Proteomics & Glyco-Proteomic Analysis of Follicular Fluid

In this study, we plan to utilize ultrasensitive mass spectrometry (MS) and other
conventional proteomic approaches to identify the low and high abundant proteins present in
human FF. Additionally, we plan to use high-performance liquid chromatography (HPLC) and
Western blot techniques to evaluate the Neu5Gc and glycan array based ELISA techniques to
detect anti-Neu5Gc antibody profile in human FF. This analysis will be performed on FF
samples obtained from normal women undergoing In-Vitro Fertilization and Embryo Transfers
(IVF-ET) for a male factor alone and oocyte donors from our 3rd Party Reproduction Program
and from lean and obese women with PCOS. This study will provide information on protein,
glycoprotein, and steroid hormone expression during normal folliculogenesis and during the
pathologic condition of PCOS, which should also provide basic scientific information on
normal and abnormal oocyte development.

Human FF bathes the developing oocyte. Previous studies indicate that the FF contains
cytokines, steroidal and protein hormones, and growth factors. The presence of proteins with
such significant biological properties implies a paracrine and autocrine role for the FF in
promoting normal oocyte development. Furthermore, the presence of any antigenic sialic acid
Neu5Gc and the presence of antibodies targeting these antigenic glycoconjugates (glycolipid
and glycoproteins decorated with sialic acid) may interfere with oocyte development,
hormonal expression, fertilization, and possibly implantation. Here we hypothesize that an
exhaustive proteomic and glyco-proteomic characterization of human FF is essential for a
thorough understanding of its biological significance. We also hypothesize that PCOS may
have differential expression of the FF protein and glyco-protein milieu, and that the
expression may differ further between lean and obese women with PCOS. PCOS represents a
heterogeneous disorder. The severity of hyperandrogensim, metabolic and menstrual
disturbance, and obesity is variable with up to 40% not clinically expressing signs of
classic hyperandrogenism. On the other hand, these atypical, often lean, PCOS women can have
impaired glucose tolerance and diabetes. Reports suggest that these lean PCOS women have
altered serum IGFBP-1, a characteristic endocrine feature of patients with obese PCOS, and
related to hyperinsulinemia and/or obesity. The lean phenotype of PCOS and its significance
is unclear but may represent a cryptic or unexpressed form of PCOS or may be a prelude to
individuals who will later manifest clinical signs of obese/overweight PCOS. Changes in
expression may be expected because of the different amounts of steroidal hormones and
inflammatory markers in the FF derived from women with PCOS.

Inclusion criteria: Inclusion Criteria All

1. Female patients undergoing controlled ovarian hyperstimulation (COH), transvaginal
oocyte aspiration (TVA), and Saline Infused Sonography (SIS) with UL collection

2. Age <35 y/o at time of in vitro fertilization (IVF) cycle

3. Normal ovarian function defined Day 3 Follicular Stimulating Hormone (FSH) <8 pg/ml
or Anti-Mullerian Hormone (≥ 1.0 ng/ml)

Inclusion Criteria Controls:

1. Female patients undergoing COH and TVA donating her oocytes

2. Female patients undergoing COH and TVA for male factor infertility only (i.e. no
female causes of infertility)

3. Normal menstrual cycles

Inclusion Criteria Lean PCOS:

1. Diagnosis of PCOS by Rotterdam Criteria

2. BMI ≤ 25 kg/m2 Inclusion Criteria Classic PCOS

1. Diagnosis of PCOS by Rotterdam Criteria 2. BMI > 30 kg/m2

Exclusion criteria:

1. Age ≥ 35 y/o

2. Female partners with infertility associated diagnosis (i.e. tubal factor, cervical
factor, endometriosis)

3. Unexplained infertility