Oocyte Banking for

Reproductive Preservation:
What we have done and
where we are going
Jason Barritt, PhD
Reproductive Medicine Associates
of New York
 Disclosure

ExtendFertility:
Scientific Advisory Board
 Presentation outline

• Introduction

• History of oocyte cryopreservation (OC)

• Research studies at RMA New York

• Where are we going

 Reproductive Medicine Associates
of New York
Embryology Physicians
Andrea DeVenuta Alan Copperman
Marlena Duke Lawrence Grunfeld
RoseMarie Moschini Tanmoy Mukherjee
Charity Moyer Benjamin Sandler
Flora Poleshchuk Jeff Klein
Mark Simpkins Martha Luna
Richard Slifkin Eric Flisser
Lora Valluzzo Jane Ruman
Natan Bar-Chama
Research Jonathan Schiff
Lesley Chuang Caryn Selick
 Introduction

• Human oocyte is the largest cell

• Full of water
• Highly organized cytoplasmic
architecture
• Chromosomal complement is at a very
sensitive state
• Its membrane hates ice
 Preserving the oocyte
• Slow Freeze – Rapid Thaw
• Dehydration
• Cryoprotectant
• Controlled slow freeze – with seeding
• Rapid thaw

• Vitrification
• Rapid dehydration
• Extremely increased cryoprotectant
• Extremely fast cooling
• Extremely fast warming
Oocyte Cryopreservation

The early years

Pregnancy after human oocyte
cryopreservation

Chen (1986, Lancet)

• first successful attempt reported

• dimethyl sulphoxide (DMSO)

• slow cooling / rapidly warming

• 80% survival of 40 oocytes post-thaw

• 30 oocytes inseminated

• 83% fertilized ; 60% cleavage

• twin pregnancy
Cryopreservation of mouse and human
oocytes using 1,2-propanediol and the
configuration of the meiotic spindle
Gook et al. (1993)

• 0.1M sucrose
• 1,2-propanediol (1,2-PrOH)
• Slow freeze / rapid thaw
• 171 human oocytes cryopreserved and thawed
• 64% survival
• Meiotic spindle not significantly different following
cryopreservation
Fertilization of human oocytes
following cryopreservation; normal
karyotypes and absence of stray
chromosomes

Gook et al. (1994)

• Slow freeze/ rapid thaw (0.1 M sucrose and 1,2-PrOH)

• 73% survival
• 46% fertilization following standard IVF insemination
• No stray chromosomes in 137 oocytes analyzed
• 4 normal karyotypes following normal fertilizations
Intracytoplasmic sperm injection and
embryo development of human
oocytes cryopreserved using 1,2-
propanediol

Gook et al. (1995)

• Slow freeze/ rapid thaw (0.1 M sucrose and 1,2-PrOH)

• Use of ICSI for slightly increased fertilization (50%)
• 84% embryo cleavage rate
• Development to the blastocyst stage with a 66% hatching
rate
Birth of a healthy female after
intracytoplasmic sperm injection of
cryopreserved human oocytes
Porcu et al. (1997)

• Oocyte slow freeze / rapid thaw (0.1 M sucrose and 1,2-PrOH)

with Intracytoplasmic Sperm Injection (ICSI)
• 33% survival
• 2 normally fertilized ; 1 cleaved
• One 4-cell embryo transferred on day 2
• A single gestation developed
• Amniocentesis demonstrated a normal female karyotype
• A healthy female infant was born at the 38th week of gestation
Clinical experience and applications of
oocyte cryopreservation
Porcu, et al. (2001)

• 23 patients with 338 oocytes cryopreserved / thawed

(0.1 M sucrose and 1,2-PrOH)
• 60% Survival rate
• 64% normal fertilization following ICSI; 91% cleavage rate
• 3 pregnancies
• additional 16 pregnancies in follow-up study
Report of the first FROZEN/FROZEN pregnancy
Report of successful use of Testicular and Epididymal sperm
Oocyte cryopreservation: a three-year
follow up of sixteen births
Winslow et al. (2001)

• 33 patients
• slow freezing / rapid thawing (0.1M Sucrose and 1,2-PrOH)
• 324 cryopreserved/thawed with 69% survival
• ICSI with 80% normal fertilization, with 95% cleavage rate
• 26% pregnancy rate and 13% implantation rate
• 6 singletons, three sets of twins and one set of triplets were
born
• Normal birth weight for each gestation
“Based on a limited number of births, our oocyte
cryopreservation protocol appears to be relatively efficient
and free of health concerns.”
The births of five Spanish babies from
cryopreserved donated oocytes
Fosas et al. (2003)

• Seven Donors
• 88 oocytes frozen using 0.3 M Sucrose and 1,2-PrOH
• 90% survived
• All ICSI with 73% fertilization
• 26 embryos transferred
• Four pregnancies (57%) ; 5 children born
The pregnancy rate with frozen oocytes was similar to that
obtained using fresh oocytes from donors (~50%)
Human oocyte cryopreservation as an
adjunct to IVF-embryo transfer cycles
Boldt et al. (2003)

Method 1:
• 1.5 M PrOH with 0.1 M sucrose, seeding at -7°C, and stepwise
dilution of cryoprotectant/sucrose post-thaw
• 7 patients, poor survival rate (12%) and no pregnancies
Method 2:
• 1.5 M PrOH and 0.2 M sucrose, Na-depleted media, seeding at
-6°C, and use of high sucrose (0.5 M) stepwise dilution post-thaw
• 16 cycles, better survival rate (74%), fertilization rate (59%), but
ONLY 11 transfers, with 4 pregnancies and 5 healthy infants
“Using Na-depleted media along with other alterations in freezing and
thawing procedures provide excellent survival and pregnancy rates.“
What we decided to do…
 IRB Approved Clinical Trial

Reproductive Medicine Associates of New York

EMDSerono Inc.
Report of four donor-recipient oocyte
cryopreservation cycles resulting in
high pregnancy and implantation rates

Jason Barritt, Martha Luna, Marlena Duke, Lawrence Grunfeld,

Tanmoy Mukherjee, Benjamin Sandler, and Alan B. Copperman.

Department of Obstetrics and Gynecology and Department of

Reproductive Endocrinology and Infertility, Mount Sinai School of
Medicine; Reproductive Medicine Associates of New York

Fertility and Sterility, 2007; 87 : 189. e13–7

OBJECTIVE
To determine the clinical potential of donor oocyte banking by
cryopreservation and subsequent thaw technique for oocyte
recipient patients
DESIGN
Institutional Review Board (IRB) approved prospective study of
donor oocyte cryopreservation

MATERIALS AND METHODS

• Four anonymous oocyte donors underwent ovarian
hyperstimulation
• The oocytes were subsequently thawed, fertilized and transferred
to 4 recipient patients
• slow freeze/rapid thaw protocol (0.3M sucrose and 1,2-PrOH)
• Oocyte Freeze and Oocyte Thaw (Medicult, Denmark)
• Oocytes that survived were inseminated using ICSI
• Resulting embryos were transferred into recipient patients on the

third day post-insemination

RESULTS
 Number Mean (SD) Rate

Metaphase 19.7 (9.5) 100%

II Frozen 79
Thawed and 68 17 (6.9)
Survived 86%
Fertilization 61 15.3 (6.9)
2PN 90%
3PN 1 0.3 (0.5) 1.5%

1PN 0 0 0%

0PN 3 0.8 (1.5) 4.4%

Degenerate 3 0.8 (1.5) 4.4%

Cleaved 56 14 (6.8)
Embryos 92%
 5.75 100%
Embryos
Transferred
23

6
Implantation 1.5
26%

Clinical 3
Pregnancy
0.75
75%

5 Healthy Live Births

CONCLUSIONS

 Thehigh cryosurvival, fertilization, implantation, and

pregnancy rates demonstrate that the slow freeze/rapid
thaw protocol has the potential to provide good clinical
outcomes for oocyte cryopreservation

 Strongly recommend that all programs initiate their

own trials, involving thawing, fertilizing, and transferring
prior to offering this procedure clinically
What we decided to do
next…
Long Term Fertility Preservation
Through Oocyte Cryopreservation:
an IRB approved study

Jason Barritt, Martha Luna, Marlena Duke, Lora Valluzzo,

Benjamin Sandler, and Alan B. Copperman.

Reproductive Medicine Associates of New York; Reproductive

Endocrinology, Mount Sinai School of Medicine, New York, NY

(May 2009)
OBJECTIVE
Clinical trial for long-term female fertility preservation
The relationship between cycle cancellation, patient age and the
oocytes available for cryopreservation
DESIGN
IRB approved clinical trial following informed consent, medical
evaluation and psychological counseling
MATERIALS AND METHODS
• Data from women presenting for elective oocyte cryopreservation
were reviewed retrospectively
• Variables included cancellation rate, patient age, number of
retrieved oocytes, number of cryopreserved oocytes, and oocyte
maturity at cryopreservation
• Characteristics of the variables across different age groups
(A<35, B=35-37, C=38-40, D>40) were analyzed
RESULTS
• 212 patient consultations
• 95 patients initiated cycles (45%)
• 126 attempted cycles
• 16 cycles were canceled (12.7%)
• Canceled mean age: 38.6
• Canceled mean Day 3 FSH: 15.5
• 110 completed cryopreservations
• Mean age: 37.9
• Mean Day 3 FSH: 9.0
• Mean number of cryopreserved oocytes: 12.5
 Age Group
A B C D
<35 35-37 38-40 >40

Cycles
Cryopreserved 13 38 45 14

Age –
mean ± SD 31.5 ± 4.5 36.9 ± 0.9 39.4 ± 0.8 41.7 ± 2.4

Cryopreserved
oocytes per
cycle – 13.3 ± 6.5 14.1 ± 7.4 12.2 ± 6.4 8.2 ± 3.3
mean ± SD
 Maturity of Oocytes Cryopreserved by Age Group

10
9
8
Mean Oocytes

7 A <35
6
B 35-37
5
4 C 38-40
3 D >40
2
1
0
M2 M1 GV
 A B C D+E
Age Group
<35 35-37 38-40 >40
Mature MII 8.8 ± 6.2 9.3 ± 6.0 7.6 ± 4.8 5.1 ± 2.3
oocytes –
mean ± SD
Immature MI 3.0 ± 1.9 3.7 ± 2.8 3.0 ± 2.9 2.6 ± 1.8
oocytes –
mean ± SD
Immature GV 1.5 ± 2.1 1.1 ± 3.3 1.6 ± 2.3 0.6 ± 0.5
oocytes –
mean ± SD

Cycles with
36.4% 39.5% 22.2% 7.1%
≥ 10 MII
oocytes
 A B C D+E
Age Group
<35 35-37 38-40 >40
Day 3 FSH 10.0 ± 5.0 8.2 ± 2.6 9.8 ± 4.4 7.8 ± 2.7

BAFC 10.0 ± 5.3 8.7 ± 3.7 7.8 ± 3.6 7.1 ± 1.6

Total 3826.9 ± 3885.2 ± 4117.2 ± 4580.4 ±

Gonadotropin
Units 883.6 1.68.8 927.6 756.6
 Age BAFC Day Total Total Oocy Cryo
3 days of Gonadot tes M2
FSH stim ropin retrie oocytes
Units ved

Ocp/lup 36.5 NA 6.5 ± 11.0 ± 2612.5 ± 16.0 8.3 ± 3.2

ron ± 2.4 2.3 1.0 1525.3 ± 4.6
(n=3)
Antagon 37.8 8.4 ± 8.8 ± 11.7 4093.8 ± 13.0 8.2 ±5.5
ist ± 3.4 3.8 3.8 ± 1.2 960.4 ± 7.1
(n=97)
Microfla 39.0 7.3 ± 12.3 12.3 4185.0 ± 8.7 ± 5.7 ± 2.9
re ± 3.1 3.5 ± 3.0 ± 1.1 576.4 3.8
(n=10)
Overall 37.9 8.3 ± 9.0 ± 11.7 4061.7 ± 12.7 8.0 ± 5.3
± 3.4 2.8 3.8 ± 1.2 970.9 ± 6.9
 Age Matched Infertile Control Comparison
Age Group A Age Group B Age Group C Age Group D
<35
OC vs. OC Contro OC Contr OC OC Contro
Control (n=15) l (n=47 ol (n=68 Contro (n=12) l
(n=80) ) (n=235 ) l (n=60)
Age 31.6± 31.4± 36.8 )
36.7± 39.4 (n=3400
39.5± 42.4± 1 42.4± 1
4.7 3.1 ± 0.9 0.8 ± 0.8 )
.8 .1 .0
FSH 9.7± 7.9± 8.3± 9.3± 3 10.7 9.3± 4. 7.2± 1. 10.1±
(IU/L) 5.2 3.7 2.6 .6 ± 5. 0 8 3.2
9
BAFC 8.8± 5 8.0± 4 9.3± 8.4± 3 8.2± 7.4± 3. 7.4± 1. 6.3± 3.
.4 .6 3.5 .7 4.0 7 5 7
Peak E2 1728 2289 1951 1971 2043 1867± 1685± 1486±
(pg/ml) ± 719 ± 107 ± 85 ± 996 1044 962 768 897
* 6* 5
Mean # 12.13 18.0± 13.8 13.0± 14.8 12.4± 8.4± 3. 10.6±
oocyte ± 6.3* 10.7* ± 7. 7.2 ± 11 6.7 4 6.4
retrieved 5 .1
Follicles 9.8± 13.7± 11.3 11.4± 11.1 9.5± 5. 8.1± 3. 7.8± 4.
>14mm 5.4* 7.0* ± 4. 5.0 ± 7. 4 2 6
9 0
Cancellat 1 0 2 0 13 48 3 10
ion (6.7%) (4.3% (19.1 (14.1% (25%) (16.7%
) %) ) )
Age Matched Infertile Control Comparison
Age Group A <35 Age Group B Age Gro

OC vs. Control OC Control OC Control OC

(n=15) (n=80) (n=47) (n=235) (n=68)

Age 31.6± 4.7 31.4± 3.1 36.8± 0.9 36.7± 0.8 39.4± 0.

FSH (IU/L) 9.7± 7.9± 8.3± 2.6 9.3± 3.6 10.7± 5

5.2 3.7
BAFC 8.8± 5.4 8.0± 4.6 9.3± 3.5 8.4± 3.7 8.2± 4.

Peak E2 1728± 2289± 1951± 85 1971± 996 2043 10

(pg/ml) 719* 1076* 5
Mean # 12.13± 18.0± 13.8± 7.5 13.0± 7.2 14.8± 1
oocyte 6.3* 10.7* 1
retrieved
Follicles 9.8± 13.7± 11.3± 4.9 11.4± 5.0 11.1± 7
>14mm 5.4* 7.0*
Cancellation 1 0 2 (4.3%) 0 13
(6.7%) (19.1%)
CONCLUSIONS

• 55% of initial consultations did not attempt a cycle

• 13% cycle cancellation is possibly due to the

increased age of our patient population ; high FSH
(~15) suggested a diminished ovarian reserve

• Mean number of oocytes retrieved was 12.7, however

only 27% of cycles yielded ≥10 mature oocytes for
cryopreservation

• When broken down by age group, there is a decline

in total oocytes available for cryopreservation,
especially above 40 years of age
CONCLUSIONS
• Patient age significantly affects the cancellation rate,
and both the quantity and quality of the oocytes available
for cryopreservation

• Patients seek oocyte cryopreservation after the age at

which this elective procedure could most optimally serve

• Education and counseling of the younger female

population must be further developed for the greatest
benefit
Patient Education
Oocyte Cryopreservation
Modern times
$2500 / egg (min. 3)

~12 transfers / year

~40% pregnancy rate

~8 births
Frozen Egg Donation Number of Frozen Eggs You Pregnancy Success Rate
Will Receive

6 eggs for one cycle

Frozen Eggs –One Cycle 58%

Donor Egg Bank Fees Pre-testing/ Transfer Costs Total Cost

$17,243
$15,000 $2,243*

Frozen Egg Donation Number of Frozen Eggs You Pregnancy Success Rate
Will Receive

Frozen Eggs- Two cycles* 6 eggs per cycle. Total of two 58%
cycles, 12 total eggs*
Donor Egg Bank Fees Pre-testing/ Transfer Costs Total Cost

$27,243
$25,000 $2,243**
  Type of
Egg
Donation
# of eggs Success
Rate-1
attempt
st
Success
Rate-2
attempt
nd
Donor
Egg
Bank
Additional fees Trans Total
for donor IVF
cycle
fer
Costs
Cost to
recipient
                                                                                                                                                                                                                  

Frozen
with
                  
guarantee
8 eggs/batch
(guaranteed 2

batches; 16 total
50%** 50% ** (using
2nd batch of 8
eggs
fees

$18,750 N/A $3,00

0-
$500
0
$21,750
-
$23,750

eggs)*
Frozen 50% N/A $12,750 N/A $3,00 $15,750
without
8 eggs/batch 0- -
guarantee 1 batch only $500
$17,750
0

Traditional 10-20 50% 30% (using $15,500 $9,000-$12,000 $3,00 $27,500-

frozen 0- $32,500
embryos) $5,00
0
 Reproductive Medicine Associates
of New York
Embryology Physicians
Andrea DeVenuta Alan Copperman
Marlena Duke Lawrence Grunfeld
RoseMarie Moschini Tanmoy Mukherjee
Charity Moyer Benjamin Sandler
Flora Poleshchuk Jeff Klein
Mark Simpkins Martha Luna
Richard Slifkin Eric Flisser
Lora Valluzzo Jane Ruman
Natan Bar-Chama
Research Jonathan Schiff
Lesley Chuang Caryn Selick