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In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity

Open AccessPublished:December 07, 2021DOI:https://doi.org/10.1016/j.rbmo.2021.11.015

      Abstract

      Research question

      Is it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue?

      Design

      A total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back.

      Results

      Every woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes.

      Conclusions

      The authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation may only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight ‘core genes’ control primordial follicle recruitment and development.

      Keywords

      Introduction

      Beginning in 1997 a series of 142 patients (an increase from 108 in a previous report) referred for ovary tissue cryopreservation (119 of whom went through the procedure) provided an opportunity to assess the long-term efficiency of this procedure for young women about to undergo sterilizing cancer treatment (
      • Silber S.
      Unifying theory of adult resting follicle recruitment and fetal oocyte arrest.
      ,
      • Silber S.
      Ovarian tissue cryopreservation and transplantation: scientific implications.
      ;
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      ). This paper is an update, 4 years later, of the authors’ previous summary in 2017. It also presents new results with in-vitro maturation (IVM) from the preserved ovarian tissue (
      • Andersen C.Y.
      • Rosendahl M.
      • Byskov A.G.
      • Loft A.
      • Ottosen C.
      • Dueholm M.
      • Schmidt K.L.
      • Andersen A.N.
      • Ernst E.
      Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.
      ;
      • Candy C.J.
      • Wood M.J.
      • Whittingham D.G.
      Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries.
      ;
      • Deansley R.
      Immature rat ovaries grafted after freezing and thawing.
      ;
      • Demeestere I.
      • Simon P.
      • Englert Y.
      • Delbaere A.
      Preliminary experience of ovarian tissue cryopreservation procedure: alternatives, perspectives and feasibility.
      ;
      • Dittrich R.
      • Lotz L.
      • Keck G.
      • Hoffmann I.
      • Mueller A.
      • Beckmann M.W.
      • Van Der Ven H.
      • Montag M.
      Live birth after ovarian tissue autotransplantation following overnight transportation before cryopreservation.
      ;
      • 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.
      ,
      • Donnez J.
      • Silber S.
      • Andersen C.Y.
      • Demeestere I.
      • Piver P.
      • Meirow D.
      • Pellicer A.
      • Dolmans M.M.
      Children born after autotransplantation of cryopreserved ovarian tissue. A review of 13 live births.
      ,
      • Donnez J.
      • Dolmans M.M.
      • Pellicer A.
      • Diaz-Garcia C.
      • Sanchez Serrano M.
      • Schmidt K.T.
      • Ernst E.
      • Luyckx V.
      • Andersen C.Y.
      Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation.
      ;
      • Kagawa N.
      • Silber S.
      • Kuwayama M.
      Successful vitrification of bovine and human 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.
      ;
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      • Parrott D.M.
      Factors affecting the fertility of mice with orthotopic ovarian grafts.
      ;
      • Poirot C.
      • Abirached F.
      • Prades M.
      • Coussieu C.
      • Bernaudin F.
      • Piver P.
      Induction of puberty by autograft of cryopreserved ovarian tissue.
      ;
      • Revel A.
      • Laufner N.
      • Ben Meir A.
      • Lebovich M.
      • Mitrani E.
      Micro-organ ovarian transplantation enables pregnancy: a case report.
      ;
      • Revelli A.
      • Marchino G.
      • Dolfin E.
      • Molinari E.
      • Delle Piane L.
      • Salvagno F.
      • Benedetto C.
      Live birth after orthotopic grafting of autologous cryopreserved ovarian tissue and spontaneous conception in Italy.
      ;
      • Sanchez M.
      • Alama P.
      • Gadea B.
      • Soares S.R.
      • Simon C.
      • Pellicer A.
      Fresh human orthotopic ovarian cortex transplantation: long-term results.
      ;
      • Silber S.J.
      • Gosden R.G.
      Ovarian transplantation in a series of monozygotic twins discordant for ovarian failure.
      ;
      • 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 pre-mature ovarian failure.
      ,
      • Silber S.J.
      • Derosa M.
      • Pineda J.
      • Lenahan K.
      • Grenia D.
      • Gorman K.
      • Gosden R.G.
      A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.
      ,
      • Silber S.J.
      • Grudzinskas G.
      • Gosden R.G.
      Successful pregnancy after microsurgical transplantation of an intact ovary.
      ,
      • Silber S.
      • Kagawa N.
      • Kuwayama M.
      • Gosden R.
      Duration of fertility after fresh and frozen ovary transplantation.
      ,
      • Silber S.
      • Silber D.
      • Barbey N.
      Long-term function of ovarian tissue transplants.
      ;
      • Stern C.J.
      • Gook D.
      • Hale L.G.
      • Agresta F.
      • Oldham J.
      • Rozen G.
      Jobling T. First reported clinical pregnancy following heterotopic grafting of cryopreserved ovarian tissue in a woman after a bilateral oophorectomy.
      ;
      • Stoop D.
      • Cobo A.
      • Silber S.J.
      Fertility preservation for age related fertility decline.
      ).
      The interest in human ovary transplant began after Gosden and co-workers’ report of successful pregnancies in sheep in 1994 (
      • Gosden R.G.
      • Baird D.T.
      • Wade J.C.
      • Webb R.
      Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196 degrees C.
      ). However, there are only a small number of systematic reports from single centres (
      • Donnez J.
      • Dolmans M.M.
      Fertility preservation in women.
      ;
      • Donnez J.
      • Dolmans M.M.
      • Pellicer A.
      • Diaz-Garcia C.
      • Sanchez Serrano M.
      • Schmidt K.T.
      • Ernst E.
      • Luyckx V.
      • Andersen C.Y.
      Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation.
      ;
      • Gellert S.E.
      • Pors S.E.
      • Kristensen S.G.
      • Bay-Bjørn A.M.
      • Ernst E.
      • Yding Andersen C.
      Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort.
      ;
      • Lotz L.
      • Dittrich R.
      • Hoffmann I.
      • Beckmann M.W.
      Ovarian Tissue Transplantation: Experience From Germany and Worldwide Efficacy.
      ;
      • Meirow D.
      • Ra'anani H.
      • Shapira M.
      • Brenghausen M.
      • Derech Chaim S.
      • Aviel-Ronen S.
      • Amariglio N.
      • Schiff E.
      • Orvieto R.
      • Dor J.
      Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria.
      ;
      • Pretalli J.B.
      • Frontczak Franck S.
      • Pazart L.
      • Roux C.
      • Amiot C.
      DATOR Group
      Development of Ovarian Tissue Autograft to Restore Ovarian Function: Protocol for a French Multicenter Cohort Study.
      ; Shapira et al., 2017,
      • Shapira M.
      • Dolmans M.M.
      • Silber S.
      • Meirow D.
      Evaluation of ovarian tissue transplantation: results from three clinical centers.
      ). Furthermore, there is no series from the USA except for that from the current authors (
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      ). Moreover, there has been no systematic study of the mechanisms of ovarian function and longevity that can be obtained from this extensive experience.
      The purpose of this report is to the update the results of the author's study in the USA and include what has been learned from in-vitro gametogenesis about IVM, i.e. obtaining competent metaphase II (MII) oocytes at the same time as cryopreservation of the ovarian tissue. Unique to this report are: (i) results from vitrification as well as slow freezing; (ii) the birth of six healthy infants from women with leukaemia; (iii) the relative simplicity within the series of IVM from ovary tissue; and (iv) the success rate from just one centre using the same techniques (
      • Donnez J.
      • Dolmans M.M.
      Fertility preservation in women.
      ;
      • Greve T.
      • Clasen-Linde E.
      • Andersen M.T.
      • Andersen M.K.
      • Sorensen S.D.
      • Rosendahl M.
      • Ralfkiaer E.
      • Andersen C.Y.
      Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.
      ;
      • Meirow D.
      • Ra'anani H.
      • Shapira M.
      • Brenghausen M.
      • Derech Chaim S.
      • Aviel-Ronen S.
      • Amariglio N.
      • Schiff E.
      • Orvieto R.
      • Dor J.
      Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria.
      ; Shapira et al., 2017,
      • Shapira M.
      • Dolmans M.M.
      • Silber S.
      • Meirow D.
      Evaluation of ovarian tissue transplantation: results from three clinical centers.
      ;
      • Stoop D.
      • Cobo A.
      • Silber S.J.
      Fertility preservation for age related fertility decline.
      ). Here, the authors report the latest update of a single large series of cryopreserved transplants from one centre in the USA, carried out with the same technique assessed uniformly over long-term follow-up, and studied carefully to elucidate the mechanisms of ovarian longevity and failure. The authors also wished more recently to see if mature oocytes could be obtained easily in vitro from the removed ovarian tissue, prior to freezing, and thus avoid the need for hormonal stimulation.

      Materials and methods

       Participants

      Over a period from 1997 to 2020 (24 years), 119 female patients (out of 142 who were initially consulted) between age 1 and 42 years underwent ovary tissue freezing for fertility preservation by either slow freezing or vitrification, and the 13 most recent also underwent IVM with vitrification of the resultant MII oocytes. Of these 119 cases, 85 related to cancer, 8 were for threatened premature ovarian failure, 13 for social reasons, and 13 for a variety of conditions including Turner's syndrome, multiple sclerosis, endometriosis, aplastic anaemia, a daughter born with no ovary or massive bilateral ovarian teratoma. All patients underwent slow freezing prior to September 2007, and all subsequent participants underwent vitrification of their ovarian tissue.
      The most recent 13 cases of ovary tissue freeze also underwent IVM of oocytes retrieved during the ovarian dissection. All patients were counselled in detail with the advice that the transplant might not ever be performed or might not function. All underwent international review body consent.

       Cryopreservation and transplant surgery

      The technique for slow freeze and thaw of ovarian tissue has not changed since the original description by Gosden and colleagues in 1994 (
      • Gosden R.G.
      • Baird D.T.
      • Wade J.C.
      • Webb R.
      Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196 degrees C.
      ;
      • 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 pre-mature ovarian failure.
      ). Slow freezing was the approach initially used when this programme began in 1997, and it has been previously described in detail (
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      ). The thaw for slow freezing has, however, not been well described in the literature. Thawing is performed rapidly (100°C/min) in a warm bath after first holding the cryovial in air for 30 seconds. Serial transfer to the thawing solutions of 1.0M cryoprotectants, 0.5M cryoprotectant, 0.2M sucrose and then standard media is undertaken for 5 min for each step. As there is usually redundant tissue under the cortex after thawing with slow-freeze cases, the tissue is trimmed under an operating microscope before transplantation, as previously described in great detail (
      • 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.
      ;
      • Gosden R.G.
      • Baird D.T.
      • Wade J.C.
      • Webb R.
      Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196 degrees C.
      ;
      • Newton H.
      • Aubard Y.
      • Rutherford A.
      • Sharma V.
      • Gosden R.
      Low temperature storage and grafting of human ovarian tissue.
      ;
      • 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 pre-mature ovarian failure.
      ,
      • Silber S.
      • Pineda J.
      • Lenahan K.
      • DeRosa M.
      • Melnick J.
      Fresh and cryopreserved ovary transplantation and resting follicle recruitment.
      ). The purpose of this trimming is to avoid interference with revascularization, or diffusion of nutrients to the transplanted cortex, and thus prevent ischaemic damage.
      Since 2007, the authors have exclusively used vitrification, because in-vitro viability analysis studies have shown no oocyte loss with vitrification, but a 40% oocyte loss with slow freezing (
      • Kagawa N.
      • Silber S.
      • Kuwayama M.
      Successful vitrification of bovine and human ovarian tissue.
      ;
      • Keros V.
      • Xella S.
      • Hultenby K.
      • Pettersson K.
      • Sheikhi M.
      • Volpe A.
      • Hreinsson J.
      • Hovatta O.
      Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue.
      ;
      • Silber S.
      • Kagawa N.
      • Kuwayama M.
      • Gosden R.
      Duration of fertility after fresh and frozen ovary transplantation.
      ). Nonetheless, slow freezing seems equally robust for ovarian tissue cryopreservation because the loss of half of a huge number of eggs (200,000) would not be likely to affect the clinical result. The authors’ practice exclusively adopted vitrification in 2007 because it was simpler and more feasible for the series. The technique for vitrification of ovary tissue has been well described in the literature and has also not changed (
      • Kagawa N.
      • Silber S.
      • Kuwayama M.
      Successful vitrification of bovine and human ovarian tissue.
      ;
      • Keros V.
      • Xella S.
      • Hultenby K.
      • Pettersson K.
      • Sheikhi M.
      • Volpe A.
      • Hreinsson J.
      • Hovatta O.
      Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue.
      ;
      • Silber S.
      • Kagawa N.
      • Kuwayama M.
      • Gosden R.
      Duration of fertility after fresh and frozen ovary transplantation.
      ). The transplant technique has not changed either since the team's first fresh transplant in 2004, or the frozen transplants described in 2018 (
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      ) (Figure 1, Figure 2).
      Figure 1
      Figure 1Ovary transplant tissue quilting. Reproduced from
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      .
      Figure 2
      Figure 2Ovary transplant. Reproduced from
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      .
      The pieces of thawed cortex are first quilted together with 9-0 monofilament nylon under an operating microscope and continuous pulsatile irrigation with ice cold media. This quilted ovarian cortex is then transplanted onto the residual ovary medulla after the oocyte-depleted cortex has been resected. Any haemorrhages on the medulla are controlled with micro-bipolar forceps and pressure stitches of 9-0 nylon to avoid micro-haematoma formation under the graft. The graft was placed orthotopically to allow natural conception from ovary retrieval by the fallopian tube.

       In-vitro maturation

      After the ovarian cortex had been dissected from the medulla (Figure 3) and divided into slices for cryopreservation, the ‘spent’ medium in which the dissection took place was examined for free, loose cumulus complexes, which usually contain immature germinal vesicle oocytes. The protocol of the Denmark group was followed closely for this dissection (
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ). These cumulus complexes were then placed in culture with widely varying concentrations of FSH and human chorionic gonadotrophin (HCG) or LH, and the cumulus was then stripped at between 24 and 44 h. The reason that a variety of media and gonadotrophin concentrations were employed is based on the authors’ previously published data from in-vitro gametogenesis in mice (
      • Hayashi K.
      • Galli C.
      • Diecke S.
      • Hildebrandt T.B.
      Artificially produced gametes in mice, humans and other species.
      ). The group wished to see whether IVM could proceed in various media because the oocytes might already be able in vivo to mature. Note that no prior hormonal stimulation was administered. Any oocytes that had advanced by then to mature MII stage were then vitrified in standard fashion (
      • Kuwayama M.
      • Vajta G.
      • Kato O.
      • Leibo S.P.
      Highly efficient vitrification method for cryopreservation of human oocytes.
      ;
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Prasath E.B.
      • Chan M.L.
      • Wong W.H.
      • Lim C.J.
      • Tharmalingam M.D.
      • Hendricks M.
      • Loh S.F.
      • Chia Y.N.
      First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.
      ,
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ,
      • Segers I.
      • Bardhi E.
      • Mateizel I.
      • Van Moer E.
      • Schots R.
      • Verheyen G.
      • Tournaye H.
      • De Vos M.
      Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.
      ;
      • Uzelac P.S.
      • Delaney A.A.
      • Christensen G.L.
      • Bohler H.C.
      • Nakajima S.T.
      Live birth following in vitro maturation of oocytes retrieved from extracorporeal ovarian tissue aspiration and embryo cryopreservation for 5 years.
      ).
      Figure 3
      Figure 3Dissection of ovarian cortex from the medulla.
      For IVM culture, a variety of concentrations of FSH and HCG (LH) were employed. Those concentrations were: (i) 75 mIU/mL FSH with only 10 mIU/ mL HCG with ordinary cleavage media; (ii) 150 mIU/ml FSH with only 20 mIU/mL HCG with ordinary cleavage media; (iii) 75 mIU/mL FSH with 1000 mIU/mL HCG in cleavage media; (iv) 75 mIU/mL FSH with 1000 mIU/mL HCG in Sage IVM media; and (v) 75 mIU/mL HCG in Sage IVM media. This variety of ordinary medium was used because of previous results with in-vitro gametogenesis demonstrating the stages of IVD, IVG and IVM, postulating that many of these germinal vesicles would already have completed the full phase of IVD and IVG in vivo in the ovary. The authors suspected that no special IVM media would be needed and that almost any concentration of HCG would suffice.

       Postoperative follow-up

      All the women underwent monthly hormone monitoring and recording of the return of menstrual cycling for many years, from the time of the transplant to the time of writing this paper, occasionally missing a few months. All women were free to get pregnant via intercourse. IVF and hormonal stimulation were not used with any of the participants. All cases were approved by the IRB of St. Luke's Hospital in St. Louis, Missouri, USA (IRB No. 2016.003, approved 27 April 2021). Informed consent was obtained from all patients or their guardians according to the IRB. This is a prospective series.

      Results

      All 17 transplant cases to date (as already reported for the first 13) showed a return of spontaneous menses from 4 to 5 months after transplantation, plus a return of FSH to normal concentrations, with regular cycling. As FSH returned to normal, anti-Müllerian hormone (AMH) rose to high concentrations, and then fell to very low ones after a further 4–8 months, as previously noted with the first 13 participants. All the grafts functioned from 2 years to as long as 8 years after surgery, and eight are still functioning at the time of writing. All recipients were between 19 and 31 years of age at the time of freezing, with a median age of 24 years (Table 1). Eleven of these 17 had undergone slow freezing, and 6 had undergone vitrification.
      Table 1Overall results with frozen ovary tissue transplants beginning in 1997 to the present, now updated from 2017
      Date of transplantAge at transplantation (years)Age at freeze (years)DiagnosisPregnantLive birth or ongoingTime until pregnancy (days)MiscarriagesDuration of ovarian function (months)
      6 March 20072624Premature ovarian failureYesFemale17423 (ended)
      13 January 20093120Hodgkin's lymphomaYesMale27229 (ended)
      9 June 20092924Premature ovarian failureYes276119 (ended)
      17 June 20113320Hodgkin's lymphomaNo38 (ended)
      12 October 20123331Multiple sclerosisYesFemale48167 (ended)
      29 March 20133225Premature ovarian failureYesFemale24326 (ended)
      5 April 20133330Brain cancerYesMale66561 (ended)
      12 April 20132518LeukaemiaYesMale50293 (still functioning)
      Female998
      Male157
      Female2082
      Male2497
      1 October 20132928Synovial sarcomaNo69 (ended)
      7 October 20133924LeukaemiaYesFemale128787 (still functioning)
      21 July 20152825LeukaemiaNo65 (still functioning)
      5 August 20153221Hodgkin's lymphomaYesFemale34365 (still functioning)
      18 September 20143620Hodgkin's lymphomaYesFemale47372 (ended)
      Female (2)908
      31 August 20183226Hodgkin's lymphomaYes210128 (still functioning)
      Female292
      7991
      Male964
      9 December 20193728Synovial sarcomaNo12 (still functioning)
      27 February 20193933Large B-cell lymphomaYes240121 (still functioning: never stopped having periods)
      Male645
      13 August 20193623Hodgkin's lymphomaYesFemale21017 (still functioning: never stopped having periods)
      Totals: 17 participants; 19 babies; 13 women pregnant with delivery (76%); 12 female and 6 male offspring; 6 vitrifications; 11 slow freezes.
      Thirteen transplants resulted in spontaneous pregnancy and the delivery of at least one live healthy infant (76%). In one case, five singletons so far have resulted from just one participant from one transplant, two singletons from another woman, and in a third case, two singletons and one spontaneous set of twins from just one patient. There have thus been a total of 19 live, healthy infants without IVF in these 17 cases. There have been a total of 22 pregnancies, three of which miscarried before 3 months (14%) (Table 1). Ovarian function is still continuing in 3 of the 6 cases involving vitrified tissue and 5 of the 11 slow-freeze cases (Tables 2 and 3).
      Table 2Vitrified ovary tissue transplants, now updated from 2017
      Date of transplantAge at transplantation (years)Age at freeze (years)DiagnosisPregnantLive birth or ongoingTime until pregnancy (days)MiscarriagesDuration of ovarian function (months)
      12 October 20123331Multiple sclerosisYesFemale48167 (ended)
      5 April 20133330Brain cancerYesMale66561 (ended)
      1 October 20132928Synovial sarcomaNo69 (ended)
      21 July 20152825LeukaemiaNo65 (still functioning)
      27 February 20193933Large B-cell lymphomaYes240121 (still functioning: never stopped having periods)
      Male645
      Totals: 6 participants; 3 babies; 3 women pregnant; 1 miscarriage.
      Table 3Slow-freeze ovary tissue transplant, now updated from 2017
      Date of transplantAge at transplantation (years)Age at freeze (years)DiagnosisPregnantLive birth or ongoingTime until pregnancy (days)MiscarriagesDuration of ovarian function (months)
      6 March 20072624Premature ovarian failureYesFemale17423 (ended)
      13 January 20093120Hodgkin's lymphomaYesMale27229 (ended)
      9 June 20092924Premature ovarian failureYes276119 (ended)
      17 June 20113320Hodgkin's lymphomaNo38 (ended)
      29 March 20133225Premature ovarian failureYesFemale24326 (ended)
      12 April 20132518LeukaemiaYesMale50293 (still functioning)
      Female998
      Male157
      Female2082
      Male2497
      5 August 20153221Hodgkin's lymphomaYesFemale34365 (still functioning)
      18 September 20143620Hodgkin's lymphomaYesFemale47372 (ended)
      Female (2)908
      7 October 20133924LeukaemiaYesFemale128787 (still functioning)
      31 August 20183226Hodgkin's lymphomaYes210128 (still functioning)
      Female292
      7991
      Male964
      13 August 20193623Hodgkin's lymphomaYesFemale21017 (still functioning: never stopped having periods)
      Totals: 11 participants; 19 babies; 10 women pregnant; 3 miscarriages.
      Three of the transplants were for leukaemia, for which the oncologists involved gave approval. The basis for approval was that the ovary had been removed and frozen after the patient was in her first remission, before the bone marrow transplant, and that stains for residual cancer cells were negative (
      • Greve T.
      • Clasen-Linde E.
      • Andersen M.T.
      • Andersen M.K.
      • Sorensen S.D.
      • Rosendahl M.
      • Ralfkiaer E.
      • Andersen C.Y.
      Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.
      ). Two of these three resulted in spontaneous pregnancy, with the delivery of a total of six healthy infants (Table 4). There has been no recurrence of the leukaemia, and in fact no recurrence in any of the cases involving cancer. All three women who had leukaemia still have ovarian function. These were among the first cases of success in terms of leukaemia patients having babies from transplanting their frozen tissue, although the first such case was published in 2017 (
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
      • Fan Y.
      • Castleman L.
      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      ). One of the participants who had had leukaemia now had five live births from her transplanted ovary tissue. No women has had a recurrence of leukaemia.
      Table 4Ovary tissue freeze transplants in leukaemia, now updated from 2017
      Date of transplantAge at transplantation (years)Age at freeze (years)DiagnosisPregnantLive birth or ongoingTime until pregnancy (days)MiscarriagesDuration of ovarian function (months)
      12 April 20132518Acute myeloid leukaemiaYesMale50293 (still functioning)
      Female998
      Male157
      Female2082
      Male2497
      7 October 20133924Acute lymphocytic leukaemiaYesFemale128787 (still functioning)
      21 July 20152825Acute myeloid leukaemiaNo65 (still functioning)
      Totals: 3 participants; 6 babies (67%); 2 women pregnant with delivery.
      The most recent 13 cases involved IVM of the cumulus complexes recovered in the media after cortico-medullary dissection. The number recovered varied widely – between 3 and 37 cumulus complexes depending on the age of the women and whether there had been any prior chemotherapy. Additional details are provided in Table 5. The rate of maturation to an MII oocyte (Figure 4, Figure 5) varied from a low of 19% to a high of 56% (average 35%). The average values for other centres publishing on IVM has varied between 30% and 39% (
      • Andersen C.Y.
      • Rosendahl M.
      • Byskov A.G.
      • Loft A.
      • Ottosen C.
      • Dueholm M.
      • Schmidt K.L.
      • Andersen A.N.
      • Ernst E.
      Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.
      ;
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      ,
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      ;
      • Hikabe O.
      • Hamazaki N.
      • Nagamatsu G.
      • Obata Y.
      • Hirao Y.
      • Hamada N.
      • Shimamoto S.
      • Imamura T.
      • Nakashima K.
      • Saitou M.
      • Hayashi K.
      Reconstitution in vitro of the entire cycle of the mouse female germ line.
      ;
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
      • Hayashi K.
      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ;
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Rosendahl M.
      • Schmidt K.T.
      • Ernst E.
      • Rasmussen P.E.
      • Loft A.
      • Byskov A.G.
      • Andersen A.N.
      • Andersen C.Y.
      Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique.
      ;
      • Schmidt K.T.
      • Rosendahl M.
      • Ernst E.
      • Loft A.
      • Nyboe Andersen A.
      • Dueholm M.
      • Ottosen C.
      • Yding Andersen C.
      Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: The Danish experience.
      ;
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ). Maturation of germinal vesicle to MII oocytes was detected between 24 and 48 h of exposure to the HCG-containing media. For most participants, the number of mature oocytes was what would be obtained from ovarian stimulation, as is apparent from Table 5. Surprisingly, the success of IVM was not related to the media or to the concentration of gonadotrophin in the media. A variety of media and concentrations were intentionally used in light of now-established mechanisms of in-vitro oogenesis, to see if this understanding could be used for a simplification of IVM (Hamazaki et al., 2021a, 2021b;
      • Hayashi K.
      • Saitou M.
      Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.
      ,
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      ,
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      ;
      • Hikabe O.
      • Hamazaki N.
      • Nagamatsu G.
      • Obata Y.
      • Hirao Y.
      • Hamada N.
      • Shimamoto S.
      • Imamura T.
      • Nakashima K.
      • Saitou M.
      • Hayashi K.
      Reconstitution in vitro of the entire cycle of the mouse female germ line.
      ;
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
      • Hayashi K.
      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ).
      Table 5Summary of in-vitro maturation
      Patient's age (years)Reason for cryopreservationCulture medium base usedFSH/HCGNumber of cumulus complexesMetaphase II oocytes frozenIn-vitro maturation rate (%)Total OTF pieces
      29Large B-cell lymphoma of the cervixQuinn's Cleavage75 mIU/ml FSH + 10 mIU/ml HCG25124821
      35Triple-negative breast cancer: BRCA positiveQuinn's Cleavage75 mIU/ml FSH + 1000 mIU/ml HCG111917
      41Fertility Preservation (social)Medicult IVM75 mIU/ml FSH + 1000 mIU/ml HCG326717
      18Large B-cell lymphomaMedicult IVM75 mIU/ml FSH + 1000 mIU/ml HCG1022020
      18Ewing's sacromaQuinn's Cleavage150 mIU/ml FSH + 20 mIU/ml HCG34102921
      32Fertility preservation for Turner's syndrome: daughterQuinn's Cleavage75 mIU/ml FSH + 10 mIU/ml HCG2493810
      18Breast cancerQuinn's Cleavage75 mIU/ml FSH + 10 mIU/ml HCG822516
      33Breast cancerQuinn's Cleavage75 mIU/ml FSH + 1000 mIU/ml HCG2651910
      25Breast cancerSage IVM75 mIU/ml FSH + 1000 mIU/ml HCG1453610
      14Non-Hodgkin's lymphomaSage IVM75 mIU/ml FSH + 100 mIU/ml HCG37143820
      19Hodgkin's lymphomaQuinn's Cleavage75 mIU/ml FSH + 100 mIU/ml HCG40143522
      13RhabdomyosarcomaQuinn's Cleavage75 mIU/ml FSH + 100 mIU/ml HCG25145616
      22Fertility preservation for premature ovarian failure: sisterQuinn's Cleavage75 mIU/ml FSH + 100 mIU/ml HCG44173822
      Average in-vitro maturation rate, 35% of metaphase II oocytes vitrified; average number of ovary pieces vitrified, 17.
      HCG, human chorionic gonadotrophin.
      Figure 4
      Figure 4(A) Day 0: compact appearance of cumulus complexes at the time of dissection. (B) Day 1: spreading of cumulus complexes after 24 h in culture with human chorionic gonadotrophin (HCG) or LH. (C) Day 1: metaphase II oocytes after 24 h in culture with HCG or LH.
      Figure 5
      Figure 5Three normal metaphase II oocytes, one metaphase I oocyte and one degenerated oocyte, resulting from germinal vesicle oocytes from cultured ovarian tissue, on day 2.

      Discussion

      Since the report by Gosden and colleagues with frozen ovary tissue autologous transplantation in sheep, and the first reported cases in 2005 in humans, with spontaneous pregnancy and healthy deliveries, there has been intense interest in preserving the fertility of cancer survivors (Anderson and Cameron, 2007; Bleyer, 1990;
      • 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.
      ;
      • Gosden R.G.
      • Baird D.T.
      • Wade J.C.
      • Webb R.
      Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196 degrees C.
      ;
      • Lee S.J.
      • Schover L.R.
      • Partridge A.H.
      • Patrizio P.
      • Wallace W.H.
      • Hagerty K.
      • Beck L.N.
      • Brennan L.V.
      • Oktay K.
      • Society American Society of Clinical Oncology American
      of Clinical Oncology recommendations on fertility preservation in cancer patients.
      ;
      • Maheshwari A.
      • Porter M.
      • Shetty A.
      • Bhattacharya S.
      Women's awareness and perceptions of delay in childbearing.
      ;
      • 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.
      ;
      • 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 pre-mature ovarian failure.
      ;
      • Young J.L.
      • Ries LG
      • Silverberg E.
      • Horm J.W.
      • Miller R.W.
      Cancer incidence, survival, and mortality for children younger than age 15 years.
      ). The first ovary freezing for cancer patients at the current authors’ centre was in 1997. Thawed tissue did not begin to be transplanted back until 10 years later, in 2007.
      There have now been over several hundred infants born around the world from ovary tissue transplantation in cancer survivors, with no reports of the transmission of cancer except possibly one case from an ovarian cancer (
      • Donnez J.
      • Dolmans M.M.
      Fertility preservation in women.
      ;
      • Gellert S.E.
      • Pors S.E.
      • Kristensen S.G.
      • Bay-Bjørn A.M.
      • Ernst E.
      • Yding Andersen C.
      Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort.
      ;
      • Lotz L.
      • Dittrich R.
      • Hoffmann I.
      • Beckmann M.W.
      Ovarian Tissue Transplantation: Experience From Germany and Worldwide Efficacy.
      ;
      • Meirow D.
      • Ra'anani H.
      • Shapira M.
      • Brenghausen M.
      • Derech Chaim S.
      • Aviel-Ronen S.
      • Amariglio N.
      • Schiff E.
      • Orvieto R.
      • Dor J.
      Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria.
      ;
      • Pretalli J.B.
      • Frontczak Franck S.
      • Pazart L.
      • Roux C.
      • Amiot C.
      DATOR Group
      Development of Ovarian Tissue Autograft to Restore Ovarian Function: Protocol for a French Multicenter Cohort Study.
      ;
      • Rowell E.
      • Duncan F.
      • Laronda M.
      ASRM removes the experimental label from Ovarian Tissue Cryopreservation (OTC): pediatric research must continue.
      ; Shapira et al., 2017,
      • Shapira M.
      • Dolmans M.M.
      • Silber S.
      • Meirow D.
      Evaluation of ovarian tissue transplantation: results from three clinical centers.
      ;
      • Stern C.J.
      • Gook D.
      • Hale L.G.
      • Agresta F.
      • Oldham J.
      • Rozen G.
      Jobling T. First reported clinical pregnancy following heterotopic grafting of cryopreserved ovarian tissue in a woman after a bilateral oophorectomy.
      ). All patients at the current authors’ centre were taken at no charge, which may explain why the groups was able to accumulate the only such series in the USA. This current report of relatively robust results in a small but well-studied series might generate more enthusiasm in the USA to help these patients. However, it is noteworthy that out of 119 women who had ovary tissue frozen, only 17 so far (14%) have requested to have the tissue thawed and transplanted back. It is often 10–20 years before they return, even though most have survived their cancer.
      The unique features of this report include: (i) the robustness of results with this technique; (ii) the inclusion of both slow freezing and vitrification; (iii) some of the first successful results with three leukaemia patients, who delivered six healthy infants; (iv) a well-studied series from just one centre (the only such centre in the USA); and (v) the very recent addition of IVM from ovarian tissue to freeze MII oocytes at the same time as cortical tissue cryopreservation. The advantages of ovary cryopreservation over oocyte vitrification for cancer patients include no delay of cancer treatment, the avoidance of ovarian stimulation, and a resumption of endocrine function. As these results (and those reported by other groups) with IVM demonstrate, one might consider the option to dispense with ovarian stimulation, and go right to oophorectomy, with no delay in cancer treatment. Surprisingly, there might therefore possibly be no need for stimulation to obtain mature oocytes (
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Prasath E.B.
      • Chan M.L.
      • Wong W.H.
      • Lim C.J.
      • Tharmalingam M.D.
      • Hendricks M.
      • Loh S.F.
      • Chia Y.N.
      First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.
      ;
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ,
      • Segers I.
      • Bardhi E.
      • Mateizel I.
      • Van Moer E.
      • Schots R.
      • Verheyen G.
      • Tournaye H.
      • De Vos M.
      Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.
      ;
      • Uzelac P.S.
      • Delaney A.A.
      • Christensen G.L.
      • Bohler H.C.
      • Nakajima S.T.
      Live birth following in vitro maturation of oocytes retrieved from extracorporeal ovarian tissue aspiration and embryo cryopreservation for 5 years.
      ). Mature oocytes may possibly be directly obtained from the excised ovarian tissue. Other groups have preceded these current efforts at IVM from excised ovarian tissue and achieved pregnancy with healthy infants (
      • De Vos M.
      • Grynberg M.
      • Ho T.M.
      • Yuan Y.
      • Albertini D.F.
      • Gilchrist R.B.
      Perspectives on the development and future of oocyte IVM in clinical practice.
      ;
      • Prasath E.B.
      • Chan M.L.
      • Wong W.H.
      • Lim C.J.
      • Tharmalingam M.D.
      • Hendricks M.
      • Loh S.F.
      • Chia Y.N.
      First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.
      ,
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ,
      • Segers I.
      • Bardhi E.
      • Mateizel I.
      • Van Moer E.
      • Schots R.
      • Verheyen G.
      • Tournaye H.
      • De Vos M.
      Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.
      ;
      • Uzelac P.S.
      • Delaney A.A.
      • Christensen G.L.
      • Bohler H.C.
      • Nakajima S.T.
      Live birth following in vitro maturation of oocytes retrieved from extracorporeal ovarian tissue aspiration and embryo cryopreservation for 5 years.
      ;
      • Vesztergom D.
      • Segers I.
      • Mostinckx L.
      • Blockeel C.
      • De Vos M.
      Live births after in vitro maturation of oocytes in women who had suffered adnexal torsion and unilateral oophorectomy following conventional ovarian stimulation.
      ).
      It might at first seem puzzling why IVM from ovarian tissue suddenly seems so easy at many different centres, when it has been difficult in the past. There are several potential reasons for this. First, this group was not trying to mature primordial follicles (although that may become possible in the future with the ‘core genes’) (
      • Hamazaki N.
      • Kyogoku H.
      • Araki H.
      • Miura F.
      • Horikawa C.
      • Hamada N.
      • Shimamoto S.
      • Hikabe O.
      • Nakashima K.
      • Kitajima T.S.
      • Ito T.
      • Leitch H.G.
      • Hayashi K.
      Reconstitution of the oocyte transcriptional network with transcription factors.
      ). Culturing germinal vesicle oocytes that have already become meiotically competent by in-vivo IVD and IVG would not be expected to be difficult. In addition, it is far easier to obtain many germinal vesicle oocytes with cortical dissection rather than with a needle.
      This study and the results of in-vitro gametogenesis reveal the limited role of the ovulatory cycle and ovarian stimulation in oocyte development other than to remove the oocyte from the ovary (
      • Andersen C.Y.
      • Rosendahl M.
      • Byskov A.G.
      • Loft A.
      • Ottosen C.
      • Dueholm M.
      • Schmidt K.L.
      • Andersen A.N.
      • Ernst E.
      Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.
      ;
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      , 2013, 2013;
      • Hikabe O.
      • Hamazaki N.
      • Nagamatsu G.
      • Obata Y.
      • Hirao Y.
      • Hamada N.
      • Shimamoto S.
      • Imamura T.
      • Nakashima K.
      • Saitou M.
      • Hayashi K.
      Reconstitution in vitro of the entire cycle of the mouse female germ line.
      ;
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Rosendahl M.
      • Schmidt K.T.
      • Ernst E.
      • Rasmussen P.E.
      • Loft A.
      • Byskov A.G.
      • Andersen A.N.
      • Andersen C.Y.
      Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique.
      ;
      • Schmidt K.T.
      • Rosendahl M.
      • Ernst E.
      • Loft A.
      • Nyboe Andersen A.
      • Dueholm M.
      • Ottosen C.
      • Yding Andersen C.
      Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: The Danish experience.
      ). Intrinsic tissue pressure along with eight ‘core genes’ has been shown with in-vitro gametogenesis to be the initiating mechanism at work to control primordial follicle recruitment and development to antral follicle status (
      • Hikabe O.
      • Hamazaki N.
      • Nagamatsu G.
      • Obata Y.
      • Hirao Y.
      • Hamada N.
      • Shimamoto S.
      • Imamura T.
      • Nakashima K.
      • Saitou M.
      • Hayashi K.
      Reconstitution in vitro of the entire cycle of the mouse female germ line.
      ;
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
      • Hayashi K.
      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ;
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Prasath E.B.
      • Chan M.L.
      • Wong W.H.
      • Lim C.J.
      • Tharmalingam M.D.
      • Hendricks M.
      • Loh S.F.
      • Chia Y.N.
      First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.
      ;
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ,
      • Segers I.
      • Bardhi E.
      • Mateizel I.
      • Van Moer E.
      • Schots R.
      • Verheyen G.
      • Tournaye H.
      • De Vos M.
      Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.
      ;
      • Uzelac P.S.
      • Delaney A.A.
      • Christensen G.L.
      • Bohler H.C.
      • Nakajima S.T.
      Live birth following in vitro maturation of oocytes retrieved from extracorporeal ovarian tissue aspiration and embryo cryopreservation for 5 years.
      ; Winkler-Crepaz et al., 2016;
      • Woodruff T.K.
      • Shea L.D.
      A new hypothesis regarding ovarian follicle development: ovarian rigidity as a regulator of selection and health.
      ;
      • Xiao S.
      • Zhang J.
      • Romero M.M.
      • Smith K.N.
      • Shea L.D.
      • Woodruff T.K.
      In vitro follicle growth supports human oocyte meiotic maturation.
      ). Likewise an in-vivo cortical tissue pressure gradient may be a major regulator of primordial follicle recruitment and ovarian longevity (
      • Silber S.
      Unifying theory of adult resting follicle recruitment and fetal oocyte arrest.
      ;
      • Silber S.
      • Pineda J.
      • Lenahan K.
      • DeRosa M.
      • Melnick J.
      Fresh and cryopreserved ovary transplantation and resting follicle recruitment.
      ). Primordial follicle arrest in the highly compact ovarian cortex is thought to be a possible key to saving the oocyte from disappearing after the fetal initiation of meiosis and the continuation all the way through meiosis and subsequent apoptosis (
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      ,
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      , 2013;
      • Hikabe O.
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      • Nagamatsu G.
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      Reconstitution in vitro of the entire cycle of the mouse female germ line.
      ;
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
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      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ). It may also be a key to the gradual recruitment every month of a limited number of oocytes in the adult to develop over 4 months into gonadotrophin-sensitive antral and graafian follicles, which spares the resting oocytes from sudden total depletion (
      • Woodruff T.K.
      • Shea L.D.
      A new hypothesis regarding ovarian follicle development: ovarian rigidity as a regulator of selection and health.
      ). This pressure theory is supported clinically by the changes in AMH and FSH observed and previously reported and discussed in the current group's fresh and frozen ovary tissue transplants (
      • Silber S.
      • Pineda J.
      • Lenahan K.
      • DeRosa M.
      • Melnick J.
      Fresh and cryopreserved ovary transplantation and resting follicle recruitment.
      , 2016,
      • Silber S.J.
      • DeRosa M.
      • Goldsmith S.
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      • Melnick J.
      Cryopreservation and transplantation of ovarian tissue: results from one center in the USA.
      ).
      Perhaps the most striking scientific postulate about ovarian function resulting from this series is the high rate of maturation of germinal vesicle oocytes from ovary tissue to MII in less than 2 days, with no ovarian stimulation. The success of IVM did not correlate in this series with the specifics of the media used or the concentration of gonadotrophin. This might have been expected from the early work of Edwards and of Cha and colleagues, as well as Hayashi's group (
      • Cha K.Y.
      • Koo J.J.
      • Ko J.J.
      • Choi D.H.
      • Han S.Y.
      • Yoon T.K.
      Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program.
      ;
      • Edwards R.
      Meiosis in Ovarian Oocytes of Adult Mammals.
      ;
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      ,
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      , 2013). The success of IVM appears to be intrinsic to the cumulus, in which the germinal vesicle oocyte has already in vivo developed meiotic competence, and because the germinal vesicle oocytes have already achieved meiotic competence, by in-vivo exposure to the eight ‘core genes’, and in-vivo FSH (
      • Hamazaki N.
      • Kyogoku H.
      • Araki H.
      • Miura F.
      • Horikawa C.
      • Hamada N.
      • Shimamoto S.
      • Hikabe O.
      • Nakashima K.
      • Kitajima T.S.
      • Ito T.
      • Leitch H.G.
      • Hayashi K.
      Reconstitution of the oocyte transcriptional network with transcription factors.
      ).
      On average the ovarian cortex of a young woman contains about 200,000 oocytes. Every month about 1000 are recruited from ‘resting’ follicles in the cortex, and these require 4–5 months thereafter to become sensitive to gonadotrophins and enter the ovulatory cycle. In-vitro gametogenesis studies in mice have termed this phase ‘IVD’, i.e. in-vitro differentiation to germinal vesicle stage. This phase of “PPT” (primordial to primary follicle transition) can proceed in vitro with merely eight ‘core genes’. In-vitro gametogenesis uncovers three distinct phases of oocyte development: IVD, IVG and IVM. IVD represents the non-gonadotrophin-sensitive growth from either recruited oocytes or pluripotent stem cell to germinal vesicle oocytes, which, as in antral follicles in vivo, are now sensitive to gonadotrophin. This takes 3 weeks in mice, but closer to 3 or 4 months in humans (
      • 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.
      ;
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      , 2013;
      • Silber S.
      Unifying theory of adult resting follicle recruitment and fetal oocyte arrest.
      ;
      • Silber S.
      • Pineda J.
      • Lenahan K.
      • DeRosa M.
      • Melnick J.
      Fresh and cryopreserved ovary transplantation and resting follicle recruitment.
      , 2016). IVD in vivo is a constantly occurring process, as also is IVG. There is a continuous exposure of IVG-ready oocytes in the intact in-vivo ovary to FSH. This process usually takes about 10–12 days in both mice and humans. Oocytes that have completed IVG and are not exposed to HCG in vitro, or to the ovulatory LH surge in vivo, gradually deteriorate. It is the population of oocytes retrieved from the dissected ovarian cortex that have recently completed IVG that mature readily with exposure to HCG in a variety of concentrations (as in vivo) in ordinary culture media.
      The phase referred to as “IVG”, i.e. gonadotrophin (FSH)-induced meiotic competence of germinal vesicle oocytes, usually requires 9–12 days, similar to ovarian stimulation in human IVF. The unstimulated ovary has already been exposed to FSH in vivo. Many (possibly as many as 30–50%) of the 30 or more germinal vesicle oocytes (in cumulus complexes) recovered from the ovarian dissection have already gone through the “IVD” and “IVG” phases in vivo, and therefore are meiotically competent, having already had adequate exposure to endogenous FSH. Therefore just 1–2 days of exposure to LH or HCG is all that is needed for these specific germinal vesicle oocytes to develop to mature MII oocytes. Furthermore, the eight ‘core genes’ are all that are needed to convert stem cells to oocytes. However, these oocytes are not fully competent. Full competence requires culture with fetal granulosa cells, which can also be produced from stem cells using a more complex culture system (Yoshino et al., 2021).
      It is easy to collect many germinal vesicle oocytes from these tiny follicles when you have the ovary in hand instead of using a needle, and their intrinsic 30–40% meiotic competence is universal. So why do we even need ovarian stimulation, or even the normal ovulatory cycle? The normal ovulation cycle is not needed for meiotic competence. Ovarian stimulation for IVF and even for the normal ovulatory cycle is only required for easy oocyte retrieval or just to allow oocytes to exit the ovary.
      Without the ovarian cortex, which induces the formation of primordial follicles, fetal oocytes would continue in meiosis and be completely depleted by birth (2011;
      • Donnez J.
      • Dolmans M.M.
      Fertility preservation in women.
      ;
      • Donnez J.
      • Dolmans M.M.
      • Pellicer A.
      • Diaz-Garcia C.
      • Sanchez Serrano M.
      • Schmidt K.T.
      • Ernst E.
      • Luyckx V.
      • Andersen C.Y.
      Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation.
      ;
      • Greve T.
      • Clasen-Linde E.
      • Andersen M.T.
      • Andersen M.K.
      • Sorensen S.D.
      • Rosendahl M.
      • Ralfkiaer E.
      • Andersen C.Y.
      Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.
      ;
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      ,
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      , 2013;
      • Hikabe O.
      • Hamazaki N.
      • Nagamatsu G.
      • Obata Y.
      • Hirao Y.
      • Hamada N.
      • Shimamoto S.
      • Imamura T.
      • Nakashima K.
      • Saitou M.
      • Hayashi K.
      Reconstitution in vitro of the entire cycle of the mouse female germ line.
      ;
      • Jensen A.K.
      • Kristensen S.G.
      • Macklon K.T.
      • Jeppesen J.V.
      • Fedder J.
      • Ernst E.
      • Andersen C.Y.
      Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark.
      ;
      • Nesbit Jr, M.E.
      • Robison L.L.
      • Ortega J.A.
      • Sather H.N.
      • Donaldson M.
      • Hammond D.
      Testicular relapse in childhood acute lymphoblastic leukemia: association with pretreatment patient characteristics and treatment. A report for Childrens Cancer Study Group.
      ;
      • Ortega J.J.
      • Javier G.
      • Toran N.
      Testicular relapses in childhood acute lymphoid leukaemia (author's transl).
      ;
      • Silber S.
      • Pineda J.
      • Lenahan K.
      • DeRosa M.
      • Melnick J.
      Fresh and cryopreserved ovary transplantation and resting follicle recruitment.
      ). Nagamatsu and colleagues demonstrated that dense cortical tissue pressure caused oocyte nuclei to rotate, and this held the primordial follicles in arrest (
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
      • Hayashi K.
      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ). As they encountered less tissue pressure internally, the rotation stopped, and the primordial follicles were then recruited (
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
      • Hayashi K.
      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ).
      The dense fibrous tissue of the ovarian cortex not only controls follicle development, but also represents a relatively inhospitable location for cancer cells. A perhaps surprising correlation is that prepubertal boys with leukaemia usually have metastasis to the testis, but not to the tunica albuginea of the testis (
      • Greve T.
      • Clasen-Linde E.
      • Andersen M.T.
      • Andersen M.K.
      • Sorensen S.D.
      • Rosendahl M.
      • Ralfkiaer E.
      • Andersen C.Y.
      Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.
      ;
      • Nesbit Jr, M.E.
      • Robison L.L.
      • Ortega J.A.
      • Sather H.N.
      • Donaldson M.
      • Hammond D.
      Testicular relapse in childhood acute lymphoblastic leukemia: association with pretreatment patient characteristics and treatment. A report for Childrens Cancer Study Group.
      ;
      • Ortega J.J.
      • Javier G.
      • Toran N.
      Testicular relapses in childhood acute lymphoid leukaemia (author's transl).
      ). As the tunica albuginea of the testis is the same as the ovarian cortex, much can be learned by studying leukaemia in the male testes. After initial chemotherapy for leukaemia, when the female patient is in temporary remission, no viable leukaemia cells are detected in her ovarian cortex (
      • Greve T.
      • Clasen-Linde E.
      • Andersen M.T.
      • Andersen M.K.
      • Sorensen S.D.
      • Rosendahl M.
      • Ralfkiaer E.
      • Andersen C.Y.
      Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.
      ). The three leukaemia patients in the current series had their ovary removed and cryopreserved while they were in remission before their bone marrow transplant. There has been no recurrence of cancer in these or any of these transplants.
      The recruitment of otherwise ‘locked’ primordial follicles is regulated by a reduction in tissue pressure, but also specifically requires eight ‘core genes’ to accomplish this in vitro and in vivo (
      • Hamazaki N.
      • Kyogoku H.
      • Araki H.
      • Miura F.
      • Horikawa C.
      • Hamada N.
      • Shimamoto S.
      • Hikabe O.
      • Nakashima K.
      • Kitajima T.S.
      • Ito T.
      • Leitch H.G.
      • Hayashi K.
      Reconstitution of the oocyte transcriptional network with transcription factors.
      ;
      • Lind T.
      • Holte J.
      • Olofsson J.I.
      • Hadziosmanovic N.
      • Gudmundsson J.
      • Nedstrand E.
      • Lood M.
      • Berglund L.
      • Rodriguez-Wallberg K.
      Reduced live-birth rates after IVF/ICSI in women with previous unilateral oophorectomy: results of a multicenter cohort study.
      ;
      • Thomas-Teinturier C.
      • El Fayech C.
      • Oberlin O.
      • Pacquement H.
      • Haddy N.
      • Labbé M.
      • Veres C.
      • Guibout C.
      • Diallo I.
      • De Vathaire F.
      Age at menopause and its influencing factors in a cohort of survivors of childhood cancer: earlier but rarely premature.
      ;
      • Wallace W.H.
      • Kelsey T.W.
      Human ovarian reserve from conception to the menopause.
      ; Winkler-Crepaz et al., 2016;
      • Yasui T.
      • Hayashi K.
      • Mizunuma H.
      • Kubota T.
      • Aso T.
      • Matsumura Y.
      • Lee J.S.
      • Suzuki S.
      Factors associated with premature ovarian failure, early menopause and earlier onset of menopause in Japanese women.
      ;
      • Zhai A.
      • Axt J.
      • Hamilton E.C.
      • Koehler E.
      • Lovvorn H.N.
      3rd Assessing gonadal function after childhood ovarian surgery.
      ). These eight ‘core genes’ can also recruit stem cells or IPS cells to transform all the way to MII oocyte-like cells, but recruitment from primordial follicles (from ovarian tissue) results in normal, competent oocytes. Therefore with IVM it is quite easily possible to obtain normal MII oocytes from ovarian tissue, as this report and others demonstrate (
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Prasath E.B.
      • Chan M.L.
      • Wong W.H.
      • Lim C.J.
      • Tharmalingam M.D.
      • Hendricks M.
      • Loh S.F.
      • Chia Y.N.
      First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.
      ;
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ,
      • Segers I.
      • Bardhi E.
      • Mateizel I.
      • Van Moer E.
      • Schots R.
      • Verheyen G.
      • Tournaye H.
      • De Vos M.
      Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.
      ;
      • Uzelac P.S.
      • Delaney A.A.
      • Christensen G.L.
      • Bohler H.C.
      • Nakajima S.T.
      Live birth following in vitro maturation of oocytes retrieved from extracorporeal ovarian tissue aspiration and embryo cryopreservation for 5 years.
      ). Of course, the only way to be certain of the functional competence of these MII oocytes is if live births can be obtained. But several reports have already shown this to be the case (
      • De Vos M.
      • Grynberg M.
      • Ho T.M.
      • Yuan Y.
      • Albertini D.F.
      • Gilchrist R.B.
      Perspectives on the development and future of oocyte IVM in clinical practice.
      ;
      • Nikiforov D.
      • Junping C.
      • Cadenas J.
      • Shukla V.
      • Blanshard R.
      • Pors S.E.
      • Kristensen S.G.
      • Macklon K.T.
      • Colmorn L.
      • Ernst E.
      • Bay-Bjørn A.M.
      • Ghezelayagh Z.
      • Wakimoto Y.
      • Grøndahl M.L.
      • Hoffmann E.
      • Andersen C.Y.
      Improving the maturation rate of human oocytes collected ex vivo during the cryopreservation of ovarian tissue.
      ;
      • Prasath E.B.
      • Chan M.L.
      • Wong W.H.
      • Lim C.J.
      • Tharmalingam M.D.
      • Hendricks M.
      • Loh S.F.
      • Chia Y.N.
      First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.
      ;
      • Segers I.
      • Mateizel I.
      • Van Moer E.
      • Smitz J.
      • Tournaye H.
      • Verheyen G.
      • De Vos M.
      In vitro maturation (IVM) of oocytes recovered from ovariectomy specimens in the laboratory: a promising "ex vivo" method of oocyte cryopreservation resulting in the first report of an ongoing pregnancy in Europe.
      ,
      • Segers I.
      • Bardhi E.
      • Mateizel I.
      • Van Moer E.
      • Schots R.
      • Verheyen G.
      • Tournaye H.
      • De Vos M.
      Live births following fertility preservation using in-vitro maturation of ovarian tissue oocytes.
      ;
      • Uzelac P.S.
      • Delaney A.A.
      • Christensen G.L.
      • Bohler H.C.
      • Nakajima S.T.
      Live birth following in vitro maturation of oocytes retrieved from extracorporeal ovarian tissue aspiration and embryo cryopreservation for 5 years.
      ). This paper is in that sense not original in that others have demonstrated IVM from ovarian tissue. What is perhaps a new suggestion is the relative ease with which this can be achieved using ordinary media, and that this would be expected from this group's previously published studies of in-vitro gametogenesis from stem cells in mice.
      The high success rates here with pregnancy and healthy babies after frozen ovary tissue transplantation are most likely aided by having tissue only from younger women with no prior history of infertility. Nonetheless, the live baby rate for these otherwise sterile cancer survivors and the obvious effectiveness of standard slow freezing (despite a previously demonstrated high oocyte loss compared with vitrification) for ovarian tissue cryopreservation testifies to its robustness and simplicity (
      • Aydin Y.
      • Celiloglu M.
      • Koyuncuoglu M.
      • Ulukus C.
      Follicular dynamics and apoptosis following unilateral oophorectomy.
      ;
      • Gosden R.G.
      • Telfer E.
      • Faddy M.J.
      • Brook D.J.
      Ovarian cyclicity and follicular recruitment in unilaterally ovariectomized mice.
      ;
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      ,
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      , 2013;
      • Kaaijk E.M.
      • Hamerlynck J.V.
      • Beek J.F.
      Van der Veen F. Clinical outcome after unilateral oophorectomy in patients with polycystic ovary syndrome.
      ;
      • Lass A.
      • Paul M.
      • Margara R.
      • Winston R.M.
      Women with one ovary have decreased response to GnRHa/HMG ovulation protocol in IVF but the same pregnancy rate as women with two ovaries.
      ;
      • Lind T.
      • Holte J.
      • Olofsson J.I.
      • Hadziosmanovic N.
      • Gudmundsson J.
      • Nedstrand E.
      • Lood M.
      • Berglund L.
      • Rodriguez-Wallberg K.
      Reduced live-birth rates after IVF/ICSI in women with previous unilateral oophorectomy: results of a multicenter cohort study.
      ;
      • Ortega J.J.
      • Javier G.
      • Toran N.
      Testicular relapses in childhood acute lymphoid leukaemia (author's transl).
      ;
      • Saiduddin S.
      • Rowe R.F.
      • Casida L.E.
      Ovarian follicular changes following unilateral ovariectomy in the cow.
      ;
      • Silber S.
      Unifying theory of adult resting follicle recruitment and fetal oocyte arrest.
      ;
      • Silber S.
      • Pineda J.
      • Lenahan K.
      • DeRosa M.
      • Melnick J.
      Fresh and cryopreserved ovary transplantation and resting follicle recruitment.
      ). One may be able to obtain as many mature oocytes from IVM using this tissue as one would from ovarian stimulation. Furthermore, following the lead of Greve and co-workers, this approach could even be used for patients with leukaemia (
      • Greve T.
      • Clasen-Linde E.
      • Andersen M.T.
      • Andersen M.K.
      • Sorensen S.D.
      • Rosendahl M.
      • Ralfkiaer E.
      • Andersen C.Y.
      Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells.
      ). Most leukaemia patients present with severe illness and require initial chemotherapy to go temporarily into remission before having their bone marrow transplant. This is likely to reduce the risk of transmission of leukaemia cells back to the woman after thawed ovarian tissue is transplanted back. The already recruited, developing follicles will be destroyed by this initial chemotherapy. However, the primordial follicles will be resistant (Meirow et al., 2015). Thus, the only option for leukaemia patients may be ovarian freezing and transplant.
      Ovarian tissue cryopreservation thus appears (24 years later) to be a robust method for preserving fertility, without any need to delay cancer or other treatment because of the time required for ovarian stimulation. Simple cortical tissue pressure (associated with primordial follicle nuclear rotation) has been found to be a key regulator of primordial follicle arrest, recruitment and ovarian longevity in humans, similar to mice (
      • Nagamatsu G.
      • Shimamoto S.
      • Hamazaki N.
      • Nishimura Y.
      • Hayashi K.
      Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.
      ). However, eight ‘core genes’ are also necessary to allow the primordial follicles to escape arrest and develop over 4 months to meiotically competent germinal vesicle oocytes. The ability of ovarian tissue germinal vesicle oocytes to undergo normal IVM indicates that the normal ovulatory cycle and ovarian stimulation may not always be necessary for oocyte maturation. Their only purpose may be just to grow a follicle for mono-ovulation or for egg retrieval, i.e. to get the mature oocyte out of the ovary.

      Uncited References

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      Acknowledgements

      The authors gratefully acknowledge the previous work on IVM from ovarian tissue pioneered by Dr Claus Andersen and his colleagues at the University of Copenhagen in Denmark and all that his group has taught them. They are also thankful for his demonstration that no viable leukaemic cells remain in ovary cortex after the first round of chemotherapy (
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      Biography

      Sherman Silber performed the first microsurgical vasectomy reversals, the first human testicle transplant and the first human ovary transplant. He developed testicular sperm extraction and microsurgical epididymal sperm aspiration and headed the clinical portion of the MIT (USA) team that discovered the DAZ gene. His latest research involves in-vitro gametogenesis in humans.
      Key Message
      Ovary tissue cryopreservation and orthotopic transplantation results in a 76% spontaneous pregnancy live baby rate. There has been no transmission of cancer. In-vitro maturation of oocytes from ovarian tissue is simple and robust. There is no need to delay cancer treatment for ovarian stimulation.