FIBROIDS AND IVF FIBROIDS AND IVF FIBROID AND IVF

Fibroid on IVF

1. FIBROIDS AND IVF Dep. Obgyn , Adam Malik Hospital Univeristy of North Sumatra, Indonesia Halim Fertility Center Medan, Indonesia August 2021 “ Binarwan Halim” Indonesia

2. Conflict of Interest Disclosure Binarwan Halim Halim Fertility Center Medan University of North Sumatera University of Prima I have no conflict of interest to disclosure

3. Uterine fibroids are the most common benign pelvic tumors of the female genital tract. Incidence rate approximately 25–30% and may be higher depending on race, family history and genetics. Approximately 5% – 10% of infertile women have fibroids. Their size and location determines whether fibroids affect fertility. Epidemiology ASRM. Myomas , myomectomy, and fertility. Fertil Steril 2017.

4. Uterine fibroids PATHOGENESIS Genetic factors: Chromosome (2,3,6,7,8,10,11,13,14,22) genes (MED12, HMGA2, HMGA1, FH, BHD, TSC2, PCOLCE, ORCSL, LHFPL3) Epigenetic factors: DNA methylation, histone modification, MicroRNA, (let-7, miR-21, miR-93, miR-106b, miR-200) Hormones: Estrogen, progesterone Growth factors: EGF, VEGF, PDGF, IGF, TGH- α , TGF- β , α -FGF, β -FGF, activin , myostatin )

5. FOXL2 in a transcription factor strongly implicated in a female fertility , working both in the ovary as well as in the endometrium mRNA and its protein localization do not change during endometrial cycle in eutopic endometrium from healthy individuals or patients with endometriosis The hyperexpression of FOXL2 has been supposed to be associated with activin A expression and related to the pathogenesis of endometriosis FOXL2: A possible role in fibroids pathogenesis?

6. FOXL2: A possible role in fibroids pathogenesis?

7. Reis, F et al. Best Practice & Research Clinical Obstetrics and Gynaecology xxx (2015) Hypothesis of uterine fibroid development Sex steroid hormones distruption Genetics Mutated stem cell Extracellular matrix deposition Leimyoma fibroids MMPS

8. Uterine Leiomyoma Classification Based on the concept that fibroids are primarily interstitial and gradually forced outwards or inwards: • Subserosal are in the outer wall of the uterus (55%) • Intramural are found in the muscular layers of the uterine wall (40%) • Submucosal protrude into the uterine cavity (5%) ASRM. Fibroids and Fertility. 2015.

9. SUBMUCOSAL INTRAMURAL SUBSEROSAL International Federation of Gynecology and Obstetrics (FIGO) classification system for fibroid location. (Adapted from Munro MG, Critchley HO, Broder MS, Fraser IS; for the FIGO Working Group on Menstrual Disorders. FIGO classification system (PALM-COEIN) for causes of abnormal uterine bleeding in nongravid women of reproductive age. Int J Gynecol Obstet 2011;113:3-13. Published by Elsevier Ireland Ltd. ASRM. Myomas , myomectomy, and fertility. Fertil Steril 2017.

10. Impact of Uterine Myomas on Fertility Changes in the shape of the cervix can affect the number of sperm that can enter the uterus. Changes in the shape of the uterus can interfere with the movement of the sperm or embryo. Fallopian tubes can be blocked by fibroids. They can impact the size of the lining of the uterine cavity. Blood flow to the uterine cavity can be affected. This can decrease the ability of an embryo to stick (implant) to the uterine wall or to develop. ASRM. Fibroids and Fertility. 2015.

11. FIBROIDS and Infertility Enlarged size fibroid or specific location  hinder the transport of the sperm and egg, as well as their implantation Submucosal fibroids  I nfluence levels of IL-10 and glycodelin , which support implantation and early embryonic development. Purohit et all and Yoshino et al.19 : F ibroids alter uterine contractions leading to inflammatory reaction in the uterus  latter may hinder implantation. Fibroids  change in endomyometrial junctional zone  This zone regularly consists of macrophages and NK cells that contribute decidualisation during implantation. Some studies  fibroids associated with the reduction of those cells . Mahdi, Essam E, EMJ Repro Health. 2019;5[1]:94-99.

12. Distribution of the frequency of junctional zone movement The number of patients Frequency / 3 minutes Low freq N=29 High freq N=22 Yoshino, 0., et. Al. Human Reproduction, Vol.25, No.10 pp. 2475–2479, 2010

13. FIBROIDS and Infertility Thickness of fibroid pseudo capsule (Neurovascular bundle surrounding) the considerably higher near the endometrial cavity compared to intramural and subserous fibroid  potential role in fertility. Submucosal reduction 70% of delivery rate, Intramural reduction 30% of delivery rate. Intramural > 4 cm  PR ↓↓ Laparoscopic myomectomy 45.7%, without laparoscopic 28.6% if the number of fibroids was ≤ 3. If the number of fibroid was 4-7  PR 64,7% (Kameda et al) Embolisation ↓↓PR and Miscarriage ↑↑ MRgFUS  LBR 41%, spontaneous abortion 28 % Mahdi, Essam E, EMJ Repro Health. 2019;5[1]:94-99.

14. Presence fibroids in general, regardless of localization led to a statistically significant decrease in fertility - clinical pregnancy rate (RR= 0.85; 95% ci: 0.73-0.98) - Birth rates (RR= 0.69; 95% CI: 0.59-0.82) - Increase in miscarriage rate (RR= 1.68; 95% CI: 1.37-2.05 )

15. Greatest impact in submucosal fibroids, reducing clinical pregnancy rate up tp 70% (RR= 0.36; 95% CI : 0.18-0.74) Intramural fibroids also had negative impact fertility with: - clinical pregnancy (RR= 0.81; 95% CI: 0.70-0.94) - Live birth (RR= 0.70; 95% CI: 0.58-0.85) - Miscarriage rates (RR= 1.75; 95% CI: 1.23-2.49)

16. H ammersmith and queen charlotte hospital single/multiple/size of fibroids Women with IM fibroids Control group Total no of patients 194 Included and matched 163 326 Single fibroid 69 (40%) Multiple fibroids (range 2-8) 94 (57,7%) Median largest diameter fibroids 25 mm range 10-80 mm

17. “More recently Oliveira et al (2004) and Vimercati et al (2007) indicated that interstitial myoma <4cm, not distorting the uterine cavity, do not pause threat to the pregnancy and live birth rates following IVF” - Oliveira FG et al (2004) Fertil & Steril - Vimercati et al (2007). Reprod. Biomed Online

18. PR, LB/Single, Multiple fibroids and their matched controls Christopoulos G, et. Al., BMJOG, 2016

19. “When only studies with a prospective design were considered the reduction in live birth rates was as high as 40% after 3 attempts over a 12 months period (RR 0.9, 95%CI: 0.41-0.87)” Check et al, 2002 Khalaf Y eat al (2006), Hum. Reprod . Sunkara , SK, et al, 2010, Jum . Reprod

20. Submucosal and IVF “Submucosal fibroids, in particular, significantly reduce implantation and pregnancy rates of ART” “Submucosal fibroids that distort the uterine cavity have been found to carry a relative risk of 0.3 for pregnancy and 0.28 for implantation after ART”

21. Molecular basis for fibroid influence Endometrial HOXA10 and HOXA11 mRNA expression were significantly decreased in uteri with submucosal fibroids compared to fibroids with intramural fibroids The moleculat basis by which submucosal fibroids may adversely affect reproduction includes a global decrease in endometrial HOX gene expression, not simply a focal change over the myoma This may explain the reproductive dysfunction observed with submucosal myomas ( Racknow & Taylor H, 2010)

22. Role of HOXA10 and HOXA11 Midluteal phase (time of implantation) HOXA 10mRNA expression unregulated in both endometrial and stromal cells in women Endometrial HOXA10 expression in the stroma stays relatively constant throughout the menstrual cycle, while the glands are the location of the midsecretory increase in HOXA10 expression Estrogen and progesterone each upregulate HOXA10 expression HOXA10 has diverse effects on several aspects of adult endometrial development such as stromal decidualisation & leukocyte infiltration

23. Myometrium Endometrium Estrogen & Progesterone HOXA10 Genes involved in implantation β 3 integrin, EMX2, and IGFBP-1 & BTEB1 stromal decidualisation & leukocyte infiltration Fibroid

24. “HOXA10 and LIF expression did not increase in endometrial cells from women with leiomyomas when treated with recombinant human BMP-2 while similar treatment increase HOXA10 and LIF in cells from women without leiomyomas. These findings suggest that TGF- β 3, secreted from leiomyomas, impairs the BMP-2 signaling in the endometrium necessary for implantation”. (Doherty and Taylor – 2016)

25. A complex & diverse molecular interaction is required for optimal endometrial receptivity resulting in successful pregnancy implantation The suppression or absence of certain molecules ( HOXA10) essential for endometrial receptivity leads to implantation failure Fibroids & polyps play a role in the molecular alteration of the cascade necessary for endometrial receptivity Surgical management of the underlying pathology remains optimal current therapy Gene or stem cell therapy targeting the expression of key endometrial genes may be the future therapy to improve implantation rates Conclusion

26. Furthermore HOXA10 regulates downstream target genes that are also involved in implantation such as β 3 integrin, that are also involved in implantation such as β 3 integrin, EMX2 and IGFBP-1 and BTEB1 Transforming-growth-factor-beta 3 (TGF- β 3), a growth factor known to be produced by leiomyomas in far greater amounts than normal myometrium, has detrimental effects on signaling pathways necessary for endometrial receptivity TGF- β 3 was shown to decrease the receptivity of human endometrium to BMP-2 by (Bone morphogenetic protein 2) decreasing the expression of BMP receptors

27. Clinical pregnancy rates with submucosal fibroids (common odds ratio (OR) = 0.3; 95% Confidence Interval (CI): 0.1 – 0.7) Clinical pregnancy rates with intramural fibroids (common OR of 0.8 (95% CI 0.6–0.9) Same negative impact of submucosal fibroid on delivery rates No impact of subserosal fibroids

28. Yoshino, 0., et. Al. Human Reproduction, Vol.25, No.10 pp. 2475–2479, 2010 Infertile women with fibroid (n=51) Low freq (n=29) High freq (n=22) Pregnancy (n=10) Pregnancy (n=0) Myomectomy (n=11) Myomectomy (n=17) First MRI Infertility treatment for 4 months

29. Pregnancy rate was higher in the low frequency group Low freq High freq Total number of patiens 29 22 Number of pregnancy 10 (34%) (0%) Yoshino, 0., et. Al. Human Reproduction, Vol.25, No.10 pp. 2475–2479, 2010 P: 0,005

30. 1 → 0 no 0 → 0 no 0 → 0 no 0 → 0 no 0 → 0 no 0 → 0 no 0 → 0 no Low freq Before After Pregnancy 6 → 0 yes 6 → 1 yes 5 → 3 no 5 → 0 no 4 → 1 yes 4 → 1 yes 4 → 0 no 4 → 0 no 4 → 0 no 4 → 0 no 3 → 1 no 3 → 0 yes 3 → 1 no 3 → 0 yes 3 → 0 no Before After Pregnancy High freq P<0,05 Pregnancy 6/15 (40%)

31. Up to 25% of women with unexplained infertility have endometrial polyps on hysteroscopy Rackow et. Al.: In this case-control study of women undergoing IVF: The presence of endometrial polyps was associated with significant decrease in HOXA10 & HOXA11 expression compared with controls (0.334x control; p=0,016) & (0.183x control; p=0,03) respectively Polyp size and number did not alter results

32. The role of estrogens in fibroid tissues Estrogens ER- α ERs have an increased sensitivity Increased cell proliferation Pro-inflammatory affect Increased angiogenesis Regulate growth factors expression Increase growth and development of uterine fibroid

33. The role estrogens in fibroid tissues Estrogens ER- α ERs have an increased sensitivity Increased cell proliferation Pro-inflammatory affect Increased angiogenesis Regulate growth factors expression Progesterone PR AB Increase cells growth Increased cell proliferation Regulate growth factors expression Increased local production of ECM inhibition od apoptosis

34. Estrogen, aromatase and inflammation Arachidonic acid COX-1 Constitutive COX-2 Inducible Physiological prostaglandin production Pathological prostaglandin production Normal function Inflammation, pain, fever

35. Effect on fertilization: Distortion of pelvic architecture Uterine contractility Distortion of the cervix Distortion or occlusion of tubal ostia Uterine fibroids and infertility: possible mechanism Effect on implantation : Implantation failure or gestation discontinuation Alternation of the endometrial profile Persistence of intrauterine blood and clots Focal endometrial vascular disturbance Endometrial inflammation Secretion of vasoactive substances Enhanced endometrial androgen environment

36. ART first choice Patients with subserous myoma /s Patients with single small (<3cm) interstitial myoma not distorting the uterine cavity

37. Myomectomy followed b y ART In the presence of associated factors as advanced maternal age, long term infertility, and/or tubal or male factor infertility myomectomy followed by ART is performed in: Patients with single or multiple myoma /s distorting the uterine cavity s ingle or multiple interstitial myoma /s (>3cm) particularly with previous failure of ART

38. ART/Myomectomy/ET An infertile woman with poor ovarian reverse, in her late thirties or early forties, having multiple large fibroids distorting the uterine cavity may have OHS, OPU, and vertification on her embryos prior to myomectomy

39. Steps for laparoscopic myomectomy Misoprostol 400Microgram PR Tranexamic acid 1gr IV Vasopressin (20 units: 60mls Saline)

40. Steps for laparoscopic myomectomy Misoprostol 400Microgram PR Tranexamic acid 1gr IV

41. Does myomectomy decrease abnormal uterine peristalsis and increase pregnancy rate? Yoshino, 0., et. Al. Human Reproduction, Vol.25, No.10 pp. 2475–2479, 2010

42. Effects of increased peristalsis on the endometrium Speculated mechanism IGFBP-1 → disorganizes decidualization Interleukin-8 inflammation → inflammation Neutrophil chemokines MMO-1 tissue degradation → tissue degradation   IMPLANTATION FAILURE ADVERSE PREGNANCY OUTCOME

43. Decellularized Matrix Transplantation (DMT) METHOD Hireoka , Takehiro , et. Al. JCI Insight · June 2016

44. Regenerated uteri contribute sucessful pregnancy Pups from recipient mice

45. A macroscopic image of the transplantation site of Decellularized Matrix Transplantation (DMT) Yellow arrow, a 10-0 nylon suture thread; dotted line, a boundary between DUM and UT. Scale bar: 2 mm

46. Should polyps be resected prior to IVF Up to 70% of polyps persist after 1 year conservative management It is recommended to resect polyps prior to fertility treatment Direct visualization and resection of polyps via hysteroscopy is the gold standard for diagnosis and concomitant treatment Perez-Medina et al 2005. polyp resection is associated with higher pregnancy rates compared to diagnostic hysteroscopy (63 Versus 28%) Bosteels et al, Cochrane review 2015: hysteroscopic polypectomy results in a greater than four-fold increase in clinical pregnancy among subfertile or infertile women who subsequently undergo IUI

47. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization Subjects: women morphologically normal uterus Method: Uterine contractility assessment by US just before day2 ET Fanchin , et. Al. 1998. Human Reproduction vol.13 no.7 pp.1968–1974, 1998

48. Uterine fibroids and infertility: the endometrial perspective

49. NGF (Nerve Growth Factor) has been proposed as one of the key factors responsible not only for promotion of nerve fiber growth but also for the creation of new painful afferents to the central nervous system Synaptophisin is present in neuroendocrine cells and in virtually all neurons in the brain and spinal cord that participate in synaptic transmission MAP2 (microtubule-associated protein 2) is a neuron specific protein that stabilizes microtubules in the dendrites of postmitotic neurons] Inflammation and Pain

50. Neurogenic factors mRNA expression in adenomyosis . Nerve growth factor (A), SYN (B), and MAP2 (C) mRNA expression in healthy controls (Healthy), eutopic endometrium of women with adenomyosis (E), and adenomyotic nodule (A). Fold change (y-axis) represents mRNA expression normalized to HPRT. **P < .01, ***P < .001. MAP2 indicates microtubule-associated protein 2; mRNA, messenger RNA; NGF, nerve growth factor; SYN, synaptophysin .

51. NGF, SYP, and MAP-2 mRNA expression in DIE or OMA lesions, in eutopic endometrium, and in healthy control (proliferative phase). Fold change (y-axis) represents NGF, SYP, or MAP-2 expression normalized to HPRT1, relative to healthy endometrium considered to be equal to 1. The expression of NGF was significantly stronger in the lesions than in the eutopic endometrium and healthy endometrium. SYP mRNA expression levels for DIE were the highest. MAP-2 mRNA expression levels for both OMA and DIE were the highest. Data represent value obtained by DDCt method. ***P<.001.

52. Potential causes of decreased fertility and pregnancy loss in patients with intramural fibroids. (IMF- intramural fibroid, PC- fibroid pseudocapsule , JZ- uterine junctional zone, ES- endometrium, ER- estrogen receptor, PR- progesterone receptor, uNK - uterine natural killer cells.) Pier and Bates Fertility Research and Practice  ( 2015 ) 1:12. DOI 10.1186/s40738-015-0005-2.

53. Importance of Fibroid Size during IVF Oliveira et al. found that a detrimental impact was seen in the presence of relatively larger fibroids. The clinical pregnancy rates were lower after IVF/ICSI in women with intramural or subserosal fibroids of 4.1-6.9 cm compared with women with no fibroids or fibroids ≤4 cm.

54. Comparison of mean size and location of intramural fibroids and pregnancy outcomes according to several studies. Patients with intramural fibroids 4.0 cm had lower pregnancy rates than patients with intramural fibroids ≤ 4.0 cm. There were no statistical differences related to delivery rates (31.5% vs. 32%, respectively) Premature delivery rates for singleton gestations were 10% vs. 8%, respectively

55. Non- cavitary distorting intramural myomas The Egyptian IVF-ET Center in a prospective study by our group of 406 infertile patients including 39 patients with non- cavitary distorting myomas undergoing ARRT it was found that uterine myomas <7cm and not encroaching on the cavity did not affect implantation of miscarriage rates Ramzy  AM,  Sattar  M,  Amin  Y,  Mansour  RT, Serour GI, Aboulghar MA. Uterine myomata and outcome of assisted reproduction. Hum Reprod   1998

56. ART Prospective Study Non- Cavitary distorting Fibroid Women with IM or SS myoma No. 119 Control No. 119 LBR 18% LBR 13% Or 1,45 (95%, ci: 0,71-2,94) The presence of asymptomatic small fibroid (<50mm) did not affect ART outcomes Somigliana et al, 2011 Hum Reprod , 26: 834-839

57. Retrospective Cohort Study first IVF/ICSI cycle With fibroids 249 Without fibroids 249 Delivery rate 30.5% 33.7% A significant negatice effect on delivery rate when largest tumour diameter was >2.85cm Yan et. Al., 2014. Fertil&Steril 101:716-721

58. The presence of IM fibroids not distorting the uterine cavity appears to negatively affect clinical pregnancy and live birth rates in patients undergoing their first IV/ICSI style

59. The deleterious effect of fibroids was significant in women with two or more fibroids and in women with fibroids of   ≥30mm in diameter. conversely, in women with single fibroids of <30mm in diameter. No significant difference in pregnancy outcome was identified

60. If this trial fail or end in a miscarriage, she may be counseled about the possible beneficial effect of removal of IM before repeat trial

61. Journal of Minimally Invasive Gynecology  2019 261369-1375DOI: (10.1016/j.jmig.2019.02.008) Copyright © 2019 Terms and Conditions

62. Key takeways Key issues to be addressed ( ie misperception) Explain the benefits and efficacy of different contraceptive methods Is the use of IUD/IUS appropriate safe for women who never give birth to a child? Is there a link between LARC and higher breast cancer risk? Will it increase my chance of getting PID after IUD? STI testing prior to placement? Optimal choice/decision ( ie scientific, rationale) Convenient Safe Reversible Lighter periods or amenorrhea with IUS low risk for adverse events such as ectopic pregnancy, expulsion and perforation Candidates od IUDs? Addordability ? Main counseling messages LARC methods are easy to use, safe, long lasting, quickly reversible and 20 times more effective than oral contraceptive pills The LARC methods have limited contraindications

63. Ways to remind her to take pills Associate the pill taking with a daily activity, eg . Brushing teeth, s hower, meal Phone apps to reminder Own a smart phone Usually free charge Phone must not be switched off Internet connectivity no important one downloaded Preferably not in silent mode, Customization of type of pill Need her active deactivation of alarm to acknowledge receipt Snooze reminder in case OCP not near patient

64. THANK YOU

65. Introduction Decrease in oocyte number with age

66. Age (years) Nybo Andersen AM, et al. BMJ 2000; 320:1708-1712 Miscarriage rate Newberger DS. Am Fam Physician 2000; 62: 825-832 Incidence rate of Down syndrome Age (years) Introduction Decrease in oocyte quality with age

67. Toner JP, Grainger DA, Frazier LM, Registry Res Comm SA. Clinical outcomes among recipients of donated eggs: an analysis of the US national experience, 1996-1998. Fertility and Sterility 2002; 78:1038-1045 Implantation rate Miscarriage rate Toner JP, et al. Fertil Steril. 2002 Age in recipients (years) Age in recipients (years) Introduction Implantation and miscarriage rates in ART treatment using oocyte donation

68. Uterine fibroids and infertility Submucosal myoma Intramural myomas with symptoms Subserosal myomas Intramural myomas without symptoms Surgery is required before pregnancy Keiji Kuroda, Treatment of infertility and recurrent pregnancy loss. Medical View (Japan)

69. Benaglia L, et al. PLOS ONE 2014 Uterine fibroids change in their size with pregnancy. However, there are various size and number of myomas, it is difficult to assess which fibroids are at risk for complications during pregnancy. Marret H et al. Eur J obstet Gynecol Reprod Biol. 2012 Changes in myomas after pregnancy Comparison of the growth of uterine myomas from pregnant and non-pregnant women after IVF treatment

70. Subserosal myoma Intramural myomas without symptoms Fibroid-associated complications during pregnancy Red degeneration Pressure symptoms (including retention of urine, constipation) Spontaneous abortion Preterm labor Malpresentation Intrauterine fetal retardation Bradytocia Atonic bleeding Thrombosis Surgery is required before pregnancy But huge ones may need surgery… Keiji Kuroda, Treatment of infertility and recurrent pregnancy loss. Medical View (Japan)

71. Su rg e r y Infertility treatment Time loss with pre-operative treatment including GnRH agonist administration and post-operative contraceptive Negative effect on pregnancy including implantation failure, complications during pregnancy and delivery Treatment for infertile women of late reproductive age with uterine tumours and/or ovarian endometriomas Controversial problem

72. Surgery-ART hybrid therapy

73. Informed consent Decision of surgery Discuss the No of frozen embryos Time Endo s c opic Surgery Thawed embryo transfer Liquid N2 Oocyte retrieval+ embryo cryopreservation M e t ho d s P r e -o p e ra t i v e treatment

74. Objective Objective in this study Duration : 2014 － 16 Informed consent from Dr. Keiji Kuroda Diminished ovarian reserve (AMH<1.0ng/ml) and/or advanced age (>40 years) Uterine myomas and/or ovarian endometriomas with requiring an operation Women who aimed to preserve > 3-5 frozen embryos preoperatively Indication for surgery Myoma Submucosal or intramural myomas which compress intrauterine cavity Huge subserosal myomas (> 10 cm in diameter) Ovarian endometrioma > 5cm in diameter Kuroda K, et al. J Minim Invasive Gynecol. 2019

75. Surgery-ART hybrid therapy in 2014―2016 39 cases Success group Livebirth after ET 14 cases (30 ET cycles) Failure group Implantation failure or miscarriage 24 cases (56 ET cycles) Operation without frozen embryos 1 case Included patients Operation after embryo freezing 38 cases Kuroda K, et al. J Minim Invasive Gynecol. 2019 Objective Success rate: 36.8 ％

76. Success group (14 cases) Failure group (24 cases) p-value Age, years, median (IQR) 40 (38-41) 41.5 (41-42) 0.032 Duration of infertility, years, median (IQR) 1.0 (1.0-8.0) 2.5 (1.5-4.0) NS Pregnancy history, median (IQR) Gravida Parity 0 (0-2) 0 (0) 0 (0-1) 0 (0) NS AMH, ng/mL, median (range) 2.5 (0.1-8.6) 1.3 (0.1-4.2) NS Indication for surgery, n (%) Myomas Endometriosis 12 (85.7) 6 (42.3) 24 (100) 10 (41.7) NS Results Patient characteristics IQR = interquartile range. Kuroda K, et al. J Minim Invasive Gynecol. 2019

77. Success group Failure group (14 cases) (24 cases) p -value R e s ul t s Pre-operative IVF and cryopreservation of embryos IQR = interquartile range. * Competent embryos were defined as grade 1−3 of the Veeck classification and ≥6-cell embryos in cleavage stage at day 3 after fertilization and 5- or 6-day blastocysts after fertilization, except for grade C in both the inner cell mass and the trophectoderm of the Gardner classification. Kuroda K, et al. J Minim Invasive Gynecol. 2019 Success group (14 cases) Failure group (24 cases) p -value Oocyte retrieval cycle, median (IQR) 3.0 (1.0-3.0) 2.0 (1.5-2.0) NS Number of cryopreserved embryos, median (IQR) Cleavage embryo Blastocyst 5.0 (4.0-6.0) 2.0 (0-3.0) 2.0 (1.0-5.0) 2.0 (1.0-3.0) 2.0 (1.0-2.5) 0 (0-0) < 0.001 NS < 0.001 Number of competent embryos, median (IQR) * Cleavage embryo Blastocyst 3.0 (2.0-5.0) 0.5 (0-1.0) 2.0 (1.0-4.0) 1.0 (0-1.0) 1.0 (0-1.0) 0 (0-0) < 0.001 NS < 0.001 Embryo transfer cycle, median (IQR) 3.0 (1.0-3.0) 2.0 (2.0-3.0) NS

78. Pregnancy outcomes after frozen-thawed ET Success group (14 cases) Failure group (24 cases) p -value Success group (14 cases, 30 ET cycles) Failure group (24 cases, 56 ET cycles) p -value Number of embryos transferred, median (IQR) 1.0 (1.0−1.0) 1.0 (1.0−1.0) NS Clinical pregnancy rate, n (%) 22 (73.3) 7 (10.0) 0.014 Live birth rate, n (%) 14 (46.7) (0) 0.006 Kuroda K, et al. J Minim Invasive Gynecol. 2019 Results Success group (14 cases) Failure group (24 cases) p -value Myomas Number of myomas , median (IQR) Myoma diameter, cm, median (IQR) n = 12 3.5 (1.0-9.5) 5.5 (2.0-10.0) n = 24 4.0 (1.0-8.5) 8.0 (5.5-9.5) NS NS Ovarian endometriomas Unilateral/bilateral, n Endometrioma diameter, cm, median (IQR) Revised ASRM score, median (IQR) n = 6 4 / 2 6.0 (5.0-7.0) 51.0 (30.0-120.5) n = 10 7 / 3 7.0 (5.0-8.5) 61.5 (32.0-108.0) NS NS NS Findings on laparoscopy

79. Summary The 14 women who had a successful surgery- ART hybrid therapy were significantly younger and had a larger number of cryopreserved embryos than those in failure group Kuroda K, et al. J Minim Invasive Gynecol. 2019

80. 0% 1 % 2 % 3 % 4 % 5 % 6 % 7 % 8 % 0% 5% 1 % 1 5 % 2 % 2 5 % 3 % 3 5 % 4 % 4 5 % 5 % 9 % Miscarriage rate (%) Rates of pregnancy and delivery (%) 妊娠率 / 総 ET 流産率 / 総妊娠 Decrease in pregnancy outcomes with age 44% 27% 8% 17% 34% 63% Japanese ART registry data, 2017 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Age (years) ART data in 2017 (The Japan Society of Obstetrics and Gynecology) Pregnancy/ET Miscarriage/Pregnancy

81. Cumulative livebirth rates per number of frozen embryos Age ( y e a r s) ART data in 2012 (JSOG) No of preoperative frozen embryo Pregnancy rate (/ET) M is c a r ria g e rate (/preg.) Delivery rate (/ET) 2 4 6 8 10 35 40.0% 20.3% 31.9% 53.6% 78 . 5 % 90 . % 95.4% 97 . 8 % 36 38.3% 21.6% 30.0% 51.0% 76 . % 88 . 3 % 94.3% 97 . 2 % 37 36.2% 23.2% 27.8% 47.9% 72 . 8 % 85 . 8 % 92.6% 96 . 2 % 38 33.5% 25.7% 24.9% 43.6% 68 . 2 % 82 . % 89.9% 94 . 3 % 39 30.8% 30.6% 21.4% 38.2% 61 . 8 % 76 . 4 % 85.4% 91 . % 40 27.2% 33.6% 18.1% 32.9% 54 . 9 % 69 . 7 % 79.7% 86 . 4 % 41 23.6% 39.2% 14.4% 26.6% 46 . 2 % 60 . 5 % 71.0% 78.8% 42 18.9% 43.2% 10.7% 20.3% 36 . 5 % 49 . 4 % 59.7% 67.9% 43 14.7% 49.3% 7.5% 14.4% 26 . 6 % 37 . 2 % 46.2% 53.9% 44 11.1% 57.5% 4.7% 9.2% 17 . 6 % 25 . 2 % 32.1% 38.3% 45 7.9% 62.6% 3.0% 5.8% 11 . 3 % 16 . 5 % 21.3% 25.9% Cumulative pregnancy rate: >50%: blue frame, > 80%: red frame Ref: ART data in 2017 (JSOG) Modified from Kuroda K, et al. J Minim Invasive Gynecol. 2019

82. Awareness of diminishing ovarian reserve with female age For successful ‘surgery-ART hybrid therapy’, the patients need to understand the fact of decreasing ovarian reserve with age and decide the number of preoperative frozen embryos Mac Dougall K, et al. Hum Reprod. 2013 the questionnaire survey of awareness of age and infertility

83. Preimplantation genetic testing for aneuploidy (PGTA) Clinical research for new embryo testing was started in Japan (2017)

84. Preimplantation genetic testing for aneuploidy (PGTA) Pregnancy rates/ET Pregnancy rates/Oocyte retrieval PGTA may be needed for successful ‘surgery-ART hybrid therapy’? Simon AL, et al , Fertil Steril . 2018

85. C onc l usio n Successful ‘Surgery-ART hybrid therapy’ requires Pre-operative cryopreservation of an age-dependent number of embryos (Necessity of ovarian hyperstimulation, Assessment of the need for PGT-A in the future) The strong desire of patients as well as doctors for pregnancy Kuroda K, et al. J Minim Invasive Gynecol. 2019

86. Successful ‘surgery-ART hybrid therapy’ requires Establishment of ART treatment with stable pregnancy outcomes and skilled reproductive surgery C on cl u s io n Kuroda K, et al. J Minim Invasive Gynecol. 2019