Surgical approaches of drug-free

in vitro activation and laparoscopic

ovarian incision to treat patients with
ovarian infertility
Yuka Tanaka, M.D.,a Aaron J. Hsueh, Ph.D.,b and Kazuhiro Kawamura, M.D.a,b
a
Department of Obstetrics and Gynecology, International University School of Medicine, Chiba, Japan; and b Department
of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California

Objective: To demonstrate our procedures of drug-free in vitro activation (IVA) for treating patients with premature ovarian
insufficiency (POI) or diminished ovarian reserve (DOR), as well as the laparoscopic ovarian incision (LOI) procedure for treating
patients with resistant ovary syndrome (ROS).
Design: Step-by-step video demonstration of the surgical procedures.
Setting: Fertility clinic and reproductive medicine department.
Patients: Women were diagnosed with POI based on recent amenorrhea before 40 years of age or with DOR according to the Bologna
criteria, showing growth of a few antral follicles after ovarian stimulation. ROS patients were diagnosed based on amenorrhea with
hypergonadotropic hypoestrogenism but showing age-appropriate number of antral follicles under transvaginal ultrasound.
Interventions: The drug-free IVA consists of the following 4 steps: removing a part of the cortex from one or both ovaries; cutting
ovarian cortical pieces into small cubes in vitro; making pockets for ovarian tissue grafting; and grafting ovarian cortical cubes.
The LOI procedure consisted of only one step: cutting ovarian cortex in situ. Both procedures were followed by ovarian
hyperstimulation for at least 1 year. Informed consent was obtained from patients and approval was granted by the Biomedical
Ethics Committee of the International University School of Medicine and the Rose Ladies Clinic. The present clinical trial was
carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).
Main Outcome Measure: Follicle growth.
Results: These procedures can be completed within 1 hour under laparoscopic surgery. There were no complications. In 13 of 15 pa-
tients treated with drug-free IVA, increases in antral follicle numbers were found, followed by a higher number of retrieved oocytes for
in vitro fertilization. In addition to one spontaneous pregnancy, embryo transfer allowed four live births and one ongoing pregnancy.
Five additional patients and one miscarriage patient have cryopreserved embryos for future transfer. We also found follicle growth to
the preovulatory stage in seven of 11 ROS patients who have not responded to any endogenous and exogenous follicle-stimulating
hormone stimulations for follicle growth prior to LOI treatment, allowing the retrieval of mature oocytes for in vitro fertilization.
Four ROS patients became pregnant, followed by the delivery of three healthy infants and one ongoing pregnancy.
Conclusion: A drug-free IVA approach provided an infertility treatment for recent POI or DOR patients. This procedure promoted
growth of residual ovarian follicles following ovarian tissue fragmentation in vitro, leading to Hippo signaling disruption. Although
ROS patients exhibited symptoms of hypergonadotropic hypoestrogenism similar to that of POI patients, they still had multiple
secondary follicles. Hippo signaling disruption in vivo based on cutting ovarian cortex using LOI could promote follicle growth.
UMIN Clinical Trials Registration Number: UMIN000029807.
(Fertil SterilÒ 2020;114:1355–7. Ó2020 by American Society for Reproductive Medicine.)
El resumen está disponible en Español al final del artículo. Use your smartphone
to scan this QR code
Key Words: Ovary, infertility, Hippo signaling, follicle activation, laparoscopic surgery and connect to the
video for this
article now.*
Discuss: You can discuss this article with its authors and other readers at https://www.
fertstertdialog.com/posts/29830 * Download a free QR code scanner by searching for “QR
scanner” in your smartphone’s app store or app marketplace.

Received February 10, 2020; revised July 15, 2020; accepted July 16, 2020; published online September
6, 2020.
Y.T. has nothing to disclose. A.J.H. has nothing to disclose. K.K. has nothing to disclose. The present
work was supported by Grant-in-Aid for Scientific Research B (19H03801) and Challenging
Exploratory Research (18K19624), Japan.
Reprint requests: Kazuhiro Kawamura, M.D., Department of Obstetrics and Gynecology, International
University School of Medicine, Narita, Chiba, 286-8686, Japan (E-mail: kazuhironanami@gmail.
com).

Fertility and Sterility® Vol. 114, No. 6, December 2020 0015-0282/$36.00

Copyright ©2020 American Society for Reproductive Medicine, Published by Elsevier Inc.
https://doi.org/10.1016/j.fertnstert.2020.07.029

VOL. 114 NO. 6 / DECEMBER 2020 1355

VIDEO

NOW AVAILABLE ON YOUTUBE cortical tissue: a laparoscopic demonstration. Fertil Steril 2018;110:
1181–3.
https://youtu.be/Eg37o_brTGI 3. Hsueh AJ, Kawamura K, Cheng Y, Fauser BC. Intraovarian control of early fol-
liculogenesis. Endocr Rev 2015;36:1–24.
SUGGESTED READING 4. Suzuki N, Yoshioka N, Takae S, Sugishita Y, Tamura M, Hashimoto S, et al.
Successful fertility preservation following ovarian tissue vitrification in patients
1. Kawamura K, Ishizuka B, Hsueh AJ. Drug-free in-vitro activation of follicles for
with primary ovarian insufficiency. Hum Reprod 2015;30:608–15.
infertility treatment in poor ovarian response patients with decreased ovarian
5. Kawamura K, Cheng Y, Suzuki N, Deguchi M, Sato Y, Takae S, et al. Hippo
reserve. Reprod Biomed Online 2019;19:30743–6.
signaling disruption and Akt stimulation of ovarian follicles for infertility treat-
2. Lunding SA, Pors SE, Kristensen SG, Andersen CY, Jeppesen JV,
ment. Proc Natl Acad Sci USA 2013;110:117474–9.
Macklon KT, et al. Autotransplantation of fragmented ovarian

1356 VOL. 114 NO. 6 / DECEMBER 2020

 Fertility and Sterility®

Enfoques quir
urgicos de la activaci
on in vitro libre de drogas e incisi

on ov

arica laparosc

opica para tratar pacientes con infertilidad
ov

arica.
Objetivo: Demostrar nuestros procedimientos de activaci
on in vitro libre de drogas (IVA) para tratar pacientes con insuficiencia ov
arica
prematura (POI) o reserva ov
arica disminuida (DOR), así como tambi
en el procedimiento de incisi
on ov
arica laparosc
opica (LOI) para el
tratamiento de pacientes con síndrome de ovario resistente (ROS).
~o: Video demostrativo paso a paso de los procedimientos quir

Disen urgicos.
Lugar: Clínica de fertilidad y departamento de medicina reproductiva.
Pacientes: Mujeres que fueron diagnosticadas con POI en base a amenorrea previa a los 40 a~ nos de edad o con DOR de acuerdo a los
criterios de Bolonia, evidenciando crecimiento de algunos folículos antrales despu
es de la estimulaci

on ov
arica. Las pacientes con ROS
fueron diagnosticadas en base a amenorrea con hipoestrogenismo hipergonadotropico pero evidenciando un n
umero de folículos an-
trales apropiados por la edad mediante ecografía transvaginal.
Intervenciones: La IVA libre de drogas consiste en los siguientes 4 pasos: remover un poco de la corteza de uno o ambos ovarios; cor-
tando in vitro fragmentos de corteza ov
arica en peque~ nos cubos; haciendo bolsas para injertos de tejido ov
arico; y cubos de injerto de
corteza ov
arica. El procedimiento LOI consiste en un solo paso: cortando la corteza ov
arica in situ. Ambos procedimientos fueron se-
guidos de una hiperestimulaci
on ov
arica tras al menos 1 a~
no. Se obtuvieron consentimientos informados por parte de los pacientes y la
aprobaci


on fue concedida por el Comit
e de Eticas Biom
edicas de la Escuela de Medicina de la Universidad Internacional y la Cínica de
Damas Rosas. El presente ensayo clínico fue llevado a cabo de acuerdo con el C


odigo de Etica de la Asociaci
on M
edica Mundial (De-
claraci

on de Helsinki).
Medida de resultado principal: Crecimiento folicular.
Resultados: Estos procedimientos se pueden realizar en 1 hora mediante cirugía laparosc
opica. No hubo complicaciones. En 13 de 15
pacientes tratadas con IVA libre de drogas, se hall

o un incremento en el n
umero de folículos antrales, seguido por un mayor n
umero de
ovocitos obtenidos para fecundaci
on in vitro. Adem
as de un embarazo espontaneo, la transferencia embrionaria dio como resultado
cuatro reci
en nacidos vivos y un embarazo en curso. Cinco pacientes adicionales y una paciente con aborto espontaneo criopreservaron
embriones para futuras transferencias. Tambi
en se observ
o crecimiento folicular en el estadío preovulatorio en siete de 11 pacientes con
ROS que no respondieron a ninguna estimulaci
on folicular end
ogena y ex
ogena de hormonas de estimulaci
on para el crecimiento fo-
licular previo al tratamiento de LOI, permitiendo así la recuperaci
on de ovocitos maduros para la fecundaci
on in vitro. Cuatro pacientes
con ROS se embarazaron, con el consiguiente nacimiento de tres ni~ nos sanos y un embarazo en curso.
Conclusi
on: Una aproximaci
on de IVA libre de drogas proporcion
o un tratamiento de infertilidad para pacientes recientes con POI y
DOR. Este procedimiento promovi
o el crecimiento de folículos ov
aricos residuales siguido de una fragmentaci
on de tejido ov
arico in
vitro, llevando a una interrupci

on de la se~
nalizaci

on del hipot
alamo. Aunque las pacientes con ROS exhibieron síntomas de hipoestro-
genismo hipergonadotropico similar a los de las pacientes con POI, todavía tenían m
ultiples folículos secundarios. La interrupci

on de la
se~
nalizaci
on del hipot
alamo en base al corte de la corteza ov
arica utilizando LOI puedría promover el crecimiento folicular.

VOL. 114 NO. 6 / DECEMBER 2020 1357