Maternal Serum Cytokine Levels and Angiogenic Factor Levels in IVF vs Spontaneously Conceived Pregnancies

IVF subjects: Subjects will be screened for participation at the Magella Office in Long
Beach. Healthy patients with singleton pregnancies conceived with ART will be invited to
participate.

Control subjects: A group of control subjects with spontaneously conceived, singleton
pregnancies matched for age, parity, and race, will be identified at our Magella offices in
Long Beach.

Sample size: 20 IVF subjects and 20 control subjects will be enrolled. A power analysis was
performed using an alpha error of 0.05 and beta error of 0.80. Calculations were made for a
30% difference in the most studied markers during each trimester. The specific markers used
in the power analysis were VEGF, sFlt, sEng, PlGF, IL-6, IL-8, TGF-beta1, and TNF-alpha.
Using the values for the markers with the most stringent criteria it was determined that 15
patients would be required in each group to demonstrate a significant difference using a p
value of 0.05. In anticipation of a 20-25% drop out rate, 20 subjects will be enrolled in
each arm.

Specimens will be obtained at the specified intervals. Venipuncture will be performed and no
more than 20 ml of blood will be obtained.

Within 2 hours of the blood draw, the blood will be spun at 2000 rpm for 10 minutes. The
plasma layer will then be collected into 1 ml aliquots and stored frozen at -80°C. Upon
completion of specimen collection, assays will be performed by personnel who are unaware of
the outcome of the pregnancy. The markers being evaluated are not currently routinely used
in clinical practice thus preventing a retrospective analysis.

The cytokine analysis and VEGF measurements will be performed using Cytokine 29 Plex Kit
Premixed Beads, HCYTO-60K-PMX29 (Linco Research, St. Charles, MO, USA). This kit measures
various markers and proteins of the immune system to determine the degree of inflammation.
Cytokines measured with the kit include: Human IL-1, Human IL-2, Human IL-1ra, Human IL-4,
Human IL-5, Human EGF, Human IL-6, Human IL-7, Human TGF,_Human Fractalkine, Human IL-8,
Human IL-10, Human IL-12p70, Human IL-13, Human IL-15, Human IL-17, Human IL-1, Human
IFNgamma, Human G-CSF, Human GM-CSF, Human TNF, Human Eotaxin, Human MCP-1, Human sCD40L,
Human IL-12p40, Human MIP-1, Human MIP-1beta, Human IP-10, and Human VEGF.

The assays will be run according to the established protocol using the 96 well plate
provided. Briefly, each serum sample is to be incubated with cytokine microbeads for 1 hour
at room temperature. After washing two times, the beads will be incubated with detection
antibodies for 30 min. The beads will then be combined with streptavidin-phycoerythrin and
remain at room temperature for an additional 30 minutes. After two additional washings, the
beads will be resuspended in assay buffer for five minutes and then the beads are to be read
on the Luminex Instrument.

Commercially available enzyme-linked immunosorbent assays (ELISAs) for sFlt 1 (BioSource™)
(Invitrogen Corporation, Carlsbad, CA), endoglin (R&D Systems Inc, Minneapolis, MN), PIGF
(R&D Systems, Inc. Minneapolis, MN) will be used according to established protocols.
Briefly, various samples for ELISA measurement will be diluted in respective calibrator
diluent. After adding assay diluent, the diluted sample will be placed in a 96-well plate
precoated with captured antibodies directed against human sFlt-1, human endoglin, or PlGF,
and the plates were incubated for 2 hours. The wells will be washed 4 times in wash buffer
and incubated with secondary polyclonal antibody against sFlt-1, endoglin, and PIGF
conjugated to horseradish peroxidase for an additional 2 hours. The plates are then washed 4
times in wash buffer.

Substrate solution containing hydrogen peroxide and tetramethylbenzidine will then be added
to each well and incubated for 30 minutes under protection from light with stop solution was
added to each well also. The optical density will then be determined by subtracting readings
at 540 nm from the reading at 450 nm. Protein levels will be calculated using a standard
curve derived from a known concentration of respective recombinant proteins. The minimum
detectable doses in the assay for sFlt-1, endoglin, and PlGF are 3.5, 7, and 5 pg/mL,
respectively. Intraassay and interassay coefficients of variation of 3.5% and 5.5%,
respectively, for sFlt-1, 3.2% and 6.5%, respectively, for endoglin, and 6% and 11%,
respectively for PlGF.

Following delivery placental biopsies will be obtained for possible further analysis in
anticipation of extramural funding pending the results of this study. For each placenta,
three small full-thickness samples approximately 2 cm x 2 cm x 2 cm, not to include fetal
membranes, will be obtained and stored in a -80ºC freezer pending mRNA extraction. We will
follow a standard protocol for placental tissue harvest. The remainder of the placentas will
be disposed of per routine on the Labor and Delivery unit.

Additionally, umbilical artery blood will also be obtained following delivery for possible
further analysis. Within 2 hours of the blood collection, the blood will be spun at 3000 rpm
for 10 minutes. The plasma layer will then be collected into 1ml aliquots and frozen in a
-80ºC freezer.

For each subject, maternal demographic information including age, ethnicity, infertility
history, previous obstetrical history, pregnancy complications and delivery outcomes will be
collected. In addition, neonatal outcome data will be collected including delivery data,
birth weight, and complications. See attached data sheet (Appendix B).

All specimens and data from the medical record will be collected and assigned a unique case
number. Identifying information (e.g. name, birth date, and medical record number) will not
be present in this database. In order to enable us to retrieve the medical record in the
future for data verification, a key relating case numbers and medical record numbers will be
kept separately. This key will be password protected, with the password only known to the
investigators.

With the above exceptions, throughout the course of their pregnancy study participants will
receive standard prenatal care as determined by their physicians.

Data Analysis

Categorical data will be analyzed using the Fisher exact test and chi squared test. Student
t test and Mann-Whitney U tests will be used for the evaluation of parametric and non
parametric continuous data, respectively.

Inclusion Criteria:

- Healthy patients with singleton pregnancies conceived with ART and control subjects
with spontaneously conceived pregnancies. Only subjects that are already pregnant and
have confirmed singleton intrauterine pregnancies are eligible.

Exclusion Criteria:

- Subjects with hypertension, diabetes, renal disease, illicit drug use, tobacco use,
morbid obesity, collagen vascular disease, and autoimmune diseases will be excluded
from participation.

- Pregnancies that had more than one gestation viewed on ultrasound at any time will be
excluded.