Drug-free IVA (in vitro activation) as an infertility treatment for aging poor responders

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1 Drug-free IVA (in vitro activation) as an infertility treatment for aging poor responders Professor, Department of OB/GYN Director of Advanced Reproductive Medicine Research Center International University Health and Welfare School of Medicine Kazuhiro Kawamura

2 Follicle number Changes in follicle number during women s life Periodic follicle growth/follicle atresia No follicle growth <1,000 follicles birth Menarche 40 yrs menopause

3 Aging infertility Young ovary Problems Decreasing residual follicle number Aging Aging ovary Decreasing oocyte/embryo quality Solutions Donor egg, adoption Regenerate oocytes from ips/stem cells Drug-free IVA Donor egg, adoption Regenerate oocytes from ips/stem cells MT injection/nt antioxidants, GH, etc

4 Contents 1. Background of POI 1. Basic and translational studies for in vitro activation of dormant follicles (IVA) 1. Clinical application of IVA 1. Drug-free IVA

5 Primary ovarian insufficiency (POI) Diagnosis 1. Amenorrhea before 40 years of age 2. Hypergonadotropic hypogonadism Symptoms 1. Infertility 2. Estrogen deficiency-hot flashes, mood disturbances, sexual dysfunction etc. Etiology POI affects approx. 1% of women

6 Follicle number Less than 1,000 follicles in POI patients before 40 yrs. POI patients Normal women Birth Menarche 40 yrs No follicle growth <1,000 follicles

7 Specific features Lack of follicle growth and ovulation Exhaustion of ovarian follicles and few residual follicles: <1,000 follicles (undetectable AMH levels) Treatments Resistant to traditional gonadotropin treatments Egg donation is the most successful treatment option, but

8 Is it possible to activate residual dormant follicles in POI patients?

9 Contents 1. Background of POI 1. Basic and translational studies for in vitro activation of dormant follicles (IVA) 1. Clinical application of IVA 1. Drug-free IVA

10 Primordial follicle Primary follicle Among dormant follicles, ~0.1% follicles are selected to activate. Because POI has < 1,000 residual follicles, these growth factors can activate only 1 follicle. We focused on intracellular signaling system Involving in the activation. Growth factors (kit ligand, neurotrophins, BMPs, VEGF, LIF, etc.)

11 PTEN null mice Reddy et al. Science, 2008 Castrillon et al. Science, 2003 Foxo3 null mice At early stage after birth, PTEN or FOXO3 deletion led to the activation of dormant primordial follicles and resulted in depletion of follicles within weeks.

12 Phosphatidylinositol 3-kinase (PI3K) signaling pathway RTK Apoptosis, Cell-cycle arrest Nucleus Growth factors GRB2 CRK Ras PIP 2 GSK 3 BAD EIF4E p27 4EBP PTEN PI3K Protein synthesis Apoptosis, cell-cycle arrest TSC2 TSC1 TOR Akt p70s6k FOXO1 PIP 3 RHEB FOXO4 PDK1 SGK Akt SGK FOXO3 The PI3K signaling pathway begins PI3K activation by receptor tyrosine kinases (RTKs) after binding growth factors. PI3K activates AKT, which inhibits the activities of FOXO3, resulting in cell proliferation and survival. PTEN negatively regulates PI3K signaling. In primordial follicles, local factors activate dormant follicles through PI3K-Akt-Foxo3 signaling pathway, whereas PTEN acts to block the signaling.

13 Is it possible to activate residual dormant follicles in POI patients artificially by transient PTEN suppression and/or PI3K activation using drugs?

14 PTEN inhibitor A vanadyl complexed to hydroxypicolinic acid is a highly potent and specific inhibitor at nano-molar concentrations. PI3K activator A cell-permeable phospho-peptide (740Y-P) binds to the SH2 domain of p85 regulatory subunit of PI3K and activates enzyme activity. RTK SH2 Derossi et al. BBRC, 1998

15 Method--mouse 24h 48h D3 mice PTEN Inhibitor &PI3K activator Ovaries transplanted into kidney capsule Control 740YP bpv (Hopic) Culture 2 days Pups Adult ovariectomized FSH treatment Histological analyses IVF-ET hcg treatment Oocyte retrieval Mature oocytes Epigenetic analyses

16 % total follicles In vitro activation (IVA) - in vivo transplantation -- ovarian histology PTEN inhibitor+pi3k activator * * * * * primordial primary preantral antral large primordial primary preantral antral large antral Follicular dynamics at day 14 after transplantaion of activated ovaries.

17 % embryonic development In vitro activation (IVA) - in vivo transplantation - IVF-ET 0h 24h 48h ** 2-cel/mature oocytes 1 2 normal control normal control PTEN inhibitor+pi3k bpv(pic)+740y-p activator blastocyst/2-cell 96h

18 Xeno-transplantation of human ovarian fragments to activate dormant follicles: IVA, in vitro activation Ovarian cortical fragments were obtained from patients with benign ovarian tumor with informed consent from the patient and approval from local ethical human subject committee. PTEN Inhibitor Human ovarian cortical fragments Xeno-transplanted into kidney capsule Control Culture 2 days Ovariectomized SCID mice FSH treatment for 6 months hcg treatment Oocyte retrieval Mature oocytes

19 Morphology of human ovarian fragments after 6 months of xeno-transplantation control control PTEN inhibitor PTEN inhibitor

20 Contents 1. Background of POI 1. Basic and translational studies for in vitro activation of dormant follicles (IVA) 1. Clinical application of IVA 1. Drug-free IVA

21 Conventional IVA Clinical application of IVA for POI patients Cryo- Preservation Histological analyses Preparation of ovarian cortical strips for freezing Fragmentation of ovarian strips to cubes Culture of ovarian cubes with PI3K activators Ovariectomy Fresh IVA Auto-transplantation Retrieve mature eggs In vitro fertilization IRB approval: Human Subject committee of St. Marianna University and Japan Society of Obstetrics and Gynecology Embryo transfer Embryo cryopreservation

22 Localization of early follicles in ovarian cortex cortex 1-2 mm medulla

23 Dissect ovarian cortices containing residual follicles by removing medulla. Before dissection of medulla Cut into small strips (1 x 1 cm 2, 1-2 mm thickness, where residual follicles are located). (Option: Cryo-preservation by vitrification method.) After dissection of medulla Small ovarian stripes ready for use 6-8 pieces of ovarian stripes could be obtained from one POI ovary. histological analyses Using 10% of volume of each ovarian stripe, detect residual follicles.

24 Fragmentation of ovarian strips to cubes In Vitro Activation Culture of ovarian cubes Fragment 2-3 ovarian pieces into 1-2 mm 2 of cubes IVA drugs treatment (PTEN inhibitor and PI3K activator) for 2 days to activate dormant follicles

25 Before auto-transplantation, wash cultured ovarian cubes by warmed culture media alone to avoid to introduce reagents inside of body. Transplant beneath the serosa of Fallopian tubes (20-40 cubes per site). Culture of ovarian cubes Autotransplantation of activated ovarian cubes In Vitro Activation Beneath serosa of Fallopian tubes high vascularization, convenience for trans-vaginal ultrasound monitoring ease for oocyte retrieval

26 Auto-transplantation Cutting the serosa and making a pouch between serosa (arrows) and Fallopian tube (arrowhead). Grafting multiple ovarian cubes (arrows) beneath the serosa of Fallopian tubes. Wound was covered by an oxidized regeneration cellulose to avoid cube loss from the graft site.

27 Is this site the best for grafting? Remaining ovary is also good site, especially in the cases of early stage of POI.

28 Patients follow up protocols Monitor follicle growth weekly to biweekly: serum estrogen and gonadotropin levels + transvaginal ultrasound. Specific protocols for ovarian stimulation in POI patients (Zhai, Kawamura, et al. JCEM 2016). Conventional IVF-ET Retrieve mature eggs In vitro fertilization

29 Tips for ovarian stimulation after IVA In POI patients, due to absence of antral follicles before and immediately after IVA, the first sign of follicle growth is elevation of estrogen (E2) levels. Without ovarian stimulation, follicles can grow spontaneously followed by decline in FSH and LH levels based on negative feed back of E2. However, in most of cases, the decrease in LH levels is inadequate. Early luteinization Arrest of follicle growth Oocyte degeneration

30 Success rate of oocyte retrieval after IVA in different protocols Zhai, Kawamura et al JCEM 2016 without LH level suppression+spontaneous growth: 27% (3/11) LH level suppression: 100% (2/2) without LH level suppression+hmg stimulation: 0% (0/2)

31 Effects of hyper-lh on oocyte-granulosa-theca cell interactions Preculture with LH camp production after FSH stimulation Follicle growth after FSH stimulation FSH stimulation High chronic LH stimulation CYP17 GDF9 FSHR Preantral follicles exposed to high LH express low levels GDF-9 in oocyte and FSHR in granulosa cells, resulting in decreases in sensitivity of FSH stimulation and suppression of follicle growth. Orisaka Endocrinology 2013

32 How can we stimulate ovaries after IVA? 1. Normalize LH levels by supplementation of estrogen and estrogen + progesterone with induction of withdrawal bleeding. 2. After confirmation of normal LH levels (<10 miu/ml), maintain its low levels using GnRHa. (Daily GnRH AN injection is too expensive) 3. Similar to short protocol, treat patients with rfsh or pure HMG (low LH content) for >2 weeks.

33 Enrolled patients Results 83 of POI patients (37.4 ± 4.9 years of age) Duration of amenorrhea: 5.7 ± 3.5 years Ovary grafting was performed in 46 patients Follicle growth was found in 28 out of 46 patients containing residual follicles based on the histological analyses. After IVF, embryos were cryopreserved at day 2.

34 Results Thawing embryo transfer was performed in 8 patients. Others were accumulating cryopreserved embryos. 3 out of 8 patients became pregnant after embryo transfer. One miscarriage Two successful deliveries ー a male baby, 3254 ー a female baby, 2970g

35 Second babies by re-transplantation of frozenthawed cryopreserved ovaries after activation After the delivery, we performed IVA again using cryopreserved ovarian tissue. 1st patient: Dec first baby Sep successful pregnancy 2nd patient: May first baby April Second baby (female, 3091g)

36 Current clinical outcome of IVA Ovariectomy: n=152 Residual follicles based on histology: Positive: n=96 Negative: n=56 Negative N=56 Total N=152 Positive N=96 IVA grafting with residual follicles with follow up > 6 months: n=70 IVA grafting without residual follicles with follow up > 6 months: n=16

37 % of follicle growth % 45/70 6% 1/16 Residual follicle (+) Residual follicle (-) Histological analyses Our histological analyses were effective to predict IVA outcome

38 Indication for IVA treatment POI/DOR patients with residual follicles. How can we predict presence of residual follicles? Recent follicle growth after diagnosis of POI Short duration of amenorrhea Positive AMH

39 Duration of amenorrhea (Year) Follicle number per 1x1x5mm tissue Duration of amenorrhea is a good predictive factor for presence of residual follicles * (-) (+) Residual follicle >9 Duration of amenorrhea (Year)

40 Clinical application of IVA for POI patients Conventional Histological analyses Fragmentation of IVA ovarian strips to For long Cryo- cubes term Preservation amenorrhea Preparation of ovarian Culture of ovarian cubes cortical strips for freezing with PI3K activators Ovariectomy Fresh IVA For short Auto-transplantation term amenorrhea Retrieve mature eggs In vitro fertilization IRB approval: Human Subject committee of St. Marianna University and Japan Society of Obstetrics and Gynecology Embryo transfer Embryo cryopreservation

41 Results of IVF after IVA Success of oocyte retrieval : 42/46 patients (91%) 139/212 follicles (66%) [1-16 cycles] % /139 85/116 76/ / MII oocyte fertilization cleavage *high quality D2-3 embryo *Veek: G1-3

42 Over all outcome after embryo transfer IVA grafting with residual follicles with follow up > 6 months: n=124 Mean age: 38.9 years of age Embryo transfer (cleavage stage D2 or 3): n=38 Pregnancy: 15/38 (39%) Miscarriage: 5/15 (33%) 7 babies, 3 ongoing pregnancies

43 Results Reproducibility of IVA was already confirmed by China, Spain, Poland, and Mexico groups under our guidance. Kawamura et al Hum Reprod 2015 Zhai et al JCEM 2016 IVA pregnancy/delivery N=4 N=4 N=2 N=1 IVA in Poland: December 2016 IVA press conference in China: May 2015

44 International patent: STIMULATION OF OVARIAN FOLLICLE DEVELOPMENT AND OOCYTEMATURATION PCT/US2013/ Out-licensing to Ovascience. Inc Kawamura et al. PNAS 2013 Hsueh, Kawamura et al. Endocrine Rev 2014 Suzuki, Kawamura et al. Hum Reprod 2015 Yuan, Kawamura et al FASEB J 2015 Kawamura et al. Hum Reprod 2016 Kawamura and Hsueh Curr Opin Obstet Gynecol 2016 Zhai, Kawamura et al. JCEM, 2016 Kawamura et al. Reproduction, 2017 Haino, Kawamura et al. JAYAO 2017 Sato, Kawamura et al. J Gynecol Women s Health 2017 Kawamura et al. Syst Biol Reprod Med 2017

45 Temporal follicle growth in transplanted ovaries Follicle growth from primordial to preovulatory stage takes more than 4-6 months. In contrast to our expectation, we found follicle growth before 4-6 months after grafting. This result suggested that our IVA method also stimulated growth of secondary follicles in grafted ovaries. Derived from secondary follicles Derived from primordial follicles

46 Fragmentation of mouse ovaries followed by allo-transplantation facilitated ovarian growth Ovary weight (mg) Paired ovaries from D10 mice with or without fragmentation were grafted into kidney capsules of adult ovariectomized mice. At 5 days after grafting, ovaries were dissected and fixed before measurement of their weights.

47 Ovarian fragmentation followed by grafting promoted growth of secondary follicles in mice Follicle dynamics before and after grafting of intact and fragmented (three pieces) ovaries from day 10 mice were evaluated at 5 days after grafting.

48 What is the molecular mechanisms underlying follicle growth after fragmentation?

49 Hippo signaling pathway restricts cell proliferation Hippo pathway regulates cell proliferation. In normal tissue with an intact cytoskeleton (purple), hippos signaling inactivates a core transcriptional factor (Yki or YAP) and prevent it from activating proliferation genes in the nucleus. When cytoskeleton conformation is changed, Yki/YAP is free to enter the nucleus and produce CCN growth factors leading to cell proliferation.

50 Whole Pieces Whole Pieces Ovarian fragmentation decreased phospho-yap (unactivated form) in mice Paired ovaries from day 10 mice with or without cutting into 3 pieces were incubated for 1 h, followed by immunoblotting. YAP pyap GAPDH

51 Whole Pieces Whole Pieces Whole Pieces CCN2/ Ovarian fragmentation increased CCN2 proteins in mice CCN2 GAPDH 3h 4h 5h Paired ovaries from day 10 mice with or without cutting were incubated for 3 5 h before immunoblotting.

52 Increases in CCN transcripts after fragmentation of human ovarian tissues CCN2/CTGF CCN5/WISP2 * * (h) CCN3/NOV * * 4 * 3 2 * (h) (h) CCN6/WISP3 * * (h) Thawed human cortical strips were cut into pieces or left intact before incubation for different intervals followed by realtime RT-PCR analyses.

53 Roles of CTGF (CCN2) in ovary CTGF treatment of ovaries induces the expression of many genes related to cellcycle progression and cell differentiation, thereby enhancing the growth of immature follicles (Schindler R, Nilsson E, Skinner MK 2010). Ctgf cko mice exhibit severe subfertility and multiple reproductive defects including reduced numbers of preantral and antral follicles, and a trend toward increased numbers of atretic preantral and atretic antral follicles (Nagashima et al. 2011).

54 Regulation of Hippo pathway by F-actin Under conditions of low F-actin, Hippo pathway activity is high, leading to inhibition of Yki /YAP activity and inhibition of tissue growth. When F-actin levels are high, Hippo pathway activity is inhibited leading to nuclear import of Yki/YAP, and up0regulation of downstream targets to promote tissue growth. Physiologically, F-actin accumulation may be regulated by external tension cues, thereby controlling tissue growth via the Hippo pathway.

55 Ovarian fragmentation stimulated actin polymerization followed by increased F-actin levels Paired ovaries from day 10 mice were cut into three pieces or kept intact before immunoblotting analyses of F- and G-actin levels.

56 Ovarian fragmentation suppresses Hippo signaling, leading to follicle growth G-Actin Hippo pathway genes YA P YA P CCN P F-Actin YA P Degradation CCN Growth factors Anti-apoptotic factors Ovarian fragmentation led to changes in intercellular tension and facilitated the conversion of G-actin to F-actin. Subsequent disruption of Hippo signaling decreased pyap to total YAP ratios, leading to increased expression of downstream CCN growth factors. Secretion of CCN growth factors stimulated follicle growth. Secondary follicle growth

57 Contents 1. Background of POI 1. Basic and translational studies for in vitro activation of dormant follicles (IVA) 1. Clinical application of IVA 1. Drug-free IVA

58 Two-step follicle stimulation in IVA PI3K signal stimulation Preovulatory follicle Primordial follicle Primary follicle Secondary follicle Antral follicle Hippo signal disruption

59 New IVA approach targeting immature secondary follicles In early POI/DOR (poor responder) patients, is it possible to stimulate residual secondary follicles to increase number of antral follicles by disruption of Hippo signaling only?

60 Ovarian cubes culture

61 1. Original IVA (PI3K stimulation and Hippo disruption) POI patients DOR/early POI patients Cryopreservation Oophrectomy Fragmentation and PI3K stimulators treatment (2 days) Two surgeries 2. Drug-free IVA (Hippo disruption only) Partial cortex removal Fragmentation and immediate return One surgery

62 Drug-free IVA and Direct Activation Drug-free IVA Direct Activation A clinical study in progress: Successful pregnancy in 7 out of 68 aging ovarian dysfunction patients (43-48 yrs) No positive evidence up to now

63 Drug-free IVA and orthologous grafting Limitation: Only applicable to DOR/early POI patients who likely have secondary follicles Advantages: Minimal damages to ovarian blood supply No need to use Akt-stimulating drugs Avoid potential follicle loss during culture One laparoscopic surgery Spontaneous pregnancy possible IVI group, Spain (Bilbao workshop 2017) 3 spontaneous pregnancies/14 patients

64 St. Marianna University School of Medicine Bunpei Ishizuka, Nao Suzuki, Yorino Sato, Naoki Okamoto, Ikko Kawashima, Midori Tamura Seido Takae, Yodo Sugishita, Nobuhito Yoshioka, Yuta Kawagoe, Mariko Hoshina, Noriyuki Takahashi Collaborators Stanford University School of Medicine Aaron JW Hsueh Yuan Cheng Jing Li