title: Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.
abstract: OBJECTIVE
  Is ovarian tissue cryopreservation using vitrification followed by in vitro activation (IVA) of dormant follicles a potential approach for infertility treatment of patients with primary ovarian insufficiency (POI)?
  
  
  CONCLUSIONS
  Our vitrification approach followed by IVA treatment is a potential infertility therapy for POI patients whose ovaries contain residual follicles.
  
  
  BACKGROUND
  Akt (protein kinase B) stimulators [PTEN (phosphatase with TENsin homology deleted in chromosome 10) inhibitor and phosphatidyinositol-3-kinase (PI3 kinase) stimulator] activate dormant primordial follicles in vitro and ovarian fragmentation disrupts the Hippo signaling pathway, leading to the promotion of follicle growth. We treated POI patients with a combination of ovarian vitrification, fragmentation and drug treatment, followed by auto-transplantation, and reported successful follicle growth and pregnancies.
  
  
  METHODS
  Prospective clinical study of 37 infertile women with POI between 12 August 2011 and 1 November 2013. We enrolled 10 new patients since the previous publication.
  
  
  METHODS
  POI patients were originally selected based on a history of amenorrhea for more than 1 year and elevated serum FSH levels of >40 mIU/ml (n = 31) but this was later changed to >4 months, age <40 years and serum FSH levels of >35 mIU/ml (n = 6) (mean 71.8 ± 30.8, range 35.5-197.6) so as to include patients with a shorter duration of amenorrhea. Under laparoscopic surgery, ovariectomy was performed and ovarian cortices were dissected into strips for vitrification. Some pieces were examined histologically. After warming, two to three strips were fragmented into smaller cubes before culturing with Akt stimulators for 2 days. After washing, ovarian cubes were transplanted beneath the serosa of Fallopian tubes under laparoscopic surgery. Follicle growth was monitored by ultrasound and serum estrogen levels. After oocyte retrieval from mature follicles, IVF was performed.
  
  
  RESULTS
  Among 37 patients, 54% had residual follicles based on histology. Among patients with follicles, 9 out of 20 showed follicle growth in auto-grafts with 24 oocytes retrieved from six patients. Following IVF and embryo transfer into four patients, three pregnancies were detected based on serum hCG, followed by one miscarriage and two successful deliveries. For predicting IVA success, we found that routine histological analyses of ovarian cortices and shorter duration from initial POI diagnosis to ovariectomy are valid parameters.
  
  
  CONCLUSIONS
  Although our findings suggest that the present vitrification protocol is effective for ovarian tissue cryopreservation, we have not compared the potential of vitrification and slow freezing in follicle growth after grafting. We chose the serosa of Fallopian tubes as the auto-grating site due to its high vascularity and the ease to monitor follicle growth. Future studies are needed to evaluate the best auto-grafting sites for ovarian tissues. Also, future studies are needed to identify biological markers to indicate the presence of residual follicles in POI to predict IVA treatment outcome.
  
  
  CONCLUSIONS
  In POI patients, ovarian reserve, namely the pool of residual follicles, continues to diminish with age. If one ovary is cryopreserved at an earlier stage of POI, patients could undergo additional non-invasive infertility treatments before the final decision for the IVA treatment. Furthermore, in the cases of unmarried POI patients, cryopreservation of ovarian tissues allows their fertility preservation until they desire to bear children.
  
  
  BACKGROUND
  This work was supported by Grant-In-Aid for Scientific Research (Research B: 24390376, Challenging Exploratory Research: 24659722, and Innovative Areas, Mechanisms regulating gamete formation in animals: 26114510) and by research funds from the Smoking Research Foundation, and the Takeda Science Foundation. None of the authors has a conflict of interest.
  
  
  BACKGROUND
  UMIN000010828.
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan.
authors.forename: Nao
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan.
authors.forename: Nobuhito
authors.initials: N
authors.lastname: Yoshioka
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan.
authors.forename: Seido
authors.initials: S
authors.lastname: Takae
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan.
authors.forename: Yodo
authors.initials: Y
authors.lastname: Sugishita
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Tamura
authors.affiliation: IVF Namba Clinic, Osaka, Osaka 550-0015, Japan.
authors.forename: Shu
authors.initials: S
authors.lastname: Hashimoto
authors.affiliation: IVF Namba Clinic, Osaka, Osaka 550-0015, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Morimoto
authors.affiliation: Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan kazuhironanami@gmail.com.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kawamura
mesh_terms: D000328:Adult; D015925:Cryopreservation / Q000379:methods; D005260:Female; D059247:Fertility Preservation / Q000379:methods*; D006801:Humans; D007247:Infertility, Female / Q000628:therapy*; D006080:Ovarian Follicle / Q000254:growth & development / Q000637:transplantation; D010053:Ovary / Q000502:physiology / Q000637:transplantation*; D011247:Pregnancy; D011256:Pregnancy Outcome; D016649:Primary Ovarian Insufficiency / Q000628:therapy*; D011446:Prospective Studies; D058989:Vitrification
keywords: Akt stimulation; in vitro activation; infertility treatment; ovarian tissue vitrification; primary ovarian insufficiency