Hippo signaling disruption and ovarian follicle activation in infertile patients

The Hippo signaling pathway, which is important in organ size regulation, is present in organisms from the fly to mammals. Disruption of the Hippo signaling pathway leads to increased nuclear translocation of the effector Yes-associated protein (YAP), resulting in the expression of cystein-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors to increase organ sizes. Furthermore, genome-wide knockdown of genes in insect cells demonstrated that actin polymerization promoted nuclear translocation of YAP. In the mammalian ovary, we demonstrated the expression of Hippo signaling pathway genes and showed that ovarian fragmentation increased actin polymerization, leading to YAP nuclear translocation and increased expression of cystein-rich 61, CCN growth factors and BIRC apoptosis inhibitors, followed by enhanced follicle growth. Here we summarize evidence suggesting the role of mechanical stress on follicle growth in the ovary and describe recent use of ovary-damaging procedures to treat ovarian infertility. Ovarian fragmentation, together with in vitro incubation with Akt-stimulating drugs, formed the basis of an in vitro activation (IVA) therapy to treat patients with premature ovarian insufficiency, whereas ovarian fragmentation alone (drug-free IVA) was successful in treating patients with premature ovarian insufficiency with recent menses cessation. For middle-aged women with poor ovarian responses and diminished ovarian reserve, drug-free IVA was also effective in promoting follicle growth for infertility treatment. In addition, an in vivo follicle activation approach based on laparoscopic ovarian incision showed promise for patients with resistant ovary syndrome. With initial success using mechanical disruption approaches, future investigation could evaluate possibilities to refine mechanical methods and to locally administer actin polymerization-enhancing drugs for ovarian infertility treatment. The Hippo signaling pathway, which is important in organ size regulation, is present in organisms from the fly to mammals. Disruption of the Hippo signaling pathway leads to increased nuclear translocation of the effector Yes-associated protein (YAP), resulting in the expression of cystein-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors to increase organ sizes. Furthermore, genome-wide knockdown of genes in insect cells demonstrated that actin polymerization promoted nuclear translocation of YAP. In the mammalian ovary, we demonstrated the expression of Hippo signaling pathway genes and showed that ovarian fragmentation increased actin polymerization, leading to YAP nuclear translocation and increased expression of cystein-rich 61, CCN growth factors and BIRC apoptosis inhibitors, followed by enhanced follicle growth. Here we summarize evidence suggesting the role of mechanical stress on follicle growth in the ovary and describe recent use of ovary-damaging procedures to treat ovarian infertility. Ovarian fragmentation, together with in vitro incubation with Akt-stimulating drugs, formed the basis of an in vitro activation (IVA) therapy to treat patients with premature ovarian insufficiency, whereas ovarian fragmentation alone (drug-free IVA) was successful in treating patients with premature ovarian insufficiency with recent menses cessation. For middle-aged women with poor ovarian responses and diminished ovarian reserve, drug-free IVA was also effective in promoting follicle growth for infertility treatment. In addition, an in vivo follicle activation approach based on laparoscopic ovarian incision showed promise for patients with resistant ovary syndrome. With initial success using mechanical disruption approaches, future investigation could evaluate possibilities to refine mechanical methods and to locally administer actin polymerization-enhancing drugs for ovarian infertility treatment. Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/31023 Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/31023 Unlike males of the species, female mammals have a finite number of germ cells that undergo gradual depletion throughout reproductive life. Although advances in controlled ovarian hyperstimulation and in vitro fertilization (IVF) have provided new therapies for infertility (1Niederberger C. Pellicer A. Cohen J. Gardner D.K. Palermo G.D. O’Neill C.L. et al.Forty years of IVF.Fertil Steril. 2018; 110: 185-324.e5Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), middle-aged women have diminished ovarian reserve with poorer oocyte quality, leading to IVF failure. For patients with premature ovarian insufficiency (POI) who reach menopause before 40 years of age, the only option is to use donor eggs. In addition, a subgroup of patients with POI have multiple antral follicles under ultrasound examination but are unresponsive to gonadotropin treatments, exhibiting resistant ovary syndrome (ROS) (2Dewhurst C. Koos E.B. Ferreira H. The resistant ovary syndrome.Br J Obstet Gynecol. 1975; 82: 341-345Crossref PubMed Scopus (39) Google Scholar). Recent studies on ovarian Hippo signaling pathway provide the theoretical basis to disrupt this pathway and promote follicle growth. Here we discuss the present understanding of the role of Hippo signaling pathway in restraining organ growth and the use of mechanical approaches to disrupt ovarian Hippo signaling and promote follicle growth as infertility treatments. The Hippo signaling pathway is essential for organ size control, and components of this pathway are conserved in all metazoan animals (3Pan D. Hippo signaling in organ size control.Genes Dev. 2007; 21: 886-897Crossref PubMed Scopus (476) Google Scholar, 4Hergovich A. Mammalian Hippo signalling: a kinase network regulated by protein-protein interactions.Biochem Soc Trans. 2012; 40: 124-128Crossref PubMed Scopus (56) Google Scholar) (Fig. 1). Hippo signaling consists of several negative growth regulators acting in a serine kinase cascade that ultimately phosphorylate and inactivate key transcriptional coactivators, Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ). Hippo signaling kinases include mammalian sterile 20-like (MST) 1/2, Salvador homolog 1 (SAV1), and large tumor suppressor (LATS) 1/2. When Hippo signaling is disrupted, nuclear YAP acts in concert with transcriptional enhanced associate domain (TEAD) transcriptional factors to increase the expression of downstream cystein-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors (3Pan D. Hippo signaling in organ size control.Genes Dev. 2007; 21: 886-897Crossref PubMed Scopus (476) Google Scholar). These CCN proteins, in turn, stimulate cell growth, survival, and proliferation (5Holbourn K.P. Acharya K.R. Perbal B. The CCN family of proteins: structure-function relationships.Trends Biochem Sci. 2008; 33: 461-473Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The Hippo signaling pathway proteins regulate liver growth, and deletion of the SAV1 or MST1/2 gene in hepatocytes results in enlarged livers in mice (6Lu L. Li Y. Kim S.M. Bossuyt W. Liu P. Qiu Q. et al.Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver.Proc Natl Acad Sci U S A. 2010; 107: 1437-1442Crossref PubMed Scopus (513) Google Scholar, 7Lee K.P. Lee J.H. Kim T.S. Kim T.H. Park H.D. Byun J.S. et al.The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis.Proc Natl Acad Sci U S A. 2011; 107: 8248-8253Crossref Scopus (328) Google Scholar). In cardiomyocytes, conditional deletion of SAV1 leads to enlarged hearts (8Heallen T. Zhang M. Wang J. Bonilla-Claudio M. Klysik E. Johnson R.L. et al.Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size.Science. 2011; 332: 458-461Crossref PubMed Scopus (644) Google Scholar). Hippo signaling is also involved in tissue regeneration and expansion of stem cells (9Zhao B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal.Nat Cell Biol. 2011; 13: 877-883Crossref PubMed Scopus (754) Google Scholar). Actin comprises up to 5% to 10% of total soluble proteins in eukaryotic cells, and polymerization of globular actin (G-actin) to the filamentous form (F-actin) is important for cell shape maintenance, adhesion, and locomotion. Using genome-wide RNAi screening to “knock down” individual genes in cultured insect cells identified key genes capable of disrupting Hippo signaling and promoting nuclear translocation of YAP. Most identified genes were involved in actin polymerization and thus are upstream regulators of Hippo singling (10Sansores-Garcia L. Bossuyt W. Wada K. Yonemura S. Tao C. Sasaki H. et al.Modulating F-actin organization induces organ growth by affecting the Hippo pathway.EMBO J. 2012; 30: 2325-2335Crossref Scopus (305) Google Scholar, 11Fernandez B.G. Gaspar P. Bras-Pereira C. Jezowska B. Rebelo S.R. Janody F. Actin-capping protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila.Development. 2012; 138: 2337-2346Crossref Scopus (217) Google Scholar). After induction of extra F-actin formation or overexpression of an activated version of the actin polymerizing formin diaphanous (DIAPH), Hippo signaling was disrupted, leading to overgrowth in fly imaginal discs and proliferation of human HeLa cells. When Hippo signaling was disrupted, nuclear YAP or TAZ interacted with TEAD transcriptional factors to increase the expression of downstream CCN growth factors and BIRC apoptosis inhibitors (12Yu F.-X. Guan K.-L. The Hippo pathway: regulators and regulations.Genes Devel. 2013; 27: 355-371Crossref PubMed Scopus (776) Google Scholar). In diverse normal tissues, upstream Hippo signaling pathway proteins phosphorylate YAP to promote its degradation (4Hergovich A. Mammalian Hippo signalling: a kinase network regulated by protein-protein interactions.Biochem Soc Trans. 2012; 40: 124-128Crossref PubMed Scopus (56) Google Scholar). Based on initial observations showing enhanced follicle growth after fragmentation of murine ovaries and grafting, we investigated underlying cellular mechanisms (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar). Cutting ovaries induced a transient increase of actin polymerization (G-actin to F-actin) and decreased YAP phosphorylation, leading to increased nuclear YAP levels, followed by increased expression of downstream CCN growth factors and BIRC apoptosis inhibitors (14Hsueh A.J. Kawamura K. Cheng Y. Fauser B.C. Intraovarian control of early folliculogenesis.Endocrine Rev. 2015; 36: 1-24Crossref PubMed Scopus (285) Google Scholar) (Fig. 1). We further demonstrated that treatment of murine ovaries with jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, promoted the conversion of G-actin to F-actin, followed by increased nuclear YAP and expression of downstream CCN2. After short-term treatments of intact ovaries with JASP or S1P in vitro, subsequent grafting of ovaries promoted follicle growth (15Cheng Y. Feng Y. Jansson L. Sato Y. Deguchi M. Kawamura K. et al.Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP.FASEB J. 2015; 29: 2423-2430Crossref PubMed Scopus (56) Google Scholar). Among six known CCN growth factors, the expression of four of them (CCN2, 3, 5, and 6) was induced upon Hippo signaling disruption in the ovary (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar). Furthermore, treatment of ovarian explants with either one of these four CCN growth factors promoted early follicle growth (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar). Also, expression of two apoptosis inhibitors (BIRC1 and 7) was increased after ovarian fragmentation (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar). These findings demonstrated that mechanical signals induced by ovarian fragmentation could increase biochemical signals responsible for early follicle growth (Fig. 1). Using cultured human cortical fragments, recent studies confirmed that PI3K/Akt activators act synergistically with the Hippo signaling pathway disrupted by tissue fragmentation to accelerate primordial follicle recruitment (16Grosbois J. Demeestere I. Dynamics of PI3K and Hippo signaling pathways during in vitro human follicle activation.Hum Reprod. 2018; 33: 1705-1714Crossref PubMed Scopus (54) Google Scholar). Furthermore, transcriptional and histological analyses revealed that oocytes in primordial follicles were compressed by surrounding granulosa cells secreting extracellular matrix proteins, leading to a state of high mechanical pressure (17Nagamatsu G. Shimamoto S. Hamazaki N. Nishimura Y. Hayashi K. Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.Science Advances. 2019; 5eaav9960Crossref PubMed Scopus (31) Google Scholar). Similar to our fragmentation approach, primordial follicles became activated upon loosening the structure with enzymes that degraded extracellular matrix, and follicle dormancy was restored by compression with exogenous pressure (17Nagamatsu G. Shimamoto S. Hamazaki N. Nishimura Y. Hayashi K. Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.Science Advances. 2019; 5eaav9960Crossref PubMed Scopus (31) Google Scholar). Another study compared gene expression profiles between in vitro “nondormant” and in vivo dormant primordial follicles, leading to the identification of hypoxic conditions in the maintenance of follicle dormancy (18Shimamoto S. Nishimura Y. Nagamatsu G. Hamada N. Kita H. Hikabe O. et al.Hypoxia induces the dormant state in oocytes through expression of Foxo3.Proc Natl Acad Sci. 2019; 116: 12321-12326Crossref PubMed Scopus (16) Google Scholar). In addition to disrupting ovarian Hippo signaling pathway to promote follicle growth, our fragmentation of cortical strips could confer a nonhypoxia and low mechanical pressure environment to enhance primordial follicle activation and growth. Genomic and genetic studies provided support for the essential roles of the ovarian Hippo signaling system and actin polymerization in regulating follicle growth. Defects in Hippo signaling genes were associated with POI, polycystic ovary syndrome (PCOS), follicle reserve, and ovarian epithelial cell tumorigenesis in patients (14Hsueh A.J. Kawamura K. Cheng Y. Fauser B.C. Intraovarian control of early folliculogenesis.Endocrine Rev. 2015; 36: 1-24Crossref PubMed Scopus (285) Google Scholar). Three human diaphanous (DIAPH) genes are important for suppressing actin depolymerization. The coding region of DIAPH2 was disrupted in a POI family due to chromosomal translocation (19Bione S. Sala C. Manzini C. Arrigo G. Zuffardi O. Banfi S. et al.A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility.Am J Hum Genet. 1998; 62: 533-541Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). In addition, DIAPH2 was found in a genome-wide association study as a candidate gene for menopausal age (20He C. Kraft P. Chasman D.I. Buring J.E. Chen C. Hankinson S.E. et al.A large-scale candidate gene association study of age at menarche and age at natural menopause.Hum Genet. 2010; 128: 515-527Crossref PubMed Scopus (97) Google Scholar), whereas copy number variants of DIAPH2 were identified in patients with POI (21Bestetti I. Castronovo C. Sironi A. Caslini C. Sala C. Rossetti R. et al.High-resolution array-CGH analysis on 46, XX patients affected by early onset primary ovarian insufficiency discloses new genes involved in ovarian function.Hum Reprod. 2019; 34: 574-583Crossref PubMed Scopus (18) Google Scholar). For the related DIAPH3 gene, it was also found as a marker of follicle reserve and early menopause (22Schuh-Huerta S.M. Johnson N.A. Rosen M.P. Sternfeld B. Cedars M.I. Reijo Pera R.A. Genetic markers of ovarian follicle number and menopause in women of multiple ethnicities.Hum Genet. 2012; 131: 1709-1724Crossref PubMed Scopus (49) Google Scholar). Thus, loss of function or diminished expression of DIAPH proteins could lead to downstream Hippo signaling disruption and accelerated follicle loss. Among Hippo pathway genes, LATS1 null mice were subfertile and developed ovarian stromal cell tumors (23St John M.A. Tao W. Fei X. Fukumoto R. Carcangiu M.L. Brownstein D.G. et al.Mice deficient of Lats1 develop soft-tissue sarcomas, ovarian tumours and pituitary dysfunction.Nat Genet. 1999; 21: 182-186Crossref PubMed Scopus (349) Google Scholar), whereas Hippo effector YAP was implicated in PCOS in genome-wide association studies of patients from different ethnic groups (24Chen Z.-J. Zhao H. He L. Shi Y. Qin Y. Shi Y. et al.Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16. 3, 2p21 and 9q33. 3.Nat Genet. 2011; 43: 55-59Crossref PubMed Scopus (450) Google Scholar, 25Louwers Y.V. Stolk L. Uitterlinden A.G. Laven J.S. Cross-ethnic meta-analysis of genetic variants for polycystic ovary syndrome.J Clin Endocrinol Metab. 2013; 98: E2006-E2012Crossref PubMed Scopus (80) Google Scholar). Of interest, hypomethylation of the YAP promoter was found in granulosa cells of patients with PCOS, together with increased YAP expression (26Jiang L.-L. Xie J.-K. Cui J.-Q. Wei D. Yin B.-L. Zhang Y.-N. et al.Promoter methylation of yes-associated protein (YAP1) gene in polycystic ovary syndrome.Medicine. 2017; 96e5768Crossref PubMed Scopus (18) Google Scholar), presumably conferring follicle growth. Furthermore, granulosa cell–specific deletion of YAP in mice increased granulosa cell apoptosis and decreased the number of corpora lutea, leading to subfertility (27Lv X. He C. Huang C. Wang H. Hua G. Wang Z. et al.Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.FASEB J. 2019; 33: 10049-10064Crossref PubMed Scopus (19) Google Scholar). In ovarian epithelial cancer cells, overexpression of YAP was also found (28Hall C.A. Wang R. Miao J. Oliva E. Shen X. Wheeler T. et al.Hippo pathway effector Yap is an ovarian cancer oncogene.Cancer Res. 2011; 70: 8517-8525Crossref Scopus (168) Google Scholar). For downstream Hippo signaling genes, granulosa cell–specific deletion of CCN2 led to disrupted follicle development and anovulation (29Nagashima T. Kim J. Li Q. Lydon J.P. Demayo F.J. Lyons K.M. et al.Connective tissue growth factor is required for normal follicle development and ovulation.Mol Endocrinol. 2011; 25: 1740-1759Crossref PubMed Scopus (59) Google Scholar). Based on network and gene ontology analysis, CCN2 was also identified as a regulator for patients with diminished ovarian reserve (30Pashaiasl M. Ebrahimi M. Ebrahimie E. Identification of the key regulating genes of diminished ovarian reserve (DOR) by network and gene ontology analysis.Mol Biol Rep. 2016; 43: 923-937Crossref PubMed Scopus (15) Google Scholar), whereas gene copy variations for the apoptosis inhibitor BIRC1 were found in patients with POI (31Aboura A. Dupas C. Tachdjian G. Portnoi M.F. Bourcigaux N. Dewailly D. et al.Array comparative genomic hybridization profiling analysis reveals deoxyribonucleic acid copy number variations associated with premature ovarian failure.J Clin Endocrinol Metab. 2009; 94: 4540-4546Crossref PubMed Scopus (70) Google Scholar). These data implicated Hippo signaling–associated growth factor/apoptosis inhibitor in regulating folliculogenesis. With the current understanding on mechanical manipulations to disrupt ovarian Hippo signaling and promote follicle growth, one can reevaluate histological findings. As early as 1922, F. R. Girard at Stanford University performed hysterectomy with double salpingo-oophorectomy and transplanted parts of one or both ovaries into the abdominal wall of patients (32Girard F. Ovarian autotransplantation.Calif State J Med. 1922; 20: 21PubMed Google Scholar). The grafts took, and there was periodic enlargement of grafts associated with amelioration of ablation (now known as menopausal) symptoms and menstruation. Girard concluded that “better results will be obtained from multiple grafts, because a much larger surface is obtained which insures a better blood supply.” Although better graft vascularity is beneficial, grafting of small ovarian portions after cutting ovaries also likely disrupted Hippo signaling. In mice, initial success of orthotopic whole ovary transplantation (33Robertson G.A.G. Ovarian transplantation in the house mouse.Proc Soc Exp Biol Med. 1940; 44: 302-304Crossref Scopus (15) Google Scholar) was also improved by cutting ovaries in half before transplantation (34Stevens L.C. A modification of Robertson's technique of homoiotopic ovarian transplantation in mice.Transplant Bull. 1957; 4: 106-107PubMed Google Scholar). Indeed, ovarian cortices were routinely fragmented to allow better cryopreservation and grafting for fertility preservation of cancer patients (35Donnez J. Dolmans M.-M. Demylle D. Jadoul P. Pirard C. Squifflet J. et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet. 2004; 364: 1405-1410Abstract Full Text Full Text PDF PubMed Scopus (1251) Google Scholar, 36Pacheco F. Oktay K. Current success and efficiency of autologous ovarian transplantation: a meta-analysis.Reprod Sci. 2017; 24: 1111-1120Crossref PubMed Scopus (97) Google Scholar). Although less frequently used now, patients with PCOS were treated with ovarian wedge resection (37Stein I.F. Leventhal M.L. Amenorrhea associated with bilateral polycystic ovaries.Am J Obstet Gynecol. 1935; 29: 181Abstract Full Text PDF Google Scholar) and ovarian drilling by diathermy or laser (38Farquhar C. Lilford R.J. Marjoribanks J. Vandekerckhove P. Laparoscopic “drilling” by diathermy or laser for ovulation induction in anovulatory polycystic ovary syndrome.Cochrane Database Syst Rev. 2007; CD001122PubMed Google Scholar). These damaging procedures likely involved ovarian Hippo signaling disruption. Based on the essential role of the Akt signaling pathway in primordial follicle growth, we developed the IVA approach as the basis to treat ovarian infertility (39Li J. Kawamura K. Cheng Y. Liu S. Klein C. Duan E.K. et al.Activation of dormant ovarian follicles to generate mature eggs.Proc Natl Acad Sci U S A. 2010; 107: 10280-10284Crossref PubMed Scopus (245) Google Scholar). Using an inhibitor of the phosphatase and tensin homolog (PTEN) phosphatase and a PI3K activating peptide, we demonstrated that 2-day treatment of neonatal mouse ovaries containing primordial follicles increased nuclear exclusion of Foxo3 in primordial oocytes, suggesting follicle activation. After transplantation of ovaries under kidney capsules of ovariectomized hosts, treated primordial follicles developed to the preovulatory stage, with mature eggs displaying normal epigenetic changes. After IVF and embryo transfer, healthy progeny with proven fertility were delivered. Furthermore, human ovarian cortical fragments treated with the PTEN inhibitor, followed by xenotransplantation into immune-deficient mice for 6 months, allowed development of primordial follicles to the preovulatory stage with oocytes capable of undergoing nuclear maturation (39Li J. Kawamura K. Cheng Y. Liu S. Klein C. Duan E.K. et al.Activation of dormant ovarian follicles to generate mature eggs.Proc Natl Acad Sci U S A. 2010; 107: 10280-10284Crossref PubMed Scopus (245) Google Scholar). We subsequently reported that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to follicle growth and generation of mature oocytes (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar). When combining Akt stimulators treatments and ovarian fragmentation, additive follicle growth was found. We then extended these results to treatment of infertility in patients with POI by fragmenting cortical strips followed by Akt stimulators treatment and autografting under laparoscopic surgery (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar). After ovarian stimulation, retrieval of mature oocytes, IVF, and embryo transfer, a healthy baby was delivered. Subsequently, IVA was successfully used to treat patients with POI in Japan (40Suzuki N. Yoshioka N. Takae S. Sugishita Y. Tamura M. Hashimoto S. et al.Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.Hum Reprod. 2015; 30: 608-615Crossref PubMed Scopus (241) Google Scholar) and China (41Zhai J. Yao G. Dong F. Bu Z. Cheng Y. Sato Y. et al.In vitro activation of follicles and fresh tissue auto-transplantation in primary ovarian insufficiency patients.J Clin Endocrinol Metab. 2016; 101: 4405-4412Crossref PubMed Scopus (97) Google Scholar). Using a simplified drug-free IVA procedure without 2-day incubation of Akt stimulators (PTEN inhibitor and PI3K activator), two case reports indicated successful pregnancies of patients with POI with recent menses cessation (42Fabregues F. Ferreri J. Calafell J. Moreno V. Borrás A. Manau D. et al.Pregnancy after drug-free in vitro activation of follicles and fresh tissue autotransplantation in primary ovarian insufficiency patient: a case report and literature review.J Ovarian Res. 2018; 11: 76Crossref PubMed Scopus (25) Google Scholar, 43Mahajan N. Kaur J. Bhattacharya B. Naidu P. Gupta S. In vitro activation of ovary.Onco Fertil J. 2019; 2: 35Crossref Google Scholar). Using this drug-free IVA approach, a recent report showed oocyte retrieval in five of the 14 patients with POI with four successful pregnancies and a pregnancy rate of 57% per oocyte retrieval and 67% per embryo transfer (44Ferreri J. Fàbregues F. Calafell J.M. Solernou R. Borrás A. Saco A. et al.Drug-free in vitro activation of follicles and fresh tissue autotransplantation as a therapeutic option in primary ovarian insufficiency patients.Reprod Biomed Online. 2020; 40: 254-260Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). For these studies, Hippo signaling disruption was likely the underlying mechanism responsible for follicle growth. Based on the hypothesis that subfertile patients showing POR still have multiple residual follicles that could be activated by mechanical stress, we applied the drug-free IVA approach to treat patients with POR. Follicle growth was promoted after Hippo signaling disruption alone using in vitro ovarian cortical fragmentation, followed by autologous grafting under laparoscopic surgery (45Kawamura K. Ishizuka B. Hsueh A.J. Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve.Reprod Biomed Online. 2020; 40: 245-253Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In nine of 11 patients with POR treated with drug-free IVA, increases in antral follicle numbers in multiple growth waves were detected after follicle-stimulating hormone (FSH) treatment. Subsequent retrieval of mature oocytes for IVF allowed 16 embryo transfers in five patients, leading to one live birth, two ongoing pregnancies, and one miscarriage. Another patient conceived naturally. A video paper on detailed surgical procedure of drug-free IVA has been published (46Tanaka YH A.J. Kawamura K. Surgical approaches of Drug-free IVA (in vitro activation) and LOI (laparoscopic ovarian incision) to treat patients with ovarian infertility.Fertil Steril. 2020; Abstract Full Text Full Text PDF Scopus (3) Google Scholar). A recent paper used a similar drug-free IVA approach to treat patients with diminished ovarian reserve (47Lunding S.A. Pors S.E. Kristensen S.G. Landersoe S.K. Jeppesen J.V. Flachs E.M. et al.Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve.Hum Reprod. 2019; 34: 1924-1936Crossref PubMed Scopus (17) Google Scholar). Although pregnancies were achieved in 12 of 20 patients, the investigators stated that “the current study does not indicate that biopsying, fragmenting and autotransplanting of ovarian cortical tissue increase the number of recruitable follicles for IVF/ICSI after 10 weeks.” This paper was accompanied by a commentary: “Evidence That Biopsying, Fragmentation and Auto-Transplantation of Ovarian Tissue Should Be Abandoned as a Treatment of Diminished Ovarian Reserve” (48Steiner A.Z. Evidence that biopsying, fragmentation and auto-transplantation of ovarian tissue should be abandoned as a treatment of diminished ovarian reserve.Hum Reprod. 2019; 34: 1853-1854Crossref PubMed Scopus (2) Google Scholar). However, several issues remained to be addressed. First, follicle growth was monitored based on serum antimüllerian hormone (AMH) levels and ultrasound-based antral follicle counts (AFCs) for only 10 weeks. If grafts contained early but not late secondary follicles, longer durations are needed before AFC can increase. Second,because seven of 20 patients showed increases in serum AMH levels accompanied by increases in AFC, these findings suggested efficacy of the cortical fragmentation approach. However, both parameters might not be sensitive enough to detect secondary follicle growth during the short (10 weeks) monitoring. Indeed, our earlier work (41Zhai J. Yao G. Dong F. Bu Z. Cheng Y. Sato Y. et al.In vitro activation of follicles and fresh tissue auto-transplantation in primary ovarian insufficiency patients.J Clin Endocrinol Metab. 2016; 101: 4405-4412Crossref PubMed Scopus (97) Google Scholar) showed that serum AMH levels did not increase in four out of five patients with POI responding to IVA treatment showing preovulatory follicle growth. Serum AMH levels represent “leakage” of an ovarian paracrine factor into the general circulation and might not show increases when a few follicles start growth. Third, ovarian biopsies were performed randomly from either side of the ovary but always grafted back to the right side. Because the grafts were put under the peritoneal serosa underneath the right ovary, it is difficult to distinguish follicle growth between grafts and biopsied ovaries, making ultrasound monitoring results unreliable. Thus, more extensive studies are needed to extend the drug-free IVA approach and investigate the types of patients suitable for this procedure. In patients with ROS, multiple antral follicles are present in the ovary and detectable under ultrasound monitoring (2Dewhurst C. Koos E.B. Ferreira H. The resistant ovary syndrome.Br J Obstet Gynecol. 1975; 82: 341-345Crossref PubMed Scopus (39) Google Scholar). Follicles in ovaries of these patients secrete low (but detectable) levels of estrogens, unlike the negligible serum estrogens found in traditional patients with POI. However, both patients with ROS and patients with POI exhibit menopausal syndromes and are unresponsive to exogenous FSH treatment. Because in vitro fragmentation of cortical tissues promoted follicle growth for patients with POI and patients with POR, we hypothesized that patients with ROS could respond to incision of ovarian cortex in vivo to disrupt Hippo signaling and performed LOI to promote follicle growth in these patients without removing tissues outside of the body (46Tanaka YH A.J. Kawamura K. Surgical approaches of Drug-free IVA (in vitro activation) and LOI (laparoscopic ovarian incision) to treat patients with ovarian infertility.Fertil Steril. 2020; Abstract Full Text Full Text PDF Scopus (3) Google Scholar). Of interest, our data showed that LOI promoted follicle growth in seven of 11 patients with ROS, leading to retrieval of mature oocytes for infertility treatment. Thus, in vivo activation using LOI could also promote follicle growth as a potential therapeutic option. It is of interest that widely used laparoscopic ovarian drilling in vivo for patients with PCOS also promoted follicle growth and remodeled ovarian architecture (38Farquhar C. Lilford R.J. Marjoribanks J. Vandekerckhove P. Laparoscopic “drilling” by diathermy or laser for ovulation induction in anovulatory polycystic ovary syndrome.Cochrane Database Syst Rev. 2007; CD001122PubMed Google Scholar). Thus, Hippo signaling disruption using mechanical procedures could serve as the basis for treating different types of ovarian infertility. As shown in Figure 2, IVA was used to treat patients with POI with prolonged menopause, whereas drug-free IVA based on Hippo signaling disruption alone was successful in treating patients with POI with recent menses cessation and patients with POR with diminished ovarian reserve. In addition, in vivo incision was shown to be effective for patients with ROS. Furthermore, wedge resection and laparoscopic ovarian drilling are well established and effective procedures for treating patients with PCOS. The success of these ovary-damaging procedures as infertility therapies is counterintuitive to traditional thinking of organ damage but is similar to monthly ovarian remodeling. Except for neoangiogenesis associated with tumorigenesis, the ovary and uterus are the only two organs in adult mammals undergoing cyclic remodeling and neoangiogenesis. Ovaries undergo monthly cyclic changes associated with follicle growth and new blood vessel formation. This is followed by rupture of one preovulatory follicle to release the mature oocyte and subsequent tissue repair and organ remodeling. Our ovarian imaging data showed that follicles resemble fruits hanging in a tree with blood vessels as tree branches providing nutrients and FSH (49Feng Y. Cui P. Lu X. Hsueh B. Billig F.M. Yanez L.Z. et al.CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.Sci Rep. 2017; 7: 44810Crossref PubMed Scopus (47) Google Scholar). One can envision the human ovary as an organ with inner medulla and branching blood vessels and in vitro ovarian cortical fragmentation or in vivo incision altering the mechanical stress of primordial/primary and secondary follicles located in the cortical layer to disrupt Hippo signaling and promote follicle growth. Ovary-damaging procedures (fragmentation, incision, drilling, and wedge resection) induced biochemical changes (increases in CCN growth factors and apoptosis inhibitors) to promote follicle growth (Fig. 1) in patients with diverse ovarian infertility (Fig. 2), leading to natural conception or successful retrieval of mature oocytes for IVF and pregnancies. Although cutting of human cortical strips into cubes in vitro led to increased expression of several CCN growth factors (13Kawamura K. Cheng Y. Suzuki N. Deguchi M. Sato Y. Takae S. et al.Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.Proc Natl Acad Sci U S A. 2013; 110: 17474-17479Crossref PubMed Scopus (413) Google Scholar), future in vivo monitoring of ovarian secretion of CCN growth factors in treated patients is of interest. A recent study investigated deposition and remodeling of mechanical matrisome components (collagen, elastin, elastin microfibril interface-located protein 1 [EMILIN-1], fibrillin-1, and glycosaminoglycans [GAGs]) and found the localization of primordial follicles in the collagen-rich ovarian cortex, conferring a rigid physical environment that supports follicle architecture and limits follicle expansion (50Ouni E. Bouzin C. Dolmans M. Marbaix E. Pyr dit Ruys S. Vertommen D. et al.Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.Hum Reprod. 2020; 35: 1391-1410Crossref PubMed Scopus (8) Google Scholar). Future studies on the physical environment surrounding follicles in diseased ovaries are important for designing more refined treatments. For a better diagnosis of patients, recent advances in in vivo imaging tools could allow monitoring of primary to secondary follicles using near-infrared probes conjugated to FSH (51Feng Y. Zhu S.J. Antaris A.L. Chen H. Jiang L.L. Wang Y. et al.Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore.Chem Sci. 2017; 8: 3703-3711Crossref PubMed Google Scholar). Similar approaches to conjugate the Kit ligand with receptors in primordial follicles (52Zhang H. Risal S. Gorre N. Busayavalasa K. Li X. Shen Y. et al.Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.Curr Biol. 2014; 24: 2501-2508Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) could also be developed. With future adaptation of intravaginal near-infrared cameras and improvement of IVA and LOI procedures, it would be possible to select patients with POI and patients with POR with residual preantral follicles for IVA or LOI. Because rodent studies demonstrated the ability of phospholipids such as S1P to promote YAP nuclear translocation and follicle growth (15Cheng Y. Feng Y. Jansson L. Sato Y. Deguchi M. Kawamura K. et al.Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP.FASEB J. 2015; 29: 2423-2430Crossref PubMed Scopus (56) Google Scholar), future studies could also evaluate the efficacy of intraovarian injection of Hippo signaling disruption drugs to promote follicle growth. It is important to note the present Hippo signaling disruption approaches increase the number of preovulatory follicles and mature oocytes in patients, but there is no evidence the quality of mature oocytes is improved. Because occurrence of aneuploidy/poor quality of mature oocytes is presumably a random event, an increased number of mature oocytes allowed for the success of the above-mentioned procedures. Because recent single-cell transcriptomic analyses of aging monkey oocytes and granulosa cells underscored the importance of oxidative damage as the basis of ovarian senescence (53Wang S. Zheng Y. Li J. Yu Y. Zhang W. Song M. et al.Single-cell transcriptomic atlas of primate ovarian aging.Cell. 2020; 180: P585-P600Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), future approaches to improve oocyte quality by boosting ovarian mitochondrial functions could improve oocyte quality (54Bertoldo M.J. Listijono D.R. Ho W.J. Riepsamen A.H. Goss D.M. Richani D. et al.NAD(+) repletion rescues female fertility during reproductive aging.Cell Rep. 2020; 30: 1670-1681.e7Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 55Yang Q. Cong L. Wang Y. Luo X. Li H. Wang H. et al.Increasing ovarian NAD+ levels improve mitochondrial functions and reverse ovarian aging.Free Rad Biol Med. 2020; 156: 1-10Crossref PubMed Scopus (11) Google Scholar) and treatment outcomes. The authors thank Yanjie Guo for graphic assistance.