SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 109A

Scientific Abstracts

O-157
High Number of Ovarian Follicles Maintained after Ovarian
Tissue Cryopreservation in Young Females with Cancer Requiring
Gonadotoxic Treatment. Mindy S Christianson,2 Jacqueline Y Maher,2
Erik W Jenson,1 Qihong Wang,1 Jeffrey Lukish*.1 1Johns Hopkins
University School of Medicine, Baltimore, MD, United States; 2Johns
Hopkins University School of Medicine, Lutherville, MD, United States.
INTRODUCTION: For young females with cancer requiring
gonadotoxic treatment, oocyte cryopreservation is not an option if the
patient is prepubertal or time to treatment is limited. Ovarian tissue
cryopreservation has emerged as a fertility preservation modality for this
group. The objective of this study is to examine the efficacy of ovarian
tissue cryropreservation in pediatriac patients with cancer requiring
gonadotoxic chemotherapy.
METHODS: Ovarian tissue samples were harvested from 6 young
females with cancer requiring high-dose alkylating chemotherapy by
laparoscopy at the time of infusion port placement. Ovarian cortex
segments were frozen by the slow-freeze technique and stored for 90 days.
Fresh and frozen-thawed ovarian tissue fragments for each subject were
grafted into the dorsal flank muscle of 4 severe combined immunodeficient
(SCID) mice for 28 days. Assessment of follicular density and follicle
classification was carried out by histological analysis.
RESULTS: Mean patient age was 12 years (range 6-17) with 50%
prepubertal (3/6) and 50% postmenarchal (3/6). Cancer diagnoses
included Hodgkin Lymphoma (4), peripheral nerve sheath tumor (1)
and rhabdomyosarcoma (1). Graft viability of fresh grafts was 85.0%
and 79.1% for frozen grafts. Mean follicular density was 48.7 follicles/

109A

mm3 for fresh tissue grafts and 42.8 follicles/mm3 for frozen tissue. The
follicular densities between fresh and frozen-thawed ovarian grafts were
not statistically different (p=0.8). Follicular classification proportions for
fresh grafts were 57.3% primordial, 34.6% primary, 2.4% secondary, 0.5%
pre-antral, 0% antral and 5.2% unclassified. For frozen grafts, follicular
classifications were 53% primordial, 28.9% primary, 5.4% secondary, 0.6
pre-antral, 0% antral and 12% unclassified.
CONCLUSION: To our knowledge, this is the first study to evaluate
ovarian tissue cryopreservation in the pediatric population that compares
follicular density in the same patient before and after ovarian cortex
cryopreservation and xenografting. Our study demonstrates that a high
number of ovarian follicles in young females with cancer are maintained
after ovarian tissue cryopreservation. This fertility preservation modality
should be offered to patients in this age group facing gonadotoxic cancer
treatment if oocyte cryopreservation is not a viable option.

O-158
A Cargo of Amplifiable Genomic DNA is Present in Human
Preimplantation Embryo-Exported Extracellular Vesicles and
Assessable for Mutation Detection. Sofia Makieva†,2 Francesca
Spinella,1 Elisa Giacomini†,2 Laura Corti*,2 Anil Biricik,1 Francesco
Fiorentino,1 Paola Viganò.2 1Laboratori Genoma Group, Roma, Italy;
2
Reproductive Sciences Lab, Division of Genetics and Cell Biology, IRCCS
San Raffaele Scientific Institute, Milano, Italy.
INTRODUCTION: Various cell types secrete extracellular vesicles
(EVs), which deliver their cargo into target cells to alter their function(s).
We have previously characterized EVs secreted from human embryos
at critical developmental stages and demonstrated their uptake by
maternal endometrial cells, suggesting a role for EVs in embryo-maternal
communication for the establishment of implantation. We sought to
scrutinize the DNA contained within embryonic EVs to deduce whether
it could aid preimplantation genetic diagnostics (PGD) by predicting the
genetic status of the to-be-transferred embryo.
METHODS: Embryos from Assisted Reproductive Technology (ART)
patients of San Raffaele Hospital undergoing trophectoderm (TE)
biopsy for PGD, were cultured under standard conditions for 5 days
post-fertilization. The conditioned medium containing the embryonic
secretome was collected every 48 hours; between days 1-3 (D3 group)
and 3-5 (D5 group) of development. Embryos underwent TE biopsy
between day 5 and 7. EVs were isolated from the culture medium
following ultracentrifugations, characterized with Nanoparticle Tracking
Analysis and Transmission Electron Microscopy, lysed and subjected to
DNA amplification. To assess the DNA, EVs were analysed for Short
Tandem Repeat (STR) common-markers and fragmentation was examined
with TapeStation (TSA) analysis. The EV- and TE- amplified DNA was
examined with multiplex PCR analysis for direct gene mutation detection
and parental STR-mutation associated analysis. Concordance between TE
and EV analyses was deduced.
RESULTS: STR analysis (n=4) identified 8 common STRs markers in EVDNA pools from day 3 (D3) and day 5 (D5) embryo-derived EV samples
(n=50 embryos/group). TSA showed that both D3 and D5 EV-DNA were
less fragmented and more enriched compared to the free DNA found
in embryo culture medium. Parental STRs associated with congenital
deafness (D13S217 and D13S1236) and β-thalassemia (D11S4146)
were detected in D5-EVs. These analyses were conducted on 3 of 12 EV
amplified samples with a sensitivity of 25% due to technical difficulties.
The presence of the maternal mutated allele causing congenital deafness
was confirmed in the EV-DNA by direct analysis of 35delG of CX26 gene.
Concordance in the genetic analysis between EV and TE was found in 1
out of 3 investigated cases.
CONCLUSION: We confirm for the first time the presence of genomic
DNA in human embryo-exported EVs, which is less fragmented compared
to embryo-exported free DNA. Preliminary direct and indirect analysis
of embryo-derived EV-DNA to detect mutated or healthy alleles are
encouraging in a non-invasive pre-implantation diagnosis context.

Saturday Orals

contraindicated. While later stage follicles have been successfully cultured
in vitro, primary and primordial follicles, the most abundant follicles in the
ovary, have proven more resistant to in vitro culture due to the absence of
early paracrine signaling present in ovarian tissue. The goal of this study
was to examine the use of human adipose derived stem cells (hADSCs)
as a source of paracrine factors to support in vitro folliculogenesis.
METHODS: Primary and early secondary follicles measuring 90-110
um in diameter were mechanically isolated from 10-12 day old female
mouse ovaries and co-encapsulated with hADSCs in a poly-(ethylene
glycol) (PEG) hydrogel system. Follicles were cultured for 12 days.
Growth, survival, and in vitro maturation studies were performed. RNA
was isolated from hADSCs following co-encapsulation with follicles at
days 4 and 10 of culture. Reverse transcriptase qPCR (RT-qPCR) was
performed to evaluate expression of differentiation markers relative to
non-encapsulated hADSCs cultured in 2D. hADSCs encapsulated without
follicles were used as controls.
RESULTS: Primary follicles co-cultured with hADSCs showed
improvement in growth with an average diameter on Day 12 of 215 um ±
34 compared to 168 um ± 27 for follicles cultured alone (p=0.01). There
was no difference in follicle size on Day 0 of culture between the two
groups (p>1). Day 12 survival of follicles co-encapsulated with hADSCs
was 60% compared to 10% for follicles cultured alone (p=0.03). RT-qPCR
of co-cultured hADSCs compared to controls showed an 8-fold increase in
expression of POSTN (p=0.049), a marker of undifferentiated stem cells,
as well as a 5-fold decrease in expression of PPAR-g (p=0.048), a marker
of adipocyte differentiation, and a 15-fold decrease in SOX9 (p=0.038),
a marker of chondrocyte differentiation.
CONCLUSION: Primary follicles encapsulated with hADSCs in a
PEG hydrogel system showed improved growth and survival compared
to follicles cultured alone. This suggests that hADSCs provide the
paracrine factors required to support early folliculogenesis in vitro.
hADCs in co-culture with primary follicles show increased expression
of undifferentiated stem cell markers indicating that they retain stem
cell characteristics in culture and do not undergo lineage differentiation.
hADSCs have more translational application than other feeder cells,
such as mouse embryonic fibroblasts, due to their abundance, ease of
procurement, and autologous sourcing. Further studies examining the
secreted products of hADSCs co-cultured with follicles may further
elucidate the early paracrine factors that support primary folliculogenesis
leading to improved culture conditions.

Reproductive Sciences Vol. 25, Supplement 1, March 2018

Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018