Advertisement

Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve

Open AccessPublished:September 19, 2019DOI:https://doi.org/10.1016/j.rbmo.2019.09.007

      Abstract

      Research question

      The recently developed in-vitro activation (IVA) approach provides a promising infertility treatment for patients with premature ovarian insufficiency. The IVA method promotes growth of residual ovarian follicles following ovarian tissue fragmentation leading to Hippo signalling disruption, together with in-vitro incubation with Akt stimulators. As poor ovarian response (POR) patients with decreased ovarian reserve (DOR) have multiple secondary follicles, this study tested whether Hippo signalling disruption alone using in-vitro ovarian cortical fragmentation, followed by autologous grafting, was sufficient to promote follicle growth.

      Design

      A case series study.

      Results

      In 9 out of 11 POR patients with DOR treated with a simplified IVA procedure, increases in antral follicle numbers in multiple growth waves were detected following FSH treatment. Subsequent injection with human chorionic gonadotrophin allowed retrieval of more mature oocytes for IVF (median antral follicle counts before and after IVA per ovarian stimulation: 1.0 versus 2.6) with 68.7% fertilization rates and 56.9% showing high-quality embryonic development. One natural conception and 16 embryo transfers in five patients resulted in one live birth, two ongoing pregnancies and one miscarriage. Three additional patients and the miscarriage patient have cryopreserved embryos for future transfer.

      Conclusions

      The present drug-free IVA approach may be suitable for POR patients with DOR, as it increased the number of antral follicles. The procedure also eliminated the need for 2-day incubation with drugs and required only one surgery. This approach could allow the retrieval of more oocytes in middle-aged women to achieve higher pregnancy rates and deserves proper evaluation in future randomized controlled trials.

      Keywords

      Introduction

      Although gonadotrophins are routinely used to stimulate folliculogenesis and generate mature oocytes in women seeking infertility treatment, some patients with poor ovarian response (POR) showed the growth of only a few antral follicles after treatment as compared with 20–30 antral follicles found in most patients (
      • Ferraretti A.P.
      • La Marca A.
      • Fauser B.C.
      • Tarlatzis B.
      • Nargund G.
      • Gianaroli L.
      ESHRE Working Group on Poor Ovarian Response Definition.
      ). Although middle-aged women with low ovarian reserve (
      • De Vos M.
      • Devroey P.
      • Fauser B.C.
      Primary ovarian insufficiency.
      ) showed poor responses to gonadotrophin treatments, some young patients also responded poorly, probably reflecting early transition into premature ovarian failure. Because POR patients (
      • Ferraretti A.P.
      • La Marca A.
      • Fauser B.C.
      • Tarlatzis B.
      • Nargund G.
      • Gianaroli L.
      ESHRE Working Group on Poor Ovarian Response Definition.
      ) or patients with diminished ovarian reserve (
      • Scott R.T.
      • Toner J.P.
      • Muasher S.J.
      • Oehninger S.
      • Robinson S.
      • Rosenwaks Z.
      Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome.
      ) showed low numbers of antral follicles after routine gonadotrophin treatment, further improvement in infertility treatments are needed.
      For patients with premature ovarian insufficiency (POI), who become amenorrhoeic before 40 years of age and suffer from ovarian infertility, egg donation has been the only option for having their own genetic offspring (
      • Huhtaniemi I.
      • Hovatta O.
      • La Marca A.
      • Livera G.
      • Monniaux D.
      • Persani L.
      • Heddar A.
      • Jarzabek K.
      • Laisk-Podar T.
      • Salumets A.
      Advances in the molecular pathophysiology, genetics, and treatment of primary ovarian insufficiency.
      ;
      • Jiao X.
      • Zhang H.
      • Ke H.
      • Zhang J.
      • Cheng L.
      • Liu Y.
      • Qin Y.
      • Chen Z.-J.
      Premature ovarian insufficiency: Phenotypic characterization within different etiologies.
      ). Several years ago, an in-vitro activation (IVA) procedure was reported, which involved fragmenting ovarian cortexes and incubating them for 2 days with follicle-activating (Akt-stimulating) drugs, before heterotopic grafting back into artificial pouches created beneath the serosa of fallopian tubes (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ;
      • Suzuki N.
      • Yoshioka N.
      • Takae S.
      • Sugishita Y.
      • Tamura M.
      • Hashimoto S.
      • Morimoto Y.
      • Kawamura K.
      Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.
      ;
      • Zhai J.
      • Yao G.
      • Dong F.
      • Bu Z.
      • Cheng Y.
      • Sato Y.
      • Hu L.
      • Zhang Y.
      • Wang J.
      • Dai S.
      • Li J.
      • Sun J.
      • Hsueh A.J.
      • Kawamura K.
      • Sun Y.
      In vitro activation of follicles and fresh tissue auto-transplantation in primary ovarian insufficiency patients.
      ). This IVA procedure promoted follicle growth and allowed the generation of mature oocytes for POI patients, leading to a number of pregnancies and deliveries. The initial IVA approach relies upon disruption of the ovarian Hippo signalling pathway (ovarian fragmentation) (
      • Hsueh A.J.
      • Kawamura K.
      • Cheng Y.
      • Fauser B.C.
      Intraovarian control of early folliculogenesis.
      ) as well as stimulation of the ovarian Akt signalling pathway (incubation with specific drugs) (
      • Li J.
      • Kawamura K.
      • Cheng Y.
      • Liu S.
      • Klein C.
      • Duan E.K.
      • Hsueh A.J.
      Activation of dormant ovarian follicles to generate mature eggs.
      ) to promote growth of primordial, primary and secondary follicles. Because Hippo signalling disruption alone is effective in promoting secondary follicle growth (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ) and POR patients have multiple secondary follicles, this study tested the possibility of promoting follicle growth in POR patients with DOR by using a simplified, drug-free IVA approach involving only one surgery.

      Materials and methods

       Patient characteristics

      POR patients were recruited with DOR based on the Bologna criteria (
      • Ferraretti A.P.
      • La Marca A.
      • Fauser B.C.
      • Tarlatzis B.
      • Nargund G.
      • Gianaroli L.
      ESHRE Working Group on Poor Ovarian Response Definition.
      ), showing the growth of few antral follicles following FSH treatment or with low ovarian reserve who failed to achieve pregnancy following three or IVF cycles. Their serum gonadotrophin and oestrogen levels were monitored, together with autoimmune antibodies and karyotypes. Informed consent was obtained from patients and the study was approved by the Biomedical Ethics Committee of the Rose Ladies Clinic (29 June 2017, reference number RLC-005). The present clinical trial was registered under number UMIN000029807 and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).

       Hormonal measurements

      Prior to IVA treatment, serum concentrations of FSH and oestradiol were ascertained based on fully automated measurements using Passfirst FSH and oestradiol assays (LSI Medicine, Tokyo, Japan). The measuring range of the analyses was 0.2–200 mIU/ml and 20–2000 pg/ml, respectively. Serum anti-Müllerian hormone (AMH) levels were measured using the Access AMH Assay (Beckman Coulter, Brea, CA, USA) with a detection limit of 0.02 ng/ml.

       Pre-IVA hormonal treatments

      Before IVA, patients received oestrogen replacement therapy to maintain serum oestradiol between 40 and 90 pg/ml, in order to suppress circulating LH and FSH levels, followed by 10–14 days of oestrogen plus progesterone. Serum LH and FSH concentrations were monitored to make sure that LH concentrations were less than 10 mIU/ml before surgery. This oestrogen–progesterone treatment was discontinued before autologous grafting to induce withdrawal bleeding soon after the surgery. Oestrogen replacement continued throughout IVA treatment, including ovarian stimulation using recombinant FSH or purified urinary FSH, as described below.

       Drug-free IVA and autografting

      As shown in Figure 1a, the original IVA approach for infertility therapy in POI patients involved removing one entire ovary for the preparation of ovarian strips before vitrification. This was followed by thawing strips for fragmentation into small cubes and incubation with PI3K-stimulating drugs for 2 days (
      • Li J.
      • Kawamura K.
      • Cheng Y.
      • Liu S.
      • Klein C.
      • Duan E.K.
      • Hsueh A.J.
      Activation of dormant ovarian follicles to generate mature eggs.
      ) before heterologous grafting (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ). For POR patients with DOR, drug-free IVA was performed (Figure 1b). Part of the cortex from one or both ovaries or one whole ovary (in the case of patients with shrunken ovaries), was removed under general anaesthesia by laparoscopy. As POR patients have ovaries of medium size, only partial removal of cortical tissues from one or both ovaries was performed for most patients. In general, cortical fragments of the larger ovary were removed and immediately transferred to the adjacent laboratory in an incubator with a constant temperature of 37°C. For Hippo signalling disruption, ovarian cortex was dissected to remove residual medulla tissues before cutting into strips (10 × 10 mm, 1–2 mm thick). After dissecting a small part from all strips for histology (5 × 1 × 1 mm3 per strip) to determine the presence of residual follicles, tissue strips were then cut into small cubes (1 × 1 × 1 mm3) before returning to the contralateral ovary by making a tunnel between cortex and medulla from an incision of the cortex. If the originally dissected cortex was large, some cubes were also inserted back into the remaining ovary. The orthotropic grafting procedure was similar to earlier reports (
      • Donnez J.
      • Dolmans M.-M.
      • Demylle D.
      • Jadoul P.
      • Pirard C.
      • Squifflet J.
      • Martinez-Madrid B.
      • Van Langendonckt A.
      Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.
      ;
      • Meirow D.
      • Levron J.
      • Eldar-Geva T.
      • Hardan I.
      • Fridman E.
      • Zalel Y.
      • Schiff E.
      • Dor J.
      Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.
      ;
      • Schmidt K.L.
      • Andersen C.Y..
      • Loft A.
      • Byskov A.G.
      • Emst E.
      • Andersen A.N.
      Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation.
      ;
      • Silber S.J.
      • Lenahan K.M.
      • Levine D.J.
      • Pineda J.A.
      • Gorman K.S.
      • Friez M.J.
      • Crawford E.C.
      • Gosden R.G.
      Ovarian transplantation between monozygotic twins discordant for premature ovarian failure.
      ). Depending on the availability of additional cubes and surgery progress, some cubes were also grafted beneath the serosa of both fallopian tubes, similar to the original IVA approach (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ). The grafting procedure was further improved by using an applicator (Kitazato Corp., Shizuoka, Japan) to transplant multiple ovarian tissue cubes. Some ovarian strips were cryopreserved for future use using a vitrification method (
      • Suzuki N.
      • Yoshioka N.
      • Takae S.
      • Sugishita Y.
      • Tamura M.
      • Hashimoto S.
      • Morimoto Y.
      • Kawamura K.
      Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.
      ).
      Figure 1
      Figure 1Comparison of original and drug-free IVA approaches. (a) The original IVA approach was applied to POI patients with long duration of amenorrhoea and involved removing one ovary under laparoscopic surgery with or without cryopreservation of cortical strips. Cortical strips were fragmented into small cubes to disrupt ovarian Hippo signalling, followed by incubation for 2 days with Akt-stimulating drugs. Ovarian cubes were autografted back under a second surgery. Patients with follicle growth were then treated with IVF–embryo transfer. (b) For the drug-free IVA based on Hippo signalling disruption alone, partial ovarian cortical removal was performed in POR patients with DOR. Cortical fragments were immediately cut into cubes and autografted back to patients during the same laparoscopic surgery. The grafts were returned to the contralateral ovary or both ovaries, but could also be placed beneath serosa of Fallopian tubes. Patients either opted to attempt natural conception or were treated with artificial insemination with husband’s spermatozoa or IVF–embryo transfer. DOR = decreased ovarian reserve; IVA = in-vitro activation; POR = poor ovarian response.

       Post-grafting hormonal treatments

      After surgery, patients took oestrogen plus progesterone for 10–14 days to initiate withdrawal bleeding. After confirmation of adequate serum LH levels (<10 mIU/ml), nasal spray of a gonadotrophin-releasing hormone (GnRH) agonist (buserelin acetate; Mochida Pharma, Tokyo, Japan) was used to maintain serum LH concentrations, together with daily injections with recombinant FSH or purified urinary FSH (225–300 IU) based on basal concentrations of FSH to maintain elevated serum FSH (>25–30 mIU/ml). The use of nasal spray ensured serum LH concentrations were easily controlled so that they were not too low during FSH injections. Serum LH concentrations were maintained at 1–9 mIU/ml by adjusting the amount of daily GnRH agonists. FSH doses were higher than those used for regular IVF stimulation protocols due to poorer vasculature at the graft site. Serum oestradiol, FSH and LH measurement, as well as ultrasound monitoring for all patients, continued for 1 year.

       Monitoring of follicle growth and oocyte retrieval

      During weekly monitoring of follicles under transvaginal ultrasound and serum oestradiol concentrations following ovarian stimulation, some POR patients showed small antral follicles whereas other patients showed no follicles at the initiation of ovarian stimulation. For patients with follicles, daily FSH treatments continued for 10–14 days until follicles reached 14–18 mm in diameter. In patients without detectable follicles, FSH-stimulated elevation of serum oestradiol levels showed variable delays. The first sign of follicle growth was elevation of serum oestradiol and small antral follicles usually became detectable when serum oestradiol levels reached >50 pg/ml (after subtraction of basal concentrations resulting from exogenous oestrogens). When growing follicles reached 14–18 mm in diameter, patients were injected with 10,000–20,000 IU HCG to induce oocyte maturation before oocyte retrieval. Doses of HCG used were higher than regular IVF stimulation protocols due to poor vasculature of grafts. When no increases in serum oestradiol levels were detected after 3 weeks of GnRH agonist and FSH treatments, hormonal therapies were discontinued. Patients were then treated for 10–14 days with oestrogen plus progesterone to induce withdrawal bleeding. After bleeding, the ovarian stimulation protocol using GnRH agonist and FSH was repeated. In some cases, ovarian stimulation was performed immediately after oocyte retrieval if patients had small antral follicles, similar to the luteal phase ovarian stimulation reported earlier (
      • Kuang Y.
      • Hong Q.
      • Chen Q.
      • Lyu Q.
      • Ai A.
      • Fu Y.
      • Shoham Z.
      Luteal-phase ovarian stimulation is feasible for producing competent oocytes in women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment, with optimal pregnancy outcomes in frozen-thawed embryo transfer cycles.
      ). Thus, in some cycles, follicle growth was evident more than once per month. Waves of follicular growth could be observed up to 1 year after autografting. After detection of pre-ovulatory follicles and HCG stimulation, attempting natural conception, artificial insemination by husband’s spermatozoa or oocyte retrieval followed by IVF were selected by individual patients after consultation with doctors. This was dependent upon number of retrieved mature oocytes, the patient’s age, as well as personal or financial situations.

       Oocyte retrieval, IVF and pregnancy

      When oocyte retrieval was performed and routine IVF was successful, all embryos at cleavage stage (D2 or D3 of culture) based on the Gardner or Veeck criteria (
      • Gardner D.K.
      • Lane M.
      • Stevens J.
      • Schlenker T.
      • Schoolcraft W.B.
      Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer.
      ;
      • Veeck L.L.
      ) were cryopreserved using a vitrification kit (Kitazato Corp., Shizuoka, Japan). Number of embryos obtained was determined based on anticipated effective duration (1 year) of IVA treatment (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ) and the known decreases in egg quality in older patients (
      • Navot D.
      • Bergh R.
      • Williams M.A.
      • Garrisi G.J.
      • Guzman I.
      • Sandler B.
      • Grunfeld L.
      Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility.
      ). Culturing embryos to blastocyst stage to select high-quality embryos was not recommended for POR patients, because only limited numbers of embryos could be obtained and prolonged culture does not improve embryo quality in vitro. For embryo transfer, one or two vitrified embryos were warmed and transferred when uterine conditions were optimal under hormone replacement cycles using oestrogen followed by progesterone treatment. After embryo transfer, patients received luteal support using vaginal tablets (Lutinus; Ferring Pharma, Tokyo, Japan). Pregnancy was determined by measuring the level of serum HCG-beta at 2 weeks after embryo transfer.

       Histological analyses and follicle counting

      Using a uniform volume of specimens (5 × 1 × 1 mm3 per strip) from ovarian cortical strips, histological analyses were performed to detect residual follicles in POR ovaries, as described previously (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ). After serial section of tissues at 5 µm, followed by staining with haematoxylin and eosin, numbers of primordial, primary, secondary and degenerated follicles were counted and expressed as total number per patient.

      Results

       Patient characteristics

      Eleven POR patients ranging in age from 30 to 45 years (median 34), all showing oligomenorrhoea (Table 1). After routine gonadotrophin treatment, their median antral follicle count (AFC) was 1, ranging from 0 to 4, consistent with the Bologna criteria (
      • Ferraretti A.P.
      • La Marca A.
      • Fauser B.C.
      • Tarlatzis B.
      • Nargund G.
      • Gianaroli L.
      ESHRE Working Group on Poor Ovarian Response Definition.
      ). In two separate tests at least 8 weeks apart, median serum oestradiol levels were 0 pg/ml (range 0–31), whereas median serum FSH levels were 47.3 mIU/ml (range 13.4–84.9) in the early follicular phase of the menstrual cycle, consistent with the DOR criteria (
      • Levi A.J.
      • Raynault M.F.
      • Bergh P.A.
      • Drews M.R.
      • Miller B.T.
      • Scott Jr, R.T.
      Reproductive outcome in patients with diminished ovarian reserve.
      ;
      • Scott R.T.
      • Toner J.P.
      • Muasher S.J.
      • Oehninger S.
      • Robinson S.
      • Rosenwaks Z.
      Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome.
      ). Median serum AMH level was 0.04 ng/ml (range 0–0.8) with four patients showing non-detectable levels. Autoimmune antibody values were abnormally elevated in three out of eight patients, although data from three patients were not available. In addition, no patients had iatrogenic causes for infertility whereas karyotype abnormalities were not available in seven patients.
      Table 1Baseline characteristics of 11 POR patients with DOR who underwent drug-free IVA treatment
      Age at surgery (years)34 (30–45)
      Oligomenorrhoea11
      Serum FSH levels (mIU/ml)47.3 (13.4–84.9)
      Serum oestradiol levels (pg/ml)0 (0–31)
      Serum AMH levels (ng/ml)0.04 (0–0.8)
      Non-detectable in four patients.
      AFC1 (0–4)
      Autoimmune antibody3
      The data were not available in three patients.
      Iatrogenic0
      Karyotype abnormality0
      The data were not available in four patients.
      Values reported as median (range) or number. FSH and oestradiol levels were calculated using a mean value of two measurements.
      AFC = antral follicle count; AMH = anti-Müllerian hormone; DOR = decreased ovarian reserve; IVA = in-vitro activation; POR = poor ovarian response.
      a Non-detectable in four patients.
      b The data were not available in three patients.
      c The data were not available in four patients.
      Follicle counting using tissue samples prepared from dissected ovarian cortical strips as described below, indicated presence or absence of follicles at different stages (Table 2). Although Patients 4 and 9 showed no histological signs of follicles and Patient 11 showed only degenerate follicles using limited samples following partial cortical removal, antral follicles were detected under ultrasound for all patients. Of interest, histological counting showed the presence of multiple follicles in Patient 2. This patient also showed around eight antral follicles per cycle.
      Table 2IVA surgery and histology result in each patient
      Surgery type (whole or partial)Number of samples used for histologyHistology (n of each stage follicle in total samples)
      Patient 1Partial (Lt)2Pd (0), Pi (1), Sec (3), Deg (2)
      Patient 2Partial (both)2Many follicles
      Patient 3Partial (both)2Pd (2), Pi (2), Sec (0), Deg (9)
      Patient 4Partial (both)2None
      Patient 5Whole (Lt)6Pd (0), Pi (1), Sec (0), Deg (5)
      Patient 6Partial (both)1Pd (1), Pi (0), Sec (0), Deg (0)
      Patient 7Whole (Rt)7Pd (6), Pi (2), Sec (4), Deg (4)
      Patient 8Whole (Rt)5Pd (8), Pi (9), Sec (1), Deg (14)
      Patient 9Partial (Lt)1None
      Patient 10Whole (Rt)9Pd (4), Pi (1), Sec (0), Deg (18)
      Patient 11Partial (Lt)3Pd (0), Pi (0), Sec (0), Deg (3)
      Surgery: Whole = one whole ovariectomy; Partial = partial removal of ovarian cortex.
      Histology: Pd = primordial follicle; Pi = primary follicle; Sec = secondary follicle; Deg = degenerated follicle.

       Drug-free IVA and orthologous grafting: Hippo disruption only

      Figure 2 shows representative photographs of different steps of the drug-free IVA surgery. Ovarian cortex was removed from an ovary of a patient (Figure 2a). After in-vitro fragmentation, cortical cubes were grafted back to the same ovary using an applicator to facilitate bulk transfer of 15–20 cubes (Figure 2b, c). After grafting, the wound and incision were covered by an absorbable adhesion to avoid adhesion of ovary to surrounding tissues and to prevent cube loss from grafted sites (Figure 2d).
      Figure 2
      Figure 2Images of IVA laparoscopic surgery showing (a) removal of ovarian cortex from a POR ovary, (b and c) grafting of cortical cubes into a tunnel between cortex and medulla from an incision of the cortex using an applicator, and (d) coverage of incision site using an absorbable adhesion barrier to avoid adhesion of ovary with surrounding tissues and cube loss from grafted sites.

       Hormonal stimulation, follicle growth, oocyte retrieval and IVF–embryo transfer

      As depicted in Figure 3, the top left corner for each patient indicates AFC before drug-free IVA on two or more occasions whereas numbers in red ovals indicate increases in AFC after IVA for each stimulated or spontaneous cycle. Among 11 patients, all but Patients 3 and 5 responded to the IVA treatment by showing increases in antral follicle numbers per cycle (median values before and after IVA: 1.0 versus 2.6). Although follicle growth was found under exogenous FSH treatment, follicle growth was also detected even without this treatment (Figure 3, grey ovals). In Patients 6 and 7, ovarian stimulation was performed immediately after oocyte retrieval. Thus, in some cycles, follicle growth was evident more than once per month. When numbers of antral follicles per cycle after IVA was compared with those before the surgery, all but Patients 3 and 5 showed increases in AFC (Table 3).
      Figure 3
      Figure 3Follicle growth and oocyte retrieval in 11 POR patients with DOR after drug-free IVA treatment. Follicle growth dynamics were monitored for 1 year at different durations after drug-free IVA. Numbers in the top left-hand side boxes represent mean follicle count per cycle after gonadotrophin stimulation before the IVA surgery. Ovals with a number indicate antral follicle count (AFC) after IVA. Those showing increases in follicle numbers as compared with counts before IVA are indicated in red ovals. Blue ovals with a number indicate AFC per cycle after IVA without gonadotrophin stimulation. Right upward arrows indicate follicle growth. Pt = patient.
      Table 3Changes in number of growing follicles and retrieved oocytes before and after drug-free IVA
      Patient age (years)AFC per cycle before IVA (mean ± SD)No. of cyclesAFC per cycle after IVA (mean ± SD)No. of cyclesNumber of retrieved oocytes per cycle before IVA (mean ± SD)No. of cyclesNumber of retrieved oocytes per cycle after IVA (mean ± SD)No. of cyclesAccumulated oocyte number retrieved after IVAClinical outcome
      Patient 1382.3 ± 1.533.6 ± 1.671.0 ± 013.6 ± 1.1518
      Patient 2301.3 ± 0.436.2 ± 3.05015.3 ± 2.1421NC
      Patient 3381.0 ± 031.0 ± 041.0 ± 011.0 ± 033CE
      Patient 4340.9 ± 0.973.5 ± 2.1100.8 ± 0.952.8 ± 2.0925PM, CE
      Patient 5431.0 ± 041.0 ± 031.0 ± 011.0 ± 033
      Patient 6341.0 ± 021.7 ± 0.7101.0 ± 021.1 ± 0.3910P
      Patient 7452.7 ± 1.534.2 ± 1.7132.5 ± 0.723.0 ± 1.41339
      Patient 8451.0 ± 022.6 ± 0.78NANA1.4 ± 0.5710
      Patient 9301.0 ± 021.1 ± 0.47NANA1.0 ± 055CE
      Patient 10301.3 ± 0.542.9 ± 1.391.0 ± 012.3 ± 1.049P
      Patient 11311.0 ± 0.751.5 ± 0.581.5 ± 0.721.1 ± 0.478CE
      As oocyte retrieval was performed for every follicle cycle or growth wave, number of oocytes retrieved per cycle was calculated for comparison before and after IVA.
      AFC = antral follicle count; CE = cryopreserved embryos; NA = not applicable; NC = naturally conceived; P = pregnancy via IVF; PM = pregnancy via IVF followed by miscarriage.
      A recent paper used a procedure similar to our drug-free IVA approach to treat patients with diminished ovarian reserve (DOR) (Lunding et al. Hum Reprod 2019). Although pregnancies were achieved in 12 out of 20 patients, it was concluded 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”. Because the authors monitored follicle growth based on serum AMH levels and ultrasound-based antral follicle counts (AFC) for only 10 weeks, it is likely that the duration of monitoring was too short. If grafts contain early but not late secondary follicles, longer durations are needed before AFC could increase.
      Although 7 to 20 patients showed increases in serum AMH levels, our earlier work (
      • Zhai J.
      • Yao G.
      • Dong F.
      • Bu Z.
      • Cheng Y.
      • Sato Y.
      • Hu L.
      • Zhang Y.
      • Wang J.
      • Dai S.
      • Li J.
      • Sun J.
      • Hsueh A.J.
      • Kawamura K.
      • Sun Y.
      In vitro activation of follicles and fresh tissue auto-transplantation in primary ovarian insufficiency patients.
      ) (Figure 3) showed that serum AMH levels did not increase in 4 out of 5 patients responding to IVA treatment with 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 started growth. Also, biopsies were performed randomly from either side of the ovary and 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.
      Mature oocytes were obtained from all patients after oocyte retrieval. During different cycles, patients might decide to attempt to conceive naturally or undergo artificial insemination using husband’s spermatozoa (AIH) instead of oocyte retrieval. Therefore, the number of retrieved oocytes per oocyte retrieval cycle in patients before and after IVA treatment were compared. Cycles of natural conception and AIH were not included. As shown in Table 3, all but three patients (Patients 3, 5 and 11) showed an increased number of retrieved oocytes per cycle. As multiple cycles or follicle growth waves were found after IVA treatment, patients have 3 to 39 accumulated oocytes after IVA treatment (Table 3). Indeed, more than 10 oocytes were retrieved from 6 out of 11 patients during 1 year of monitoring. Five patients had a total of 16 embryo transfers.
      For all oocytes retrieved, a 68.7% successful fertilization rate was achieved with 56.9% of oocytes developing into high-quality embryos (Table 4). High-quality embryos were obtained from all patients except Patients 1 and 5. After IVF–embryo transfer, three patients became pregnant, with one pregnancy ending in miscarriage, while another patient conceived naturally (Table 3). Presently, one healthy baby was delivered after IVF and two pregnancies (one spontaneous and one IVF) are progressing (6 and 9 months of gestation). The hormonal data and AFC (before surgery) of three patients with delivery/ongoing pregnancy were as follows: Patient 2, FSH 13.4 mIU/ml, AFC 1.3, AMH 0.8 ng/ml; Patient 6, FSH 26.9 mIU/ml, AFC 1.0, AMH 0.03 ng/ml; Patient 10, FSH 62.9 mIU/ml, AFC 1.3, AMH 0 (undetectable) ng/ml. In addition, three additional patients and the miscarriage patient have cryopreserved embryos ready for transfer (Tables 3 and 4). When focusing on three patients above 40 years of age, Patients 7 and 8 (45 years of age) accumulated 39 and 10 oocytes but only a total of three oocytes were retrieved from Patient 5 (44 years of age) (Table 3). Although Patients 7 and 8 had five and two high-quality embryos, respectively, not all embryo transfers were successful. Also, although all three retrieved oocytes for Patient 5 were fertilized, they did not develop into high-quality embryos.
      Table 4Clinical outcome of IVF–embryo transfer in 11 patients after drug-free IVA
      Fertilization rate
      2PN embryos/mature MII oocytes.
      (%)
      68.7 ± 26.6
      High-quality embryo rate
      Embryos with Grade 1–3 by Veeck criteria and Grade 3–5AA, AB, BA by Gardner criteria/2PN embryos.
      (%)
      56.9 ± 33.8
      Number of patients with pregnancy4
      Number of patients with miscarriage1
      Number of patients with cryopreserved embryos ready for embryo transfer4
      Values reported as mean ± SD or number.
      2PN = two-pronuclear; IVA = in-vitro activation; MII = metaphase II.
      a 2PN embryos/mature MII oocytes.
      b Embryos with Grade 1–3 by Veeck criteria and Grade 3–5AA, AB, BA by Gardner criteria/2PN embryos.

      Discussion

      The present data showed that follicles from ovaries of POR patients with DOR could also be activated to grow following drug-free IVA involving cortical fragmentation and autografting. All these damaging procedures probably involve the disruption of ovarian Hippo signalling (
      • Hsueh A.J.
      • Kawamura K.
      • Cheng Y.
      • Fauser B.C.
      Intraovarian control of early folliculogenesis.
      ). As the step of 2-day Akt drug incubation was omitted during the present IVA procedure, ovarian cubes were grafted immediately after fragmentation, thus avoiding potential follicle loss during prolonged culture. Orthologous grafting probably allowed better follicle growth in the ‘ovarian niche’ whereas the immediate grafting avoided culture (2 days) loss of follicles of the original IVA protocol. Furthermore, only one instead of two laparoscopic surgeries was required, thus minimizing patient discomfort and costs. The surgery could also be performed in an outpatient facility because only short-term bed rest is required. As orthotopic grafting was performed, some patients could potentially become pregnant naturally without egg retrieval and IVF–embryo transfer.
      Most POR patients with DOR who underwent drug-free IVA showed follicle growth within several weeks, suggesting the presence of residual secondary follicles and their rapid growth into antral follicles. Consistent with previous findings in POI patients indicating serum AMH levels are not predictive of follicle growth after IVA (
      • Kawamura K.
      • Cheng Y.
      • Suzuki N.
      • Deguchi M.
      • Sato Y.
      • Takae S.
      • Ho C.H.
      • Kawamura N.
      • Tamura M.
      • Hashimoto S.
      • Sugishita Y.
      • Morimoto Y.
      • Hosoi Y.
      • Yoshioka N.
      • Ishizuka B.
      • Hsueh A.J.
      Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
      ;
      • Zhai J.
      • Yao G.
      • Dong F.
      • Bu Z.
      • Cheng Y.
      • Sato Y.
      • Hu L.
      • Zhang Y.
      • Wang J.
      • Dai S.
      • Li J.
      • Sun J.
      • Hsueh A.J.
      • Kawamura K.
      • Sun Y.
      In vitro activation of follicles and fresh tissue auto-transplantation in primary ovarian insufficiency patients.
      ), the present data indicated that POR patients with low or undetectable serum AMH levels also responded to IVA treatment, allowing the retrieval of multiple oocytes.
      Ages of POR patients studied here ranged from 30 to 45 years. Some of these patients could represent a condition of transient ovarian failure and so are referred to as POR patients with DOR. Among older patients, Patients 7 and 8 (45 years of age) accumulated 39 and 10 oocytes after IVA but only a total of three oocytes were retrieved from Patient 5 (43 years of age). Although the number of cases is limited in this study, it is important to note that the IVA approach increases number of mature oocytes retrieved but does not improve age-related decline in egg quality, especially increases in aneuploidy, in POR patients as reported earlier (
      • Kailasam C.
      • Keay S.
      • Wilson P.
      • Ford W.
      • Jenkins J.
      Defining poor ovarian response during ivf cycles, in women aged < 40 years, and its relationship with treatment outcome.
      ;
      • Ulug U.
      • Ben-Shlomo I.
      • Turan E.
      • Erden H.F.
      • Akman M.A.
      • Bahceci M.
      Conception rates following assisted reproduction in poor responder patients: A retrospective study in 300 consecutive cycles.
      ). Therefore, the IVA approach is more effective in younger POR patients or older women possessing euploid oocytes. As oocyte quality decline could be a random process, retrieval of a large number of mature oocytes after IVA in middle-aged patients may allow the possibility of successful pregnancy.
      Due to poor vasculature at graft sites, patients were injected with FSH and HCG at doses higher than regular IVF stimulation protocols. It was found to be important to maintain low levels of endogenous serum LH in patients to avoid premature luteinisation of follicles under FSH stimulation (
      • Bosch E.
      • Valencia I.
      • Escudero E.
      • Crespo J.
      • Simón C.
      • Remohí J.
      • Pellicer A.
      Premature luteinization during gonadotropin-releasing hormone antagonist cycles and its relationship with in vitro fertilization outcome.
      ). Although histological analyses and follicle counting were routinely performed using fragments of ovaries for all patients, follicle counting in patients with limited cortical tissues after partial ovariectomy might not reflect ovarian responses due to the difficulty associated with tissue availability.
      A recent case report described a 32-year-old POI patient who became amenorrhoeic at 30 years of age. Similar to the present drug-free IVA approach, the patient underwent IVA treatment without tissue culture, followed by ovarian tissue auto-transplantation into the contralateral ovary and peritoneal pockets near the ovary (
      • Fabregues F.
      • Ferreri J.
      • Calafell J.
      • Moreno V.
      • Borrás A.
      • Manau D.
      • Carmona F.
      Pregnancy after drug-free in vitro activation of follicles and fresh tissue autotransplantation in primary ovarian insufficiency patient: A case report and literature review.
      ). After surgery, GnRH agonist and HMG injections led to the growth of three pre-ovulatory follicles for oocyte retrieval. After IVF and embryo transfer, the patient became pregnant.
      Another recent case report also showed pregnancy in one POI patient following a drug-free IVA approach (
      • Mahajan N.
      • Kaur J.
      • Bhattacharya B.
      • Naidu P.
      • Gupta S.
      In vitro activation of ovary.
      ). Furthermore, a simple procedure for laparoscopic auto-transplantation of fragmented ovarian cortical tissue in DOR women has also been reported (
      • Lunding S.A.
      • Pors S.E.
      • Kristensen S.G.
      • Andersen C.Y.
      • Jeppesen J.V.
      • Macklon K.T.
      • Andersen A.N.
      • Pedersen A.T.
      Autotransplantation of fragmented ovarian cortical tissue: A laparoscopic demonstration.
      ). However, the clinical outcome of this ongoing trial remains to be published.
      Although the present approach of fragmenting ovarian cortical biopsies in vitro to disrupt ovarian Hippo signalling followed by orthotropic grafting showed promise as an infertility therapy for POR patients with DOR, the possibility of in-situ disruption of ovarian Hippo signalling to activate follicles in POI patients using ovarian biopsy and scratching was tested in a large trial (
      • Zhang X.
      • Han T.
      • Yan L.
      • Jiao X.
      • Qin Y.
      • Chen Z.-J.
      Resumption of ovarian function after ovarian biopsy/scratch in patients with premature ovarian insufficiency.
      ). Among 80 POI patients, 11 presented with ovarian function resumption spontaneously or following human menopausal gonadotrophin stimulation. After oocyte retrieval in 10 patients and IVF, two embryos were transferred to one patient and one healthy baby was born. It was concluded that ovarian biopsy and scratching could bring promising benefits for some women with POI upon future improvement in efficiency and practice criteria. Thus, the in-vitro cortical cutting approach used here appears to be more effective than the in-situ biopsy or scratching in disrupting ovarian Hippo signalling. Earlier studies have attempted to puncture (
      • Lin J.
      • Wang P.
      • Zhao J.
      • Xiao S.
      • Yu R.
      • Jin C.
      • Zhu R.
      Outcomes of in vitro fertilization cycles among patients with polycystic ovary syndrome following ovarian puncture for in vitro maturation.
      ;
      • Mio Y.
      • Toda T.
      • Tanikawa M.
      • Terado H.
      • Harada T.
      • Terakawa N.
      Transvaginal ultrasound-guided follicular aspiration in the management of anovulatory infertility associated with polycystic ovaries.
      ) or drill (
      • Ferraretti A.P.
      • Gianaroli L.
      • Magli M.C.
      • Iammarrone E.
      • Feliciani E.
      • Fortini D.
      Transvaginal ovarian drilling: A new surgical treatment for improving the clinical outcome of assisted reproductive technologies in patients with polycystic ovary syndrome.
      ;
      • Kandil M.
      • Rezk M.
      • Al-Halaby A.
      • Emarh M.
      • El-Nasr I.S.
      Impact of ultrasound-guided transvaginal ovarian needle drilling versus laparoscopic ovarian drilling on ovarian reserve and pregnancy rate in polycystic ovary syndrome: A randomized clinical trial.
      ) ovaries in PCOS patients in vivo without laparoscopic surgeries. However, these in-vivo ovary-damaging approaches showed variable degrees of success and the standardization of these approaches remains to be established.
      Ovarian cryopreservation followed by autologous transplantation for fertility preservation in cancer patients has shown promise but is still considered an experimental strategy (
      • Donnez J.
      • Dolmans M.-M.
      • Demylle D.
      • Jadoul P.
      • Pirard C.
      • Squifflet J.
      • Martinez-Madrid B.
      • Van Langendonckt A.
      Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.
      ;
      • Meirow D.
      • Levron J.
      • Eldar-Geva T.
      • Hardan I.
      • Fridman E.
      • Zalel Y.
      • Schiff E.
      • Dor J.
      Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.
      ;
      • Pacheco F.
      • Oktay K.
      Current success and efficiency of autologous ovarian transplantation: A meta-analysis.
      ). As these cases mainly involved grafting of large ovarian pieces, the approach could be improved by optimal cryopreservation of ovaries before gonadotoxic therapies. Ovarian cryopreservation could then be followed by fragmenting ovarian cortical tissues to disrupt Hippo signalling, before orthologous grafting to promote folliculogenesis.
      With recent understanding of the importance of ovarian Hippo signalling in the regulation of follicle growth, further improvement of Hippo signalling disruption approaches is anticipated. As specific drugs (e.g. sphingosine-1-phosphate) (
      • Cheng Y.
      • Feng Y.
      • Jansson L.
      • Sato Y.
      • Deguchi M.
      • Kawamura K.
      • Hsueh A.J.
      Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the hippo signaling effector yap.
      ) or physical damage (e.g. wedge resection, laser drilling, fragmentation) probably suppress ovarian Hippo signalling, local delivery of Hippo signalling-disrupting drugs or refined/standardized techniques for mechanical damage to the ovary could allow new in-vivo approaches for infertility treatments.
      As the present study involves a small group of patients, results should be interpreted with caution and further confirmation by others in a greater number of well-defined patients is awaited. Also, only randomized controlled trials comparing different procedures could conclusively demonstrate efficacy of ovarian cortex fragmentation in POR patients with DOR.

      Acknowledgements

      We thank Yorino Sato and Yuta Kawagoe for technical support in histology and IVF–embryo transfer, and Masataka Furuya for supporting IVA surgeries. We also thank Asami Onimaru and Nanami Kawamura for data collection.
      The present work was supported by Grant-in-Aid for Scientific Research B (19H03801) (to K.K.), Challenging Exploratory Research (18K19624), Japan Agency for Medical Research and Development, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Takeda Science Foundation, and Naito Foundation (to K.K.).

      References

        • Bosch E.
        • Valencia I.
        • Escudero E.
        • Crespo J.
        • Simón C.
        • Remohí J.
        • Pellicer A.
        Premature luteinization during gonadotropin-releasing hormone antagonist cycles and its relationship with in vitro fertilization outcome.
        Fertility and sterility. 2003; 80: 1444-1449
        • Cheng Y.
        • Feng Y.
        • Jansson L.
        • Sato Y.
        • Deguchi M.
        • Kawamura K.
        • Hsueh A.J.
        Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the hippo signaling effector yap.
        FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2015; 29: 2423-2430
        • De Vos M.
        • Devroey P.
        • Fauser B.C.
        Primary ovarian insufficiency.
        The Lancet. 2010; 376: 911-921
        • Donnez J.
        • Dolmans M.-M.
        • Demylle D.
        • Jadoul P.
        • Pirard C.
        • Squifflet J.
        • Martinez-Madrid B.
        • Van Langendonckt A.
        Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.
        The Lancet. 2004; 364: 1405-1410
        • Fabregues F.
        • Ferreri J.
        • Calafell J.
        • Moreno V.
        • Borrás A.
        • Manau D.
        • Carmona F.
        Pregnancy after drug-free in vitro activation of follicles and fresh tissue autotransplantation in primary ovarian insufficiency patient: A case report and literature review.
        Journal of ovarian research. 2018; 11: 76
        • Ferraretti A.P.
        • La Marca A.
        • Fauser B.C.
        • Tarlatzis B.
        • Nargund G.
        • Gianaroli L.
        ESHRE Working Group on Poor Ovarian Response Definition.
        ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: the Bologna criteria. Human Reproduction. 2011; 26: 1616-1624
        • Ferraretti A.P.
        • Gianaroli L.
        • Magli M.C.
        • Iammarrone E.
        • Feliciani E.
        • Fortini D.
        Transvaginal ovarian drilling: A new surgical treatment for improving the clinical outcome of assisted reproductive technologies in patients with polycystic ovary syndrome.
        Fertility and sterility. 2001; 76: 812-816
        • Gardner D.K.
        • Lane M.
        • Stevens J.
        • Schlenker T.
        • Schoolcraft W.B.
        Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer.
        Fertility and sterility. 2000; 73: 1155-1158
        • Hsueh A.J.
        • Kawamura K.
        • Cheng Y.
        • Fauser B.C.
        Intraovarian control of early folliculogenesis.
        Endocrine reviews. 2015; 36: 1-24
        • Huhtaniemi I.
        • Hovatta O.
        • La Marca A.
        • Livera G.
        • Monniaux D.
        • Persani L.
        • Heddar A.
        • Jarzabek K.
        • Laisk-Podar T.
        • Salumets A.
        Advances in the molecular pathophysiology, genetics, and treatment of primary ovarian insufficiency.
        Trends in Endocrinology and Metabolism. 2018;
        • Jiao X.
        • Zhang H.
        • Ke H.
        • Zhang J.
        • Cheng L.
        • Liu Y.
        • Qin Y.
        • Chen Z.-J.
        Premature ovarian insufficiency: Phenotypic characterization within different etiologies.
        The Journal of Clinical Endocrinology and Metabolism. 2017; 102: 2281-2290
        • Kailasam C.
        • Keay S.
        • Wilson P.
        • Ford W.
        • Jenkins J.
        Defining poor ovarian response during ivf cycles, in women aged < 40 years, and its relationship with treatment outcome.
        Human Reproduction. 2004; 19: 1544-1547
        • Kandil M.
        • Rezk M.
        • Al-Halaby A.
        • Emarh M.
        • El-Nasr I.S.
        Impact of ultrasound-guided transvaginal ovarian needle drilling versus laparoscopic ovarian drilling on ovarian reserve and pregnancy rate in polycystic ovary syndrome: A randomized clinical trial.
        Journal of minimally invasive gynecology. 2018; 25: 1075-1079
        • Kawamura K.
        • Cheng Y.
        • Suzuki N.
        • Deguchi M.
        • Sato Y.
        • Takae S.
        • Ho C.H.
        • Kawamura N.
        • Tamura M.
        • Hashimoto S.
        • Sugishita Y.
        • Morimoto Y.
        • Hosoi Y.
        • Yoshioka N.
        • Ishizuka B.
        • Hsueh A.J.
        Hippo signaling disruption and akt stimulation of ovarian follicles for infertility treatment.
        Proc. Natl. Acad. Sci. U S A. 2013; 110: 17474-17479
        • Kuang Y.
        • Hong Q.
        • Chen Q.
        • Lyu Q.
        • Ai A.
        • Fu Y.
        • Shoham Z.
        Luteal-phase ovarian stimulation is feasible for producing competent oocytes in women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment, with optimal pregnancy outcomes in frozen-thawed embryo transfer cycles.
        Fertility and sterility. 2014; 101: 105-111
        • Levi A.J.
        • Raynault M.F.
        • Bergh P.A.
        • Drews M.R.
        • Miller B.T.
        • Scott Jr, R.T.
        Reproductive outcome in patients with diminished ovarian reserve.
        Fertility and sterility. 2001; 76: 666-669
        • Li J.
        • Kawamura K.
        • Cheng Y.
        • Liu S.
        • Klein C.
        • Duan E.K.
        • Hsueh A.J.
        Activation of dormant ovarian follicles to generate mature eggs.
        Proc. Natl. Acad. Sci. U S A. 2010; 107: 10280-10284
        • Lin J.
        • Wang P.
        • Zhao J.
        • Xiao S.
        • Yu R.
        • Jin C.
        • Zhu R.
        Outcomes of in vitro fertilization cycles among patients with polycystic ovary syndrome following ovarian puncture for in vitro maturation.
        International Journal of Gynecology and Obstetrics. 2016; 135: 319-323
        • Lunding S.A.
        • Pors S.E.
        • Kristensen S.G.
        • Andersen C.Y.
        • Jeppesen J.V.
        • Macklon K.T.
        • Andersen A.N.
        • Pedersen A.T.
        Autotransplantation of fragmented ovarian cortical tissue: A laparoscopic demonstration.
        Fertility and Sterility. 2018; 110: 1181-1183
        • Mahajan N.
        • Kaur J.
        • Bhattacharya B.
        • Naidu P.
        • Gupta S.
        In vitro activation of ovary.
        The Onco Fertility Journal. 2019; 2: 35
        • Meirow D.
        • Levron J.
        • Eldar-Geva T.
        • Hardan I.
        • Fridman E.
        • Zalel Y.
        • Schiff E.
        • Dor J.
        Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.
        New England Journal of Medicine. 2005; 353: 318-321
        • Mio Y.
        • Toda T.
        • Tanikawa M.
        • Terado H.
        • Harada T.
        • Terakawa N.
        Transvaginal ultrasound-guided follicular aspiration in the management of anovulatory infertility associated with polycystic ovaries.
        Fertility and sterility. 1991; 56: 1060-1065
        • Navot D.
        • Bergh R.
        • Williams M.A.
        • Garrisi G.J.
        • Guzman I.
        • Sandler B.
        • Grunfeld L.
        Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility.
        The Lancet. 1991; 337: 1375-1377
        • Pacheco F.
        • Oktay K.
        Current success and efficiency of autologous ovarian transplantation: A meta-analysis.
        Reproductive Sciences. 2017; 24: 1111-1120
        • Schmidt K.L.
        • Andersen C.Y..
        • Loft A.
        • Byskov A.G.
        • Emst E.
        • Andersen A.N.
        Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation.
        Hum. Reprod. 2005; 20: 3539-3546
        • Scott R.T.
        • Toner J.P.
        • Muasher S.J.
        • Oehninger S.
        • Robinson S.
        • Rosenwaks Z.
        Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome.
        Fertility and sterility. 1989; 51: 651-654
        • Silber S.J.
        • Lenahan K.M.
        • Levine D.J.
        • Pineda J.A.
        • Gorman K.S.
        • Friez M.J.
        • Crawford E.C.
        • Gosden R.G.
        Ovarian transplantation between monozygotic twins discordant for premature ovarian failure.
        N. Engl. J. Med. 2005; 353: 58-63
        • Suzuki N.
        • Yoshioka N.
        • Takae S.
        • Sugishita Y.
        • Tamura M.
        • Hashimoto S.
        • Morimoto Y.
        • Kawamura K.
        Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.
        Human Reproduction. 2015; 30: 608-615
        • Ulug U.
        • Ben-Shlomo I.
        • Turan E.
        • Erden H.F.
        • Akman M.A.
        • Bahceci M.
        Conception rates following assisted reproduction in poor responder patients: A retrospective study in 300 consecutive cycles.
        Reproductive biomedicine online. 2003; 6: 439-443
        • Veeck L.L.
        Oocyte assessment and biological performance. 541. Annals of the New York Academy of Sciences, 1988: 259-274
        • Zhai J.
        • Yao G.
        • Dong F.
        • Bu Z.
        • Cheng Y.
        • Sato Y.
        • Hu L.
        • Zhang Y.
        • Wang J.
        • Dai S.
        • Li J.
        • Sun J.
        • Hsueh A.J.
        • Kawamura K.
        • Sun Y.
        In vitro activation of follicles and fresh tissue auto-transplantation in primary ovarian insufficiency patients.
        J. Clin. Endocrinol. Metab. 2016; 101: 4405-4412
        • Zhang X.
        • Han T.
        • Yan L.
        • Jiao X.
        • Qin Y.
        • Chen Z.-J.
        Resumption of ovarian function after ovarian biopsy/scratch in patients with premature ovarian insufficiency.
        Reproductive Sciences. 2018; 1933719118818906

      Biography

      Dr Kazuhiro Kawamura is Director of the Advanced Reproductive Medicine Research Center at the International University Health and Welfare (IUHW) School of Medicine. He is also a Professor of Obstetrics and Gynecology of IUHW School of Medicine. He received his medical and philosophy degrees from the Akita University School of Medicine.
      Key message
      Drug-free in-vitro activation (IVA) as infertility treatment for poor ovarian response patients with decreasing ovarian reserve could allow the retrieval of more oocytes to achieve higher pregnancy rates. The new approach omits the 2-day incubation of ovarian tissues with drugs used in the original IVA and requires only one surgery.