfer failures. J Assist Reprod Genet. 2012;29(7):679-85.##Oba M, Nakagawa K, Ehara K, Ishigaki N, Yama-shiro E, Shirai A, et al. Administration of the maturation trigger 35-hours before intrauterine in-semination (IUI) improves the pregnancy rate. J Fertil Implan. 2019;35:225-8.##Pelinck MJ, Hoek A, Simons AH, Heineman MJ. Efficacy of natural cycle IVF: a review of the literature. Hum Reprod Update. 2002;8(2):129-39.##Ikemoto Y, Kuroda K. Pregnancy rate by various treatment methods. In: Takeda S, Tanaka A, Ku-ro-da K, editors. Treatment for infertility and recur-rent miscarriage on the basis of clinical data. Tok-yo: Medical View; 2017. p. 136-41.##Nargund G, Fauser BCJM. Mild ovarian stimula-tion for IVF is the smartest way forward. Reprod Biomed Online. 2020;41(4):569-71.##Daya S, Gunby J, Hughes EG, Collins JA, Sagle MA, Young Lai EV. Natural cycles for in-vitro fer-tilization: cost-effectiveness analysis and factors influencing outcome. Hum Reprod. 1995;10(5): 1719-24.##Fahy UM, Cahill DJ, Wardle PG, Hull MG. In-vitro fertilization in completely natural cycles. Hum Reprod. 1995;10(3):572-5.##von Wolff M, Fäh M, Roumet M, Mitter V, Stute P, Griesinger G, et al. Thin endometrium is also associated with lower clinical pregnancy rate in unstimulated menstrual cycles: a study based on natural cycle IVF. Front Endocrinol (Lausanne). 2018;9:776.##von Wolff M, Schwartz AK, Bitterlich N, Stute P, Fäh M. Only women's age and the duration of infertility are the prognostic factors for the success rate of natural cycle IVF. Arch Gynecol Obstet. 2019;299(3):883-9.##Tomic V, Kasum M, Vucic K. Impact of embryo quality and endometrial thickness on implantation in natural cycle IVF. Arch Gynecol Obstet. 2020; 301(5):1325-30.##Kamath MS, Kirubakaran R, Mascarenhas M, Sun-kara SK. Perinatal outcomes after stimulated ver-sus natural cycle IVF: a systematic review and meta-analysis. Reprod Biomed Online. 2018;36(1): 94-101.##

Comparison of Obstetric Outcomes Between IVF cycles with Donor Oocyte and Spontaneous Conception Pregnancies: A Retrospective Cohort study 2 1 2 Background: Oocyte donation has facilitated couples to achieve pregnancy in conditions like diminished ovarian reserve, premature ovarian failure, and inheritable disorders. However, it is unclear whether pregnancy complications are due to oocyte donation per se or due to confounding factors such as maternal age or the allogenic fetus. In this retrospective comparative cohort, an attempt was made to evaluate and compare multiple obstetric and perinatal outcomes. Methods: The present study comprised all women in the age range of 20-45 years who conceived from oocyte donation (n=102) between 1/12/2011 to 30/09/2017. Control group consisted of spontaneous conception cases (n=306) in ratio of 1:3 with no previous medical or surgery comorbidity. Obstetric and perinatal outcomes were compared between two groups. Results: Mean maternal age was significantly higher in the donor oocyte IVF group (group 1; 35.13 years) as compared to spontaneous conception group (group 2; 31.75 years). Parity between the two groups was comparable. Pregnancy induced hypertension (PIH) was seen in 33.33% of cases in group 1 as compared to 7.18% in group 2. Moreover, gestational diabetes mellitus was seen in 34.31% of cases in group 1 as compared to 9.47% in group 2 (p=0.001). By the same token, there was significant difference in perinatal outcomes between the two groups. Conclusion: Oocyte donation should be treated as an independent risk factor for miscarriage, hypertensive disorder, and gestational diabetes mellitus in pregnancy. 100 107 Yadav Y Vikas Department of Obstetrics and Gynecology, School of Medical Sciences and Research, Sharda Hospital, Sharda University Campus Department of Obstetrics and Gynecology, School of Medical Sciences and Research, Sharda Hospital, Sharda University Campus India vikiyadav1789@gmail.com Malhotra M Neena Department of Obstetrics and Gynecology, All India Institute of Medical Sciences Department of Obstetrics and Gynecology, All India Institute of Medical Sciences India Mahey M Reeta Department of Obstetrics and Gynecology, All India Institute of Medical Sciences Department of Obstetrics and Gynecology, All India Institute of Medical Sciences India Singh S Neeta Department of Obstetrics and Gynecology, All India Institute of Medical Sciences Department of Obstetrics and Gynecology, All India Institute of Medical Sciences India Kriplani K Alka Department of Obstetrics and Gynecology, All India Institute of Medical Sciences Department of Obstetrics and Gynecology, All India Institute of Medical Sciences India Bleeding in first trimesterGestational diabetes mellitusOocyte donationPregnancy induced hypertension 120141.pdf Remohi J, Gartner B, Gallardo E, Yalil S, Smon C, Pellicer A. Pregnancy and birth rate after oocyte donation. Fertil Steril. 1997;67(4):717-23.##Inhorn MC. Where has the quest for conception taken us? lessons from anthropology and sociology. Reprod Biomed Soc Online. 2020;10:46-57.##Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P. The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature. 1984;307(5974):174-5.##Kavic SM, Sauer MV. Oocyte donation treats in-fertility in survivors of malignancies: ten-year ex-perience. J Assist Reprod Genet. 2001;18(3):181-3.##Michalas S, Loutradis D, Drakakis P, Milingos S, Papageorgiou J, Kallianidis K, et al. Oocyte dona-tion to women over 40 years of age: pregnancy com-plications. Eur J Obstet Gynecol Reprod Biol. 1996; 64(2):175-8.##Simchen MJ, Yinon Y, Moran O, Schiff E, Sivan E. Pregnancy outcome after age 50. Obstet Gynecol. 2006;108(5):1084-8.##Serhal PF, Craft IL. Oocyte donation in 61 patients. Lancet. 1989:1(8648):1185-7.##Blanchette H. Obstetric performance of patients after oocyte donation. Am J Obstet Gynecol. 1993; 168(6 Pt 1):1803-7.##Abdalla HI, Billett A, Kan AK, Baig S, Wren M, Korea L, et al. Obstetric outcome in 232 ovum do-nation pregnancies. Br J Obstet Gynaecol. 1998;105 (3):332-7.##Soderstrom-Anttila V, Foudila T, Hovatta O. A randomized comparative study of highly purified follicle stimulating hormone and human meno-pausal gonadotrophin for ovarian hyperstimulation in an oocyte donation programme. Hum Reprod. 1996;11(9):1864-70.##Klatsky PC, Delaney SS, Caughey AB, Tran ND, Schattman GL, Rosenwaks Z. The role of embry-onic origin in preeclampsia: a comparison of auto-logous in vitro fertilization and ovum donor preg-nancies. Obstet Gynecol. 2010;116(6):1387-92.##Levron Y, Dviri M, Segol I, Yerushalmi GM, Hou-rvitz A, Orvieto R, et al. The ‘immunologic theory’ of preeclampsia revisited: a lesson from donor oocyte gestations. Am J Obstet Gynecol. 2014;211 (4):383.e1-5.##Salha O, Sharma V, Dada T, Nugent D, Rutherford AJ, et al. The influence of donated gametes on the incidence of hypertensive disorders of pregnancy. Hum Reprod. 1999;14(9):268-73.##Sheffer-Mimouni G, Mashiach S, Dor J, Levran D, Seidman DS. Factors influencing the obstetric and perinatal outcome after oocyte donation. Hum Re-prod. 2002;17(10):2636-40.##Toner JP, Grainger DA, Frazier LM. Clinical out-comes among recipients of donated eggs: an ana-lysis of the U.S. national experience, 1996-1998. Fertil Steril. 2002;78(8):1038-45.##Wiggins DA, Main E. Outcomes of pregnancies achieved by donor egg in vitro fertilization-a com-parison with standard in vitro fertilization preg-nancies. Am J Obstet Gynecol. 2005;192(6):2002-6.##Malhotra J, Malhotra K, Talwar P, Kannan P, Singh P, Kumar Y, et al. ISAR consensus guide-lines on safety and ethical practices in In vitro fer-tilization clinics. J Hum Reprod Sci. 2021;14 (Suppl 1):S48-S68.##Paulson RJ, Boostanfar R, Saadat P, Mor E, Tourgeman DE, Slater CC, et al. Pregnancy in the sixth decade of life: obstetric outcomes in women of advanced reproductive age. JAMA. 2002;288 (18):2320-3.##Soderstrom-Anttila V, Foudila TA, Hovatta O. Ob-stetric and perinatal outcome after oocyte donation: comparison with in-vitro fertilization pregnancies. Hum Reprod. 1998;13(2):483-90.##Pados G, Camus M, Van Steirteghem A, Bonduelle M, Devroey P. The evolution and outcome of preg-nancies from oocyte donation. Hum Reprod. 1994; 9(3):538-42##Hipp HS, Gaskins AJ, Nagy ZP, Capelouto SM, Shapiro DB, Spencer JB. Effect of oocyte donor stimulation on recipient outcomes: data from a US national donor oocyte bank. Hum Reprod. 2020;35 (4):847-58.##Krieg SA, Henne MB, Westphal LM. Obstetric outcomes in donor oocyte pregnancies compared with advanced maternal age in in vitro fertilization pregnancies. Fertil Steril. 2008;90(1):65-70.##Malchau SS, Loft A, Larsen EC, Aaris Henningsen AK, Rasmussen S, Andersen AN, et al. Perinatal outcomes in 375 children born after oocyte dona-tion: a Danish national cohort study. Fertil Steril. 2013;99(6):1637-43.##Laskov I, Birnbaum R, Maslovitz S, Kupfer-min-cM, Lessing J, Many A. Outcome of singleton pregnancy in women ≥45 years old: a retrospective cohort study. J Matern Fetal Neonatal Med. 2012; 25(11):2190-3.##Le Ray C, Scherier S, Anselem O, Marszalek A, Tsatsaris V, Cabrol D, et al. Association between oocyte donation and maternal and perinatal out-comes in women aged 43 years or older. Hum Re-prod. 2012;27(3):896-901.##Tranquilli AL, Biondini V, Talebi Chahvah S, Cor-radetti A, Tranquilli D, Giannubilo S,et al. Per-inatal outcomes in oocyte donor pregnancies. J Matern Fetal Neonatal Med. 2013;26(13):1263-7.##Taglauer ES, Gundogan F, Johnson KL, Scherjon SA, Bianchi DW. Chorionic plate expression pat-terns of the maspin tumor suppressor protein in preeclamptic and egg donor placentas. Placenta. 2013;34(4):385-7.##Li DK, Wi S. Changing paternity and the risk of preeclampsia/eclampsia in the subsequent preg-nancy. Am J Epidemiol. 2000;151(1):57-62.##van der Hoorn ML, Scherjon SA, Claas FH. Egg donation pregnancy as an immunological model for solid organ transplantation. Transpl Immunol. 2011;25(2-3):89-95.##Gundogan F, Bianchi DW, Scherjon SA, Roberts DJ. Placental pathology in egg donor pregnancies. Fertil Steril. 2010;93(2):397-404##Karnis MF, Zimon AE, Lalwani SI, Timmreck LS, Klipstein S, Reindollar RH. Risk of death in pregnancy achieved through oocyte donation in patients with Turner syndrome: a national survey. Fertil Steril. 2003;80(3):498-501.##Moreno-Sepulveda J, Checa MA. Risk of adverse perinatal outcomes after oocyte donation: a sys-tematic review and meta-analysis. J Assist Reprod Genet. 2019;36(10):2017-37.##Braat DD, Schutte JM, Bernardus RE, Mooij TM, van Leeuwen FE. Maternal death related to IVF in the Netherlands 1984-2008. Hum Reprod. 2010;25 (7):1782-6.##

Triaging Women with Pregnancy of Unknown Location- the Performance of Protocols Based on Single Serum Progesterone, Serum hcg Ratios and M4 Model 2 1 2 Background: The purpose of the current study was to evaluate the ability of three protocols to triage women presenting with pregnancy of unknown location (PUL). Methods: Women with pregnancy of unknown location were recruited from Aziz Medical Centre from 1st August, 2018 to 31st July, 2020. The criterion of progesterone, human chorionic gonadotrophin (hCG) ratio, and M4 algorithm were used to predict risk of adverse pregnancy outcomes and classify women. Finally, 3 groups were established including ectopic pregnancy, failed pregnancy of unknown location, and intrauterine pregnancy (IUP). The primary outcome was to assign women to ectopic pregnancy group using these protocols. The secondary outcome was to compare the sensitivity and specificity of the three protocols relative to the final outcome. Results: Of the 288 women, 66 (22.9%) had ectopic pregnancy, 144 (50.0%) had intrauterine pregnancy, and 78 (27.1%) had failed pregnancy of unknown location. The criterion of progesterone had a sensitivity of 81.8%, specificity of 27%, negative predictive value (NPV) of 83.3%, and positive predictive value (PPV) of 25% for high risk result (ectopic pregnancy). The hCG ratio had sensitivity of 72%, specificity of 73%, NPV of 90%, and PPV of 44% for high risk result (ectopic pregnancy). However, model M4 had sensitivity of 86.4%, specificity of 91.9%, NPV of 95.8%, and PPV of 76% for high risk result. Conclusion: Based on the findings of the study, it was revealed that prediction model of M4 had the highest sensitivity, specificity, negative predictive value and positive predictive value for high risk result (ectopic pregnancy). 107 114 Rubina R Izhar Department of Gynaecology and Obstetrics, Aziz Medical Center Department of Gynaecology and Obstetrics, Aziz Medical Center Pakistan Samia S Husain Department of Gynaecology and Obstetrics, Aziz Medical Center Department of Gynaecology and Obstetrics, Aziz Medical Center Pakistan samiahusain_scorpio@hotmail.com Muhammad Ahmad MA Tahir Department of Gynaecology and Obstetrics, Aziz Medical Center Department of Gynaecology and Obstetrics, Aziz Medical Center India Syed Hasan SH Ala Dow University of Health Sciences (DUHS) Dow University of Health Sciences (DUHS) India Rahila R Imtiaz Department of Obstetrics and Gynaecology, Karachi Medical and Dental College Department of Obstetrics and Gynaecology, Karachi Medical and Dental College Pakistan Sonia S Husain Aga Khan University Hospital Aga Khan University Hospital Pakistan Sara S Talha Department of Gynaecology and Obstetrics, Aziz Medical Center Department of Gynaecology and Obstetrics, Aziz Medical Center Pakistan Ectopic pregnancyMiscarriagePrediction modelPregnancy of unknown locationResource allocationTriage methodsTriage standardsUltrasonography 120148.pdf Bobdiwala S, Al-Memar M, Farren J, Bourne T. Factors to consider in pregnancy of unknown loca-tion. Womens Health (Lond). 2017;13(2):27-33.##Fields L, Hathaway A. Key concepts in pregnancy of unknown location: identifying ectopic pregnancy and providing patient-centered care. J Midwifery Womens Health. 2017;62(2):172-9.##Fistouris J, Bergh C, Strandell A. Classification of pregnancies of unknown location according to four different hCG-based protocols. Hum Reprod. 2016; 31(10):2203-11.##Cordina M, Schramm-Gajraj K, Ross JA, Lautman K, Jurkovic D. Introduction of a single visit protocol in the management of selected patients with preg-nancy of unknown location: a prospective study. BJOG. 2011;118(6):693-7.##Condous G, Kirk E, Lu C, Van Huffel S, Gevaert O, De Moor B, et al. Diagnostic accuracy of varying discriminatory zones for the prediction of ectopic pregnancy in women with a pregnancy of unknown location. Ultrasound Obstet Gynecol. 2005;26(7): 770-5.##Condous G, Kirk E, Van Calster B, Van Huffel S, Timmerman D, Bourne T. Failing pregnancies of unknown location: a prospective evaluation of the human chorionic gonadotrophin ratio. BJOG. 2006; 113(5):521-7.##Condous G, Okaro E, Bourne T. Pregnancies of un-known location: diagnostic dilemmas and manage-ment. Curr Opin Obstet Gynecol. 2005;17(6):568-73.##Kirk E, Condous G, Van Calster B, Van Huffel S, Timmerman D, Bourne T. Rationalizing the follow-up of pregnancies of unknown location. Hum Re-prod. 2007;22(6):1744-50.##Condous G, Van Calster B, Kirk E, Haider Z, Tim-merman D, Van Huffel S, et al. Prediction of ectopic pregnancy in women with a pregnancy of unknown location. Ultrasound Obstet Gynecol. 2007;29(6): 680-7.##Barnhart KT, Sammel MD, Appleby D, Rausch M, Molinaro T, Van Calster B, et al. Does a prediction model for pregnancy of unknown location devel-op-ed in the UK validate on a US population? Hum Reprod. 2010;25(10):2434-40.##Bobdiwala S, Guha S, Van Calster B, Ayim F, Mitchell-Jones N, Al-Memar M, et al. The clinical performance of the M4 decision support model to triage women with a pregnancy of unknown loca-tion as at low or high risk of complications. Hum Reprod. 2016;31(7):1425-35.##Ooi S, De Vries B, Ludlow J. How do the M4 and M6 models perform in an Australian pregnancy of unknown location population? Aust N Z J Obstet Gynaecol. 2021;61(1):100-5.##Bobdiwala S, Saso S, Verbakel JY, Al-Memar M, Van Calster B, Timmerman D, et al. Diagnostic protocols for the management of pregnancy of unknown location: a systematic review and meta-analysis. BJOG. 2019;126(2):190-8.##van Mello NM, Mol F, Opmeer BC, Ankum WM, Barnhart K, Coomarasamy A, et al. Diagnostic value of serum hCG on the outcome of pregnancy of unknown location: a systematic review and meta -analysis. Hum Reprod Update. 2012;18(6):603-17.##Nadim B, Leonardi M, Infante F, Lattouf I, Reid S, Condous G. Rationalizing the management of pregnancies of unknown location: Diagnostic ac-curacy of human chorionic gonadotropin ratio-based decision tree compared with the risk pre-diction model M4. Acta Obstet Gynecol Scand. 2020;99(3):381-90.##Bignardi T, Condous G, Kirk E, Van Calster B, Van Huffel S, Timmerman D, et al. Viability of intrauterine pregnancy in women with pregnancy of unknown location: prediction using human cho-rionic gonadotropin ratio vs. progesterone. Ultra-sound Obstet Gynecol. 2010;35(6):656-61.##Christodoulou E, Bobdiwala S, Kyriacou C, Farren J, Mitchell-Jones N, Ayim F, et al. External vali-dation of models to predict the outcome of preg-nancies of unknown location: a multicentre cohort study. BJOG. 2020;128(3):552-62.##Nadim B, Leonardi M, Stamatopoulos N, Reid S, Condous G. External validation of risk prediction model M4 in an Australian population: Rationalis-ing the management of pregnancies of unknown location. Aust N Z J Obstet Gynaecol. 2020;60(6): 928-34.##Valdera Simbrón CJ, Hernández Rodríguez C, Ll-anos Jiménez L, Pérez García L, Plaza Arranz J, Albi González M. Management of early gestations with low beta-hCG levels after the use of assisted reproduction techniques: assessment of the M4 predictive model performance. Ultrasound Obstet Gynecol. 2021;58(4):616-24.##

The Role of Genital Mycoplasmas in Preterm Labor 2 1 2 Background: Growth of Mycoplasma in genital tract can cause problems such as infertility, pelvic inflammatory disease (PID), and preterm labor. This study was designed to evaluate the role of these bacteria in preterm labor among individuals in Gorgan city which is located in north of Iran. Methods: The study included 100 women with complaints of pain in preterm labor before 37 weeks of pregnancy (case group) and 100 women with term labor (control group) who were referred to Shahid Sayyad Shirazi Teaching Hospital in Gorgan city, north of Iran. Vaginal swabs, collected from all of these women, were evaluated for genital Mycoplasma sp. by molecular method using specific primers with poly-merization chain reaction (PCR). The comparison of results was done by conducting X 2 and p<0.05 was considered significant. Results: Genital Mycoplasma was detected in 78 cases (39%) of 200 vaginal samples. Genital Mycoplasma colonization rates in the preterm and term samples were 60% and 18%, respectively, with relative risk of 2.05 (1.78-2.37) (p=0.001). The proportion of Ureaplasma parvum (44% and 15%), Ureaplasma urealyticum (11%, 3%), and Mycoplasma homins (5%, 0%) was significantly higher in women with preterm birth (PTB) than term labor. No cases of Mycoplasma genitalum were detected in this study. Conclusion: There is a significant relationship between presence of genital Mycoplasma in vaginal secretion and the risk of preterm labor. 114 120 Saber S Alinezhad Laboratory Sciences Researcher Center, Golestan University of Medical Sciences Laboratory Sciences Researcher Center, Golestan University of Medical Sciences Iran Sepideh S Bakhshandehnosrat Department of Gynecology and Obstetrics, Golestan University of Medical Sciences Department of Gynecology and Obstetrics, Golestan University of Medical Sciences Iran Assieh Sadat AS Baniaghil Counseling and Reproductive Health Research Center, Golestan University of Medical Sciences Counseling and Reproductive Health Research Center, Golestan University of Medical Sciences Iran Sedighe S Livani Microbiology Department, Medical Faculty, Golestan University of Medical Sciences Microbiology Department, Medical Faculty, Golestan University of Medical Sciences Iran Masoud M Bazouri Microbiology Department, Medical Faculty, Golestan University of Medical Sciences Microbiology Department, Medical Faculty, Golestan University of Medical Sciences Iran Maryam M Shafipour Microbiology Department, Medical Faculty, Golestan University of Medical Sciences Microbiology Department, Medical Faculty, Golestan University of Medical Sciences Iran Naser M Behnampour Department of Public Health, Faculty of Health, Golestan University of Medical Sciences Department of Public Health, Faculty of Health, Golestan University of Medical Sciences Iran Ezzat Allah EA Ghaemi Laboratory Sciences Researcher Center, Golestan University of Medical Sciences Laboratory Sciences Researcher Center, Golestan University of Medical Sciences Iran eghaemi@yahoo.com InfertilityMycoplasmaPreterm laborUreaplasmaVaginal secretion 615.pdf Terzic M, Aimagambetova G, Terzic S, Radunovic M, Bapayeva G, Laganà AS. Periodontal pathogens and preterm birth: current knowledge and further interventions. Pathogens. 2021;10(6):730.##FG lK C, Bloom S, Spong C, Dash J, Hofman B. Williams Obstetrics. 25th ed. United States of Ame-rica: McGraw-Hill Education, 2018; 2018. 1344 p.##World Health Organization. Preterm birth [Internet]. Geneva: WHO. 2018. Available: https://www.who. int/news-room/fact-sheets/detail/preterm-birth.##Sharifi N, Khazaeian S, Pakzad R, Fathnezhad Ka-zemi A, Chehreh H. Investigating the prevalence of preterm birth in Iranian population: a systematic review and meta-analysis. J Caring Sci. 2017;6(4): 371-80.##Chawanpaiboon S, Vogel JP, Moller AB, Lum-biganon P, Petzold M, Hogan D, et al. Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. Lancet Glob Health. 2019;7(1):e37-e46.##Muhe LM, McClure EM, Nigussie AK, Mekasha A, Worku B, Worku A, et al. Major causes of death in preterm infants in selected hospitals in Ethiopia (SIP): a prospective, cross-sectional, observational study. Lancet Glob Health. 2019;7(8):e1130-e8.##You YA, Yoo JY, Kwon EJ, Kim YJ. Blood mic-robial communities during pregnancy are associated with preterm birth. Front Microbiol. 2019;10:1122.##Witt RG, Blair L, Frascoli M, Rosen MJ, Nguyen QH, Bercovici S, et al. Detection of microbial cell-free DNA in maternal and umbilical cord plasma in patients with chorioamnionitis using next generation sequencing. PloS One. 2020;15(4):e0231239.##Jang YS, Min JW, Kim YS. Positive culture rate and antimicrobial susceptibilities of Mycoplasma homi-nis and Ureaplasma urealyticum. Obstet Gynecol Sci. 2019;62(2):127-33.##Moridi K, Hemmaty M, Azimian A, Fallah MH, Abyaneh HK, Ghazvini K. Epidemiology of geni-tal infections caused by Mycoplasma hominis, M. genitalium and Ureaplasma urealyticum in Iran; a systematic review and meta-analysis study (2000–2019). BMC Public Health. 2020;20(1):1020.##Pavlidis I, Spiller OB, Demarco GS, MacPherson H, Howie SE, Norman JE, et al. Cervical epithelial damage promotes Ureaplasma parvum ascending infection, intrauterine inflammation and preterm birth induction in mice. Nat Commun. 2020;11 (1):199.##Ericson JE, Laughon MM. Chorioamnionitis: im-plications for the neonate. Clin Perinatol. 2015;42 (1):155-65.##Shahshahan Z, Hoseini N. Investigating prevalence of mycoplasma and ureaplasma infection in preg-nant women with preterm labor. Iran J Obstet Gy-necol Infertil. 2012;15(10):8-13.##Yoshida T, Maeda SI, Deguchi T, Miyazawa T, Ishiko H. Rapid detection of Mycoplasma genita-lium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum organisms in geni-tourinary samples by PCR-microtiter plate hybrid-ization assay. J Clin Microbiol. 2003;41(5):1850-5.##Ahmed ST. Antigenic ureaplasma variation and ad-aptation to ovine complement response following experimental intrauterine infection [dissertation]. [United Kingdom]: Cardiff University; 2015. 266 p.##Callahan BJ, DiGiulio DB, Goltsman DSA, Sun CL, Costello EK, Jeganathan P, et al. Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women. Proc Natl Acad Sci USA. 2017;114(37): 9966-71.##Miyoshi Y, Suga S, Sugimi S, Kurata N, Yama-shita H, Yasuhi I. Vaginal ureaplasma urealyticum or Mycoplasma hominis and preterm delivery in women with threatened preterm labor. J Matern Fetal Neonatal Med. 2022:35(5):878-83.##Mazor M, Chaim W, Horowitz S, Leiberman J, Glezerman M. Successful treatment of preterm lab-our by eradication of Ureaplasma urealyticum with erythromycin. Arch Gynecol Obstet. 1993;253(4): 215-8.##Romero R, Hassan SS, Gajer P, Tarca AL, Fadrosh DW, Bieda J, et al. The vaginal microbiota of pregnant women who subsequently have spontan-eous preterm labor and delivery and those with a normal delivery at term. Microbiome. 2014;2:18.##Kataoka S, Yamada T, Chou K, Nishida R, Mori-kawa M, Minami M, et al. Association between preterm birth and vaginal colonization by myco-plasmas in early pregnancy. J Clin Microbiol. 2006;44(1):51-5.##Rittenschober-Böhm J, Waldhoer T, Schulz SM, Stihsen B, Pimpel B, Goeral K, et al. First trimester vaginal Ureaplasma biovar colonization and pre-term birth: results of a prospective multicenter study. Neonatology. 2018;113(1):1-6.##Bartkeviciene D, Opolskiene G, Bartkeviciute A, Arlauskiene A, Lauzikiene D, Zakareviciene J, et al. The impact of Ureaplasma infections on preg-nancy complications. Libyan J Med. 2020;15(1): 1812821.##Nguyen DP, Gerber S, Hohlfeld P, Sandrine G, Witkin SS. Mycoplasma hominis in mid-trimester amniotic fluid: relation to pregnancy outcome. J Perinat Med. 2004;32(4):323-6.##Doyle RM, Alber DG, Jones HE, Harris K, Fit-zgerald F, Peebles D, et al. Term and preterm labour are associated with distinct microbial com-munity structures in placental membranes which are independent of mode of delivery. Placenta. 2014;35(12):1099-101.##Hosny AEDM, El-Khayat W, Kashef MT, Fakhry MN. Association between preterm labor and geni-tourinary tract infections caused by Trichomonas vaginalis, Mycoplasma hominis, Gram-negative bacilli, and coryneforms. J Chin Med Assoc. 2017; 80(9):575-81.##

Parenthood in Infertile Couples Attending Assisted Reproductive Technologies (ARTs) Centers: What Has Changed During the COVID-19 Pandemic? 2 1 2 Background: The COVID-19 pandemic has been shown to impact the lifestyle of couples of reproductive age and, in particular, their desire for parenthood. The purpose of this study was to carry out an evaluation on the potential changes of desire for parenthood among infertile couples waiting for assisted reproduction during the pandemic. Methods: In this multicenter cross-sectional study, the quality of sexual life in Ital-ian infertile couples was assessed and their well-being was evaluated before the pandemic and during the quarantine. All couples were asked to fill out a ques-tionnaire, in which their desire for parenthood, sexual life, and well-being were in-vestigated. Results: Out of 1650 cases, 300 patients were finally enrolled. COVID-19 nega-tively impacted the well-being of individuals, leading to significantly reduced scores of happiness, feeling energetic, and interest in life (p<0.05). Although most couples had prolonged infertility, a small number of cases (4.0%) achieved a spontaneous natural pregnancy during the lockdown, probably due to more intimacy and longer time spent together. However, major concerns about the consequential effects of the virus on pregnancy and the risk of contagion in the hospital led a small number of infertile couples (5.0%) to decide to postpone their parenting project. Conclusion: The COVID-19 pandemic may have created a further negative impact on couples, reducing their desire for parenthood. This attitude could result in a decrease in births in the near future. 120 128 Gianmartin G Cito Department of Urology, Careggi Hospital, University of Florence Department of Urology, Careggi Hospital, University of Florence Italy gianmartin.cito@gmail.com Chiara Ch Pizzarelli Institute of Functional Psychology Institute of Functional Psychology Italy Vanessa V Zurkirch Regional Referral Centre for Relational Criticalities–Tuscany Region, Careggi Hospital, University of Florence Regional Referral Centre for Relational Criticalities–Tuscany Region, Careggi Hospital, University of Florence Italy Valentina V Basile Assisted Reproductive Technology Centre, Careggi Hospital, University of Florence Assisted Reproductive Technology Centre, Careggi Hospital, University of Florence Italy Maria M Ruggiero Assisted Reproductive Technology Centre, San Rossore Private Hospital Assisted Reproductive Technology Centre, San Rossore Private Hospital Italy Maria MC Coccia Assisted Reproductive Technology Centre, Careggi Hospital, University of Florence Assisted Reproductive Technology Centre, Careggi Hospital, University of Florence Italy Pieraldo P Inaudi Obstetrics and Gynecology Unit, Centro Chirurgico Fiorentino e Centro Salute Riproduttiva Obstetrics and Gynecology Unit, Centro Chirurgico Fiorentino e Centro Salute Riproduttiva Italy Paolo P Rossi Andrology Unit, Saint Claire Hospital, University of Pisa Andrology Unit, Saint Claire Hospital, University of Pisa Italy COVID-19Life styleParentsPatientsQuality of lifeReproduction 120142.pdf Khan M, Adil SF, Alkhathlan HZ, Tahir MN, Saif S, Khan M, et al. COVID-19: A global challenge with old history, epidemiology and progress so far. Molecules. 2020;26(1):39.##Bedford J, Enria D, Giesecke J, Heymann DL, Ihek-weazu C, Kobinger G, et al. COVID-19: towards con-trolling of a pandemic. Lancet. 2020;395(10229): 1015-8.##Wang C, Pan R, Wan X, Tan Y, Xu L, McIntyre RS, et al. A longitudinal study on the mental health of general population during the COVID-19 epidemic in China. Brain Behav Immun. 2020;87:40-8.##Segars J, Katler Q, McQueen DB, Kotlyar A, Glenn T, Knight Z, et al. Prior and novel coronaviruses, Coronavirus disease 2019 (COVID-19), and human reproduction: what is known? Fertil Steril. 2020;113 (6):1140-9.##Levi-Setti PE, Borini A, Patrizio P, Bolli S, Vigi-liano V, De Luca R, et al. ART results with frozen oocytes: data from the Italian ART registry (2005-2013). J Assist Reprod Genet. 2016;33(1):123-8.##Micelli E, Cito G, Cocci A, Polloni G, Russo GI, Minervini A, et al. Desire for parenthood at the time of COVID-19 pandemic: an insight into the Italian situation. J Psychosom Obstet Gynaecol. 2020;41 (3):183-90.##Gemmell LC, Williams Z, Forman EJ. Considera-tions on the restriction of Assisted Reproductive Technology (ART) due to COVID-19. Semin Peri-natol. 2020;44(7):151288.##Esposito V, Rania E, Lico D, Pedri S, Fiorenza A, Strati MF, et al. Influence of COVID-19 pandemic on the psychological status of infertile couples. Eur J Obstet Gynecol Reprod Biol. 2020;253:148-53.##Hamidi F, Babapour F, Hamzehgardeshi Z. Infer-tility distress management in couples treated with assisted reproductive techniques (ART) in COVID-19 pandemic. J Reprod Infertil. 2020;21(4):312-3.##Turocy JM, Robles A, Hercz D, D’Alton M, For-man EJ, Williams Z. The emotional impact of the ASRM guidelines on fertility patients during the COVID-19 pandemic. Fertil Steril. 2020;114(3): e63.##Villani MT, Morini D, Spaggiari G, Simoni M, Aguzzoli L, Santi D. Spontaneous pregnancies among infertile couples during assisted reproduc-tion lockdown for COVID-19 pandemic. Androlo-gy. 2021;9(4):1038-41.##Pacchiarotti A, Frati G, Saccucci P. A surprising link with unexplained infertility: a possible Covid-19 paradox? J Assist Reprod Genet. 2020;37(11): 2661-2.##Molgora S, Fenaroli V, Acquati C, De Donno A, Baldini MP, Saita E. Examining the role of dyadic coping on the marital adjustment of couples under-going assisted reproductive technology (ART). Front Psychol. 2019;10:415.##Cocci A, Giunti D, Tonioni C, Cacciamani G, Tel-lini R, Polloni G, et al. Love at the time of the Covid-19 pandemic: preliminary results of an on-line survey conducted during the quarantine in Italy. Int J Impot Res. 2020;32(5):556-7.##Cito G, Micelli E, Cocci A, Polloni G, Russo GI, Coccia ME, et al. The impact of the COVID-19 quarantine on sexual life in Italy. Urology. 2021; 147:37-42.##Brooks SK, Webster RK, Smith LE, Woodland L, Wessely S, Greenberg N, et al. The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020;395(10227): 912-20.##Klonoff-Cohen HS, Natarajan L, Chen RV. A pro-spective study of the effects of female and male marijuana use on in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) outcomes. Am J Obstet Gynecol. 2006;194(2):369-76.##Klonoff-Cohen H. Female and male lifestyle habits and IVF: what is known and unknown. Hum Re-prod Update. 2005;11(2):179-203.##Eugster A, Vingerhoets AJ. Psychological aspects of in vitro fertilization: a review. Soc Sci Med. 1999;48(5):575-89.##Homan GF, Davies M, Norman R. The impact of lifestyle factors on reproductive performance in the general population and those undergoing infertility treatment: a review. Hum Reprod Update. 2007;13 (3):209-23.##Chen H, Guo J, Wang C, Luo F, Yu X, Zhang W, et al. Clinical characteristics and intrauterine ver-tical transmission potential of COVID-19 infection in nine pregnant women: a retrospective review of medical records. Lancet. 2020;395(10226):809-15##Elshafeey F, Magdi R, Hindi N, Elshebiny M, Far-rag N, Mahdy S, et al. A systematic scoping review of COVID-19 during pregnancy and childbirth. Int J Gynaecol Obstet. 2020;150(1):47-52.##Thomas B, Pallivalapila A, El Kassem W, Taran-num A, Al Hail F, Rijims M, et al. Maternal and perinatal outcomes and pharmacological manage-ment of Covid-19 infection in pregnancy: a sys-tematic review protocol. Syst Rev. 2020;9(1):161.##Kainz K. The role of the psychologist in the evaluation and treatment of infertility. Womens Health Issues. 2001;11(6):481-5.##Hart VA. Infertility and the role of psychotherapy. Issues Ment Health Nurs. 2002;23(1):31-41.##Tokgoz VY, Kaya Y, Tekin AB. The level of anxi-ety in infertile women whose ART cycles are post-poned due to the COVID-19 outbreak. J Psycho-som Obstet Gynaecol. 2020:1-8.##Barra F, La Rosa VL, Vitale SG, Commodari E, Altieri M, Scala C, et al. Psychological status of infertile patients who had in vitro fertilization treat-ment interrupted or postponed due to COVID-19 pandemic: a cross-sectional study. J Psychosom Obstet Gynaecol. 2020:1-8.##

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma 2 1 2 Background: The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype. Methods: The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma. Results: Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed. Conclusion: The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus. 128 135 Mohammad Hossein MH Ahmadi Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences Iran Ali Akbar AA Pourfathollah Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Iran Maryam M Rabiee Department of Obstetrics and Gynecology, Shahed University Department of Obstetrics and Gynecology, Shahed University Iran Naser N Amirizadeh Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Iran n.amirizadeh@ibto.ir Cell-free fetal DNAFetal RHD genotypingHemolytic disease of the fetus and newbornNoninvasive prenatal diagnosisRho(D) immune globulin 120140.pdf Delaney M, Matthews DC. Hemolytic disease of the fetus and newborn: managing the mother, fetus, and newborn. Hematology Am Soc Hematol Educ Prog-ram. 2015;2015:146-51.##De Haas M, Thurik F, Koelewijn J, van der Schoot CE. Haemolytic disease of the fetus and newborn. Vox Sang. 2015;109(2):99-113.##Clausen FB, Krog GR, Rieneck K, Nielsen LK, Lundquist R, Finning K, et al. Reliable test for pre-natal prediction of fetal RhD type using maternal plasma from RhD negative women. Prenat Diagn. 2005;25(11):1040-4.##Urbaniak SJ, Greiss MA. RhD haemolytic disease of the fetus and the newborn. Blood Rev. 2000;14(1): 44-61.##Hirose TG, Mays DA. The safety of RhIG in the prevention of haemolytic disease of the newborn. J Obstet Gynaecol. 2007;27(6):545-57.##Daniels G, Finning K, Martin P, Soothill P. Fetal blood group genotyping from DNA from maternal plasma: an important advance in the management and prevention of haemolytic disease of the fetus and newborn. Vox Sang. 2004;87(4):225-32.##Moise KJ, Gandhi M, Boring NH, O'Shaughnessy R, Simpson LL, Wolfe HM, et al. Circulating cell-free DNA to determine the fetal RHD status in all three trimesters of pregnancy. Obstet Gynecol. 2016;128(6):1340-6.##Rutkowski K, Nasser S. Management of hypersen-sitivity reactions to anti‐D immunoglobulin prepar-ations. Allergy. 2014;69(11):1560-3.##Lo YM, Corbetta N, Chamberlain PF, Rai V, Sar-gent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350 (9076):485-7.##Lo YD, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med. 1998;339(24):1734-8.##Faas BH, Beuling EA, Christiaens GM, von dem Borne AK. Detection of fetal RHD-specific se-quences in maternal plasma. Lancet. 1998;352 (9135):1196.##Müller SP, Bartels I, Stein W, Emons G, Guten-sohn K, Köhler M, et al. The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible. Transfusion. 2008;48(11):2292-301.##Ahmadi MH, Hantuoshzadeh S, Okhovat MA, Na-siri N, Azarkeivan A, Amirizadeh N. Fetal RHD genotyping from circulating cell-free fetal DNA in plasma of rh negative pregnant women in iran. Indian J Hematol Blood Transfus. 2016;32(4):447-53.##Moezzi L, Keshavarz Z, Ranjbaran R, Abouali-zadeh F, Behzad-Behbahani A, Abdullahi M, et al. Fetal RHD genotyping using real-time polymerase chain reaction analysis of cell-free fetal DNA in pregnancy of RhD negative women in South of Iran. Int J Fertil Steril. 2016;10(1):62.##Chan KA, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al. Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive pre-natal diagnosis. Clin Chem. 2006;52(12):2211-8.##Wang XD, Wang BL, Ye SL, Liao YQ, Wang LF, He ZM. Non‐invasive foetal RHD genotyping via real‐time PCR of foetal DNA from Chinese RhD‐ negative maternal plasma. Eur J Clin Invest. 2009; 39(7):607-17.##Orhant L, Anselem O, Fradin M, Becker PH, Beu-gnet C, Deburgrave N, et al. Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: applica-tion to the non‐invasive prenatal diagnosis of acho-ndroplasia. Prenat Diagn. 2016;36(5):397-406.##Finning KM, Martin PG, Soothill PW, Avent ND. Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD geno-typing service. Transfusion. 2002;42(8):1079-85.##Koelewijn JM, De Haas M, Vrijkotte TG, Bonsel GJ, Van Der Schoot CE. One single dose of 200 μg of antenatal RhIG halves the risk of anti‐D immunization and hemolytic disease of the fetus and newborn in the next pregnancy. Transfusion. 2008; 48(8):1721-9.##Jensen MP, Damkjær MB, Clausen FB, Ali HA, Hare KJ, Dziegiel MH, et al. Targeted Rhesus im-munoglobulin for RhD‐negative women under-going an induced abortion: a clinical pilot study. Acta Obstet Gynecol Scand. 2019;98(9):1164-71.##De Haas M, Thurik FF, Van Der Ploeg CP, Veld-huisen B, Hirschberg H, Soussan AA, et al. Sen-sitivity of fetal RHD screening for safe guidance of targeted anti-D immunoglobulin prophylaxis: pro-spective cohort study of a nationwide programme in the Netherlands. BMJ. 2016;355:i5789.##Kolialexi A, Tounta G, Mavrou A. Noninvasive fe-tal RhD genotyping from maternal blood. Expert Rev Mol Diagn. 2010;10(3):285-96.##Khosroshahi BN, Oodi A, Namjou S, Gholamali T, Amirizadeh N. RHD genotyping by molecular ana-lysis of hybrid rhesus box in RhD-negative blood donors from Iran. Indian J Hematol Blood Trans-fus. 2019;35(1):119-24.##Sadeghi-Bojd Y, Amirizadeh N, Oodi A. RHD Genotyping of Rh-Negative and Weak D Pheno-type among Blood Donors in Southeast Iran. Int J Hematol Oncol Stem Cell Res. 2021;15(4):213-20.##

A Novel Familial Case Report of Genetic Syndrome Mimicking Congenital TORCH Infections-Pseudo-TORCH Syndrome 2 2 1 2 Background: Pseudo-TORCH syndrome (PTS) is a group of autosomal recessive disorders that clinically and radiologically mimic TORCH congenital infections. The prevalence of pseudo-TORCH syndrome 2 is 1 in 1,000,000 cases worldwide. This novel disorder is extremely rare, and is generally detected by prenatal diagnosis through next generation sequencing (NGS) during pregnancy. In this study, a familial case of pseudo-TORCH syndrome 2 with novel non-sense mutation in the ubiquitin-specific peptidase 18 (USP 18) gene in the parents was reported, who are heterozygous asymptomatic carriers; however, all children have inherited a homozygous pathogenic form of USP18, which is an important negative regulator of type I interferon (IFN) signal transduction. To the best of our knowledge, this is the first case of a novel mutation of USP18 seen in a family with pseudo-TORCH syndrome 2 (PTS 2) from India Case Presentation: A 23-year-old pregnant woman with bad obstetric history, including intrauterine and neonatal mortality was referred to the Institute of Genetics in the year 2021 for clinical and genetic evaluation. Advanced clinical exome sequencing of the parents and the fetus revealed heterozygous carrier status in parents and homozygous mutation in USP 18 gene in the progeny leading to pseudo-TORCH-2 syndrome. Conclusion: The present case highlights the significance of carrier screening, prenatal diagnosis, and genetic counseling in couples with bad obstetric history for the detection of rare genetic disorders with poor prognosis. 135 139 Iram I Sehrish Institute of Genetics and Hospital for Genetic Diseases, Osmania University Institute of Genetics and Hospital for Genetic Diseases, Osmania University India Tella T Sunitha تلا سونیتا Institute of Genetics and Hospital for Genetic Diseases, Osmania University Institute of Genetics and Hospital for Genetic Diseases, Osmania University Indonesia Avvari A Srilekha Institute of Genetics and Hospital for Genetic Diseases, Osmania University Institute of Genetics and Hospital for Genetic Diseases, Osmania University India Aayushi A Gupta Institute of Genetics and Hospital for Genetic Diseases, Osmania University Institute of Genetics and Hospital for Genetic Diseases, Osmania University India Pratibha P Nallari Pratibha Nallari Institute of Genetics and Hospital for Genetic Diseases, Osmania University Institute of Genetics and Hospital for Genetic Diseases, Osmania University India Anantapur A Venkateshwari Institute of Genetics and Hospital for Genetic Diseases, Osmania University Institute of Genetics and Hospital for Genetic Diseases, Osmania University India Next generation sequencingPrenatal diagnosisPseudo-TORCH syndrome 2Type I interferon signal transductionUbiquitin-specific peptidase 18 120154.pdf Baraitser M, Brett EM, Piesowicz AT. Microceph-aly and intracranial calcification in two brothers. J Med Genet. 1983;120(3):210-2.##Sakthi Seethalakshmi P, Kumaravel KS, Sampath-kumar P, Babu T, Rani SR. Intracranial Calcifi-cations With Dandy Walker Malformation. Pediatr Oncall J. 2019;16(2):58-9.##Reardon W, Hockey A, Silberstein P, Kendall B, Farag TI, Swash M, et al. Autosomal recessive con-genital intrauterine infection-like syndrome of mic-rocephaly, intracranial calcification, and CNS dis-ease. Am J Med Genet. 1994;52(1):58-65.##Alsohime F, Martin-Fernandez M, Temsah MH, Al-abdulhafid M, Le Voyer T, Alghamdi M, et.al. JAK Inhibitor therapy in a child with inherited USP18 deficiency. N Engl J Med. 2020;382(3):256-65.##Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the in-terpretation of sequence variants: a joint consensus recommendation of the American college of medical genetics and genomics and the association for molecular pathology. Genet Med. 2015;17(5):405-24.##Abdel‐Salam GM, Zaki MS. Band-like intracranial calcification (BIC), microcephaly and malformation of brain development: a distinctive form of con-genital infection like syndromes. Am J Med Genet A. 2009;149A(7):1565-8.##Knoblauch H, Tennstedt C, Brueck W, Hammer H, Vulliamy T, Dokal I, et al. Two brothers with find-ings resembling congenital intrauterine infection-like syndrome (pseudo-TORCH syndrome). Am J Med Genet. 2003;120A(2):261-5.##O'Driscoll MC, Daly SB, Urquhart JE, Black GC, Pilz DT, Brockmann K, et al. Recessive mutations in the gene encoding the tight junction protein oc-cludin cause band-like calcification with simplified gyration and polymicrogyria. Am J Hum Genet. 2010;87(3):354-64.##Duncan CJ, Thompson BJ, Chen R, Rice GI, Gothe F, Young DF, et.al. Severe type I interferonopathy and unrestrained interferon signaling due to a homo-zygous germline mutation in STAT2. Sci Immunol. 2019;4(42):eaav7501.##Malakhov MP, Malakhova OA, Kim KI, Ritchie KJ, Zhang DE. UBP43 (USP18) specifically re-moves ISG15 from conjugated proteins. J Biol Chem. 2002;277(12):9976-81.##Yang L, Jing Y, Kang D, Jiang P, Li N, Zhou X, et al. Ubiquitin-specific peptidase 18 regulates the differentiation and function of Treg cells. Genes Dis. 2020;8(3):344-52.##Malakhova OA, Yan M, Malakhov MP, Yuan Y, Ritchie KJ, Kim KI, et.al. Protein ISGylation mo-dulates the JAK-STAT signaling pathway. Genes Dev. 2003;17(4):455-60.##Ritchie KJ, Malakhov MP, Hetherington CJ, Zhou L, Little MT, Malakhova OA, et al. Dysregulation of protein modification by ISG15 results in brain cell injury. Genes Dev. 2002;16(17):2207-12.##Meuwissen ME, Schot R, Buta S, Oudesluijs G, Tinschert S, Speer SD, et al. Human USP18 defi-ciency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J Exp Med. 2016;213(7):1163-74.##