504	 RBMO VOLUME 44 ISSUE 3 2022
1  Infertility Center of St. Louis, Saint Louis MO, USA
2  Department of Biomedical Engineering, University of Michigan, Ann Arbor MI, USA
3  St. Luke's Hospital Pathology, St. Louis MO, USA
4  Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Higashi-ku
Fukuoka, Japan
© 2021 The Author(s). Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
*Corresponding author. E-mail address: drsherm@infertile.com (S. J. Silber). https://doi.org/10.1016/j.rbmo.2021.11.015
1472-6483/© 2021 The Author(s). Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Declaration: The authors report no financial or commercial conflicts of interest.
KEYWORDS
Cancer and fertility
In-vitro oocyte maturation
Ovary tissue cryopreservation
Ovary transplantation
Primordial follicle recruitment
ARTICLE
In-vitro maturation and transplantation of
cryopreserved ovary tissue: understanding
ovarian longevity
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.
Sherman J. Silber1,
*, Sierra Goldsmith1
, Leilani Castleman1
,
Kellie Hurlbut1
, Yuting Fan2
, Jeffrey Melnick3
, Katsuhiko Hayashi4
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.
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 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.
	 RBMO VOLUME 44 ISSUE 3 2022 505
INTRODUCTION
B
eginning 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, 2015, 2016; Silber et al., 2018).
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 et al., 2008;
Candy et al., 2000; Deanesly, 1954;
Demeestere et al., 2003; Dittrich et al.,
2012; Donnez et al., 2004, 2011, 2013;
Kagawa et al., 2009; Meirow et al.,
2005; Mussett and Parrott, 1961; Poirot
et al., 2012; Revel et al., 2011; Revelli
et al., 2013; Sanchez et al., 2007; Silber
and Gosden, 2007; Silber et al., 2005,
2008a, 2008b, 2010, 2012; Stern et al.,
2013; Stoop et al., 2014).
The interest in human ovary transplant
began after Gosden and co-workers’
report of successful pregnancies in
sheep in 1994 (Gosden et al., 1994).
However, there are only a small number
of systematic reports from single centres
(Donnez and Dolmans, 2017; Donnez
et al., 2013; Gellert et al., 2018; Lotz
et al., 2019; Meirow et al., 2016; Pretalli
et al., 2019; Shapira et al., 2018, 2020).
Furthermore, there is no series from the
USA except for that from the current
authors (Silber et al., 2018). 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
and Dolmans, 2017; Greve et al., 2012;
Meirow et al., 2016; Shapira et al.,
2018, 2020; Stoop et al., 2014). 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 followup,
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 et al.,
1994; Silber et al., 2005). Slow freezing
was the approach initially used when
this programme began in 1997, and it
has been previously described in detail
(Silber et al., 2018). 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 et al., 2004;
Gosden et al., 1994; Newton et al., 1996;
Silber et al., 2005, 2015). 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
et al., 2009; Keros et al., 2009; Silber
et al., 2010). 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 et al., 2009; Keros et al.,
2009; Silber et al., 2010). The transplant
technique has not changed either since
the team's first fresh transplant in 2004,
or the frozen transplants described in
2018 (Silber et al., 2018) (FIGURES 1 AND 2).
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 oocytedepleted
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
506	 RBMO VOLUME 44 ISSUE 3 2022
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 et al., 2020).
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 et al., 2021). 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 et al., 2005; Nikiforov et al.,
2020; Prasath et al., 2014, Segers et al.,
2015, 2020; Uzelac et al., 2015).
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, United States 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 (in vitro development),
IVG, (in vitro gametogenesis) 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 18 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.
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
FIGURE 2  Ovary transplant. Reproduced from Silber et al. (2018).
FIGURE 3  Dissection of ovarian cortex from the medulla.
FIGURE 1  Ovary transplant tissue quilting. Reproduced from Silber et al., (2018).
	 RBMO VOLUME 44 ISSUE 3 2022 507
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).
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 et al., 2012). 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 et al.,
2018). One of the participants who had
had leukaemia now had five live births
from her transplanted ovary tissue.
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
(FIGURES 4 AND 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 et al., 2008;
TABLE 1  OVERALL RESULTS WITH FROZEN OVARY TISSUE TRANSPLANTS BEGINNING IN 1997 TO THE PRESENT, NOW
UPDATED FROM 2017
Date of trans-
plant
Age at trans-
plantation
(years)
Age at
freeze
(years)
Diagnosis Pregnant Live birth
or ongoing
Time until
pregnancy
(days)
Miscarriages Duration of
ovarian function
(months)
6 March 2007 26 24 Premature ovarian failure Yes Female 174 – 23 (ended)
13 January 2009 31 20 Hodgkin's lymphoma Yes Male 272 – 29 (ended)
9 June 2009 29 24 Premature ovarian failure Yes – 276 1 19 (ended)
17 June 2011 33 20 Hodgkin's lymphoma No – – – 38 (ended)
12 October 2012 33 31 Multiple sclerosis Yes Female 481 – 67 (ended)
29 March 2013 32 25 Premature ovarian failure Yes Female 243 – 26 (ended)
5 April 2013 33 30 Brain cancer Yes Male 665 – 61 (ended)
12 April 2013 25 18 Leukaemia Yes Male 502 – 93 (still functioning)
Female 998 –
Male 157 –
Female 2082 –
Male 2497 –
1 October 2013 29 28 Synovial sarcoma No – – – 69 (ended)
7 October 2013 39 24 Leukaemia Yes Female 1287 – 87 (still functioning)
21 July 2015 28 25 Leukaemia No – – – 65 (still functioning)
5 August 2015 32 21 Hodgkin's lymphoma Yes Female 343 – 65 (still functioning)
18 September 2014 36 20 Hodgkin's lymphoma Yes Female 473 – 72 (ended)
Female (2) 908 –
31 August 2018 32 26 Hodgkin's lymphoma Yes – 210 1 28 (still functioning)
Female 292 –
– 799 1
Male 964 –
9 December 2019 37 28 Synovial sarcoma No – – – 12 (still functioning)
27 February 2019 39 33 Large B-cell lymphoma Yes – 240 1 21 (still functioning:
never stopped
having periods)
Male 645 –
13 August 2019 36 23 Hodgkin's lymphoma Yes Female 210 – 17 (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.
508	 RBMO VOLUME 44 ISSUE 3 2022
TABLE 2  VITRIFIED OVARY TISSUE TRANSPLANTS, NOW UPDATED FROM 2017
Date of trans-
plant
Age at trans-
plantation
(years)
Age at
freeze
(years)
Diagnosis Pregnant Live birth
or ongoing
Time until
pregnancy
(days)
Miscarriages Duration of ovarian
function (months)
12 October 2012 33 31 Multiple sclerosis Yes Female 481 – 67 (ended)
5 April 2013 33 30 Brain cancer Yes Male 665 – 61 (ended)
1 October 2013 29 28 Synovial sarcoma No – – – 69 (ended)
21 July 2015 28 25 Leukaemia No – – – 65 (still functioning)
27 February 2019 39 33 Large B-cell lymphoma Yes – 240 1 21 (still functioning: never
stopped having periods)
Male 645 –
Totals: 6 participants; 3 babies; 3 women pregnant; 1 miscarriage.
TABLE 3  SLOW-FREEZE OVARY TISSUE TRANSPLANT, NOW UPDATED FROM 2017
Date of trans-
plant
Age at trans-
plantation
(years)
Age at
freeze
(years)
Diagnosis Pregnant Live birth
or ongoing
Time until
pregnancy
(days)
Miscarriages Duration of ovarian
function (months)
6 March 2007 26 24 Premature ovarian failure Yes Female 174 – 23 (ended)
13 January 2009 31 20 Hodgkin's lymphoma Yes Male 272 – 29 (ended)
9 June 2009 29 24 Premature ovarian failure Yes – 276 1 19 (ended)
17 June 2011 33 20 Hodgkin's lymphoma No – – – 38 (ended)
29 March 2013 32 25 Premature ovarian failure Yes Female 243 – 26 (ended)
12 April 2013 25 18 Leukaemia Yes Male 502 – 93 (still functioning)
Female 998 –
Male 157 –
Female 2082 –
Male 2497 –
5 August 2015 32 21 Hodgkin's lymphoma Yes Female 343 – 65 (still functioning)
18 September 2014 36 20 Hodgkin's lymphoma Yes Female 473 – 72 (ended)
Female (2) 908 –
7 October 2013 39 24 Leukaemia Yes Female 1287 – 87 (still functioning)
31 August 2018 32 26 Hodgkin's lymphoma Yes – 210 1 28 (still functioning)
Female 292 –
– 799 1
Male 964 –
13 August 2019 36 23 Hodgkin's lymphoma Yes Female 210 – 17 (still functioning: never
stopped having periods)
Totals: 11 participants; 19 babies; 10 women pregnant; 3 miscarriages.
TABLE 4  OVARY TISSUE FREEZE TRANSPLANTS IN LEUKAEMIA, NOW UPDATED FROM 2017
Date of
transplant
Age at trans-
plantation
(years)
Age at freeze
(years)
Diagnosis Pregnant Live birth
or ongoing
Time until
pregnancy
(days)
Miscarriages Duration of
ovarian function
(months)
12 April 2013 25 18 Acute myeloid leukaemia Yes Male 502 – 93 (still functioning)
Female 998 –
Male 157 –
Female 2082 –
Male 2497 –
7 October 2013 39 24 Acute lymphocytic leu-
kaemia
Yes Female 1287 – 87 (still functioning)
21 July 2015 28 25 Acute myeloid leukaemia No – – – 65 (still functioning)
Totals: 3 participants; 6 babies (67%); 2 women pregnant with delivery.
	 RBMO VOLUME 44 ISSUE 3 2022 509
Hayashi et al., 2011, 2012; Hikabe
et al., 2016; Nagamatsu et al., 2019;
Nikiforov et al., 2020; Rosendahl et al.,
2011; Schmidt et al., 2011; Segers et al.,
2015). Maturation of germinal vesicle
to MII oocytes was detected between
24 and 48 h of exposure to the HCGcontaining
media. For most participants,
the number of mature oocytes was
what would be obtained from ovarian
hyperstimulation, 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 and Saitou, 2013,
Hayashi et al., 2011, 2012; Hikabe et al.,
2016; Nagamatsu et al., 2019).
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 et al., 2004;
Gosden et al., 1994; Lee et al., 2006;
Maheshwari et al., 2008; Meirow et al.,
2005; Silber et al., 2005; Young et al.,
1986). 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
and Dolmans, 2017; Gellert et al., 2018;
Lotz et al., 2019; Meirow et al., 2016;
Pretalli et al., 2019; Rowell et al., 2020;
Shapira et al., 2018, 2020; Stern et al.,
2013). 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 wellstudied
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
TABLE 5  SUMMARY OF IN-VITRO MATURATION
Patient's
age
(years)
Reason for cryopres-
ervation
Culture medium
base used
FSH/HCG Number of
cumulus
complexes
Metaphase
II oocytes
frozen
In-vitro
maturation
rate (%)
Total ovarian
tissue freeze
pieces
29 Large B-cell lymphoma of
the cervix
Quinn's Cleavage 75 mIU/ml FSH + 10 mIU/ml HCG 25 12 48 21
35 Triple-negative breast
cancer: BRCA positive
Quinn's Cleavage 75 mIU/ml FSH + 1000 mIU/ml HCG 11 1 9 17
41 Fertility Preservation
(social)
Medicult IVM 75 mIU/ml FSH + 1000 mIU/ml HCG 3 2 67 17
18 Large B-cell lymphoma Medicult IVM 75 mIU/ml FSH + 1000 mIU/ml HCG 10 2 20 20
18 Ewing's sacroma Quinn's Cleavage 150 mIU/ml FSH + 20 mIU/ml HCG 34 10 29 21
32 Fertility preservation
for Turner's syndrome:
daughter
Quinn's Cleavage 75 mIU/ml FSH + 10 mIU/ml HCG 24 9 38 10
18 Breast cancer Quinn's Cleavage 75 mIU/ml FSH + 10 mIU/ml HCG 8 2 25 16
33 Breast cancer Quinn's Cleavage 75 mIU/ml FSH + 1000 mIU/ml HCG 26 5 19 10
25 Breast cancer Sage IVM 75 mIU/ml FSH + 1000 mIU/ml HCG 14 5 36 10
14 Non-Hodgkin's lymphoma Sage IVM 75 mIU/ml FSH + 100 mIU/ml HCG 37 14 38 20
19 Hodgkin's lymphoma Quinn's Cleavage 75 mIU/ml FSH + 100 mIU/ml HCG 40 14 35 22
13 Rhabdomyosarcoma Quinn's Cleavage 75 mIU/ml FSH + 100 mIU/ml HCG 25 14 56 16
22 Fertility preservation for
premature ovarian failure:
sister
Quinn's Cleavage 75 mIU/ml FSH + 100 mIU/ml HCG 44 17 38 22
Average in-vitro maturation rate, 35% of metaphase II oocytes vitrified; average number of ovary pieces vitrified, 17.
HCG, human chorionic gonadotrophin.
510	 RBMO VOLUME 44 ISSUE 3 2022
oocytes (Nikiforov et al., 2020; Prasath
et al., 2014; Segers et al., 2015, 2020;
Uzelac et al., 2015). 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
et al., 2021; Prasath et al., 2014, Segers
et al., 2015, 2020; Uzelac et al., 2015;
Vesztergom et al., 2021).
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 et al., 2021).
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 et al., 2008; Hayashi
et al., 2011, 2013, 2013; Hikabe et al.,
2016; Nikiforov et al., 2020; Rosendahl
et al., 2011; Schmidt et al., 2011). 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 et al., 2016;
Nagamatsu et al., 2019; Nikiforov et al.,
2020; Prasath et al., 2014; Segers et al.,
2015, 2020; Uzelac et al., 2015; WinklerCrepaz
et al., 2016; Woodruff and
Shea, 2011; Xiao et al., 2015). Likewise
an in-vivo cortical tissue pressure
gradient may be a major regulator of
primordial follicle recruitment and
ovarian longevity (Silber, 2015; Silber
et al., 2015). 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 et al.,
2011, 2012, 2013; Hikabe et al., 2016;
Nagamatsu et al., 2019). 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 and Shea, 2011). 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 et al., 2015,
2016, 2018).
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
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.
	 RBMO VOLUME 44 ISSUE 3 2022 511
been expected from the early work of
Edwards and of Cha and colleagues,
as well as Hayashi's group (Cha et al.,
1991; Edwards, 1962; Hayashi et al.,
2011, 2012, 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 et al., 2021).
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 et al.,
2004; Hayashi et al., 2012, 2013; Silber,
2015; Silber et al., 2015, 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 (Donnez and Dolmans, 2017;
Donnez et al., 2013; Greve et al., 2012;
Hayashi et al., 2011, 2012, 2013; Hikabe
et al., 2016; Jensen et al., 2015; Nesbit
et al., 1980; Ortea et al., 1981; Silber
et al., 2015). Nagamatsu and colleagues
demonstrated that dense cortical tissue
pressure caused oocyte nuclei to rotate,
and this held the primordial follicles
in arrest (Nagamatsu et al., 2019). As
they encountered less tissue pressure
internally, the rotation stopped, and the
primordial follicles were then recruited
(Nagamatsu et al., 2019).
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 et al., 2012; Nesbit et al.,
1980; Ortega et al., 1981). 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
et al., 2012). 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 et al., 2021; Lind et al., 2018;
Thomas-Teinturier et al., 2013; Wallace
and Kelsey, 2010; Winkler-Crepaz et
al., 2016; Yasui et al., 2012; Zhai et al.,
FIGURE 5  Three normal metaphase II oocytes, one metaphase I oocyte and one degenerated
oocyte, resulting from germinal vesicle oocytes from cultured ovarian tissue, on day 2.
512	 RBMO VOLUME 44 ISSUE 3 2022
2012). These eight ‘core genes’ can
also recruit stem cells or IPS (induced
pluripotent stem cell) 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 et al., 2020; Prasath et al.,
2014; Segers et al., 2015, 2020; Uzelac
et al., 2015). 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 et al., 2021; Nikiforov et al., 2020;
Prasath et al., 2014; Segers et al., 2015,
2020; Uzelac et al., 2015). 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 et al.,
2010; Gosden et al., 1989; Hayashi et al.,
2011, 2012, 2013; Kaaijk et al., 1999; Lass
et al., 1997; Lind et al., 2018; Ortega
et al., 1981; Saiduddin et al., 1970; Silber,
2015; Silber et al., 2015). 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 coworkers,
this approach could even be
used for patients with leukaemia (Greve
et al., 2012). 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 (Nagmatsu et al., 2019). 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 is
not necessary for 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.
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 (Greve et al., 2012).
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Received 7 July 2021; received in revised form
16 November 2021; accepted 19 November 2021.