The n e w e ng l a n d j o u r na l of m e dic i n e

Cl inic a l I m pl ic a t ions of B a sic R e se a rch

Elizabeth G. Phimister, Ph.D., Editor

Live Offspring after Testis Tissue Transplantation

Takehiko Ogawa, M.D., Ph.D.

Substantive improvements in the treatment of
pediatric diseases such as leukemia and sarcomas
have led to greater numbers of survivors, many of
whom have reduced fertility. One means of pre-
serving the reproductive potential of male patients
who have reached adolescence before they receive
chemotherapy or radiation therapy is to preserve
their sperm. However, this approach cannot be
used in prepubertal boys because spermatogen-
esis does not begin before puberty. Clinical trials
involving the cryopreservation of small pieces of
testis tissues obtained through biopsy from pe-
diatric patients before they undergo gonadotoxic
treatments have been initiated1 on the assumption
that scientific advances will deliver a means of
obtaining sperm from those tissues. The results
of a study recently reported by Fayomi et al.2 may
represent such an advance.
Using a rhesus monkey model, Fayomi et al.
nurtured frozen testis tissues to produce func-
tional sperm and obtain live offspring (Fig. 1).2
They surgically removed immature testis tissue
from five monkeys and stored the tissue in liquid
nitrogen. Several months later, but before the
monkeys had reached puberty, the tissues were
thawed; these tissues, as well as samples of freshly
obtained testis tissue, obtained from the (now
fully) castrated prepubertal monkeys, were sepa-
rately grafted subcutaneously into the original
animal at dorsal and scrotal sites (a total of eight
sites per monkey). Eight to twelve months after
grafting, and after the animals had entered pu-
berty, the grafts showed apparent increase in size,
and the investigators documented well-developed
spermatogenesis in the transplanted tissue at each
of the 39 sites that survived intact (one graft had
been physically destroyed by a monkey shortly
after engraftment). Fayomi et al. obtained live
sperm from the grafts at 26 sites (they observed no
physical or functional differences between the
grafts derived from fresh tissue and those de-
rived from cryopreserved tissue) and used sperm
derived from “cryopreserved” transplant tissue
for intracytoplasmic sperm injection. Transfer of
the resulting embryo into a recipient female mon-
key resulted in the birth of a healthy baby monkey.
Of note, after grafting, and after the animals
reached puberty, their testosterone levels rose and,
in four of the five monkeys, remained elevated
above baseline levels. This observation, together
with the normal range of the monkeys’ levels of
follicle-stimulating hormone during and after pu-
berty, indicated that a functional feedback loop
was shared by the engrafted testis tissue, the hy-
pothalamus, and the pituitary.
Two other approaches to the production of
sperm from frozen, immature testis tissues have
been attempted in animal models. One of these
approaches involves the transplantation of germ
cells.4 In this method, the testis tissues are thawed
and separated into single cells, including sper-
matogenic stem cells. Once injected into the testis
of a rhesus monkey, the stem cells colonize the
niche, repopulate the testis, and produce sperm.
However, injecting cells into the seminiferous tu-
bules is technically difficult, and colonization ef-
ficiency is quite low, making the approach im-
practical at this time. The other approach, which
involves culturing testis tissue to promote sper-
matogenesis outside the body, has been conducted
in mouse models (Fig. 1): the in vitro culture of
immature mouse testis resulted in the production
of spermatids and sperm that were then used for
intracytoplasmic sperm injection, resulting in
healthy, fertile offspring.3 This approach would,
if translated to humans, free patients from surgi-
cal procedures other than the initial biopsy. How-
ever, it has yet to be demonstrated in humans.5
The approach taken by Fayomi et al. involves
the use of an autologous graft and thereby obviates
the adverse events associated with xenografting
and heterografting, such as immunologic rejec-

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 The n e w e ng l a n d j o u r na l of m e dic i n e

Rhesus monkey
(from prepubertal to adult)

Live offspring
Prepubertal
male At sexual maturity
Normal levels of
circulating testosterone
and follicle-stimulating Intracytoplasmic
Prepubertal hormone sperm injection
male after
7 months

Sperm collected
Testis tissue removed from implanted
before gonadotoxic tissue
treatments

Testis tissue grafting

within scrotum and other
protected areas

Male mouse pup Testis tissue

removed

Cryopreserved Cryopreservation
tissue
Viable sperm
Cryopreservation
(in liquid nitrogen)
Live offspring
Intracytoplasmic
sperm injection
Testis tissue culture

Figure 1. Strategies for the Preservation of Male Fertility.

The developmental stage of prepubertal boys who receive gonadotoxic treatments presents a challenge: sperm cannot be obtained
for cryopreservation because puberty has not been reached. A study recently reported by Fayomi et al.2 involves the cryopreservation
of testis tissues of five prepubertal rhesus monkeys. After 5 to 7 months, the autologous testis tissues were subcutaneously grafted
back into the monkeys. Levels of circulating testosterone and follicle-stimulating hormone were consistent with a functional feedback
loop between transplanted testis tissue and the hypothalamus, and the grafts in each of the five monkeys yielded sperm. The birth of
a baby monkey through in vitro fertilization with the use of a sperm from one of the grafts validated the approach as a strategy for pre-
serving fertility. A previous study3 in which a mouse model was used showed that cryopreserved immature testis tissue can be cultured
for sperm production; intracytoplasmic sperm injection of these sperm resulted in healthy live-born mice.

tion, and addresses certain ethical concerns. The
site for engraftment is a consideration. If testis
tissue is to survive as a subcutaneous parcel,
intact for a year or even longer, it must be pro-
tected from physical damage. Fayomi et al. trans-
planted tissue to dorsal and scrotal sites, and the
transplantations to both sites were successful in
that the tissue survived and generated sperm that
were used to successfully produce progeny through
intracytoplasmic injection. The scrotum would
seem to be the preferred site, given that relative
to the back, it is less likely to be subject to physical
stress.
The grafting of testis tissue has other chal-
lenges in addition to that of preserving graft in-
tegrity after transplantation. Minimizing the risk

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 Clinical Implications of Basic Research

of the transplantation of premalignant or malig-
nant cells is one. Optimizing the selection of
patients is another. Preclinical studies are war-
ranted to determine whether the age of the pa-
tient at the time testis tissue is obtained has an
effect on the timing and success of in vitro matu-
ration. It is possible that hormonal treatment of
the patient, after transplantation, may be required
to achieve efficient spermatogenesis in the graft.
These and other questions remain to be addressed,
but it is clear that we are rapidly approaching the
point at which the process described by Fayomi
et al., or a variation of that process, could be con-
sidered for testing in humans if the appropriate
safety and consent mechanisms are in place.
Disclosure forms provided by the author are available at
NEJM.org.
From the Institute of Molecular Medicine and Life Science,
Yokohama City University Association of Medical Science,
Yokohama, Japan.
1. Ginsberg JP, Li Y, Carlson CA, et al. Testicular tissue cryo-
preservation in prepubertal male children: an analysis of paren-
tal decision-making. Pediatr Blood Cancer 2014;​61:​1673-8.
2. Fayomi AP, Peters K, Sukhwani M, et al. Autologous grafting
of cryopreserved prepubertal rhesus testis produces sperm and
offspring. Science 2019;​363:​1314-9.
3. Sato T, Katagiri K, Gohbara A, et al. In vitro production of
functional sperm in cultured neonatal mouse testes. Nature
2011;​471:​504-7.
4. Hermann BP, Sukhwani M, Winkler F, et al. Spermatogo-
nial stem cell transplantation into rhesus testes regenerates
spermatogenesis producing functional sperm. Cell Stem Cell
2012;​11:​715-26.
5. Komeya M, Sato T, Ogawa T. In vitro spermatogenesis: a centu-
ry-long research journey, still half way around. Reprod Med Biol
2018;​17:​407-20.
DOI: 10.1056/NEJMcibr1906768
Copyright © 2019 Massachusetts Medical Society.

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Copyright © 2019 Massachusetts Medical Society. All rights reserved.