Recurrent Pregnancy Loss

Work-up of repetitive implantation failures

Michael J. Paidas, MD Professor Co-Director, Yale Women and Children’s Center for Blood Disorders Co-Director, National Hemophilia Foundation-Baxter Clinical Fellowship at Yale Yale University School of Medicine

A Window into the Reproductive Era Research Congress Center Humanitas Clinical Institute, Via Manzoni 56, 20156 Rozzano, Milan Oct 1, 2011. Session 4: 10.20-10.40AM Pregnancy Complications USA: Overall 4,000,000 Births Per Year 1) Maternal Thromboembolism: 1 in 1,600 pregnancies

2) Preterm (< 37 wks) Delivery Rate (USA): > 13% pregnancies 3) Placenta Mediated Complications: 8% pregnancies: • Severe preeclampsia, early onset <36 wks • Abruption (decidual hemorrhage) • Intrauterine growth restriction (<5th percentile) • Late fetal loss (≥20 wks) • Early fetal loss (>9 wks) Frequency 1 in 5 pregnancies

Bourjeily G, Paidas MJ, Khalil H, Rosene-Montella K, Rodger M. Lancet. 2010 Feb 6;375(9713):500-12. Duhl AJ, Paidas MJ, et al. AJOG. 2007;197(5):457.e1-e21 Pregnancy as the stress test of life

Susceptibility / Pregnancy Mortality / vulnerability complications morbidity

• Genetics • Hypertensive • Cardiovascular • Metabolic disorders disease syndrome • Low fetal • Type 2 diabetes • Life style growth • Autoimmune factors • Preterm diseases? • Socioeconomic delivery • Cancer? status • Placental abruption • Stillbirth • Gestational diabetes

Sattar N, Greer IA., BMJ3 2002 Subsequent maternal chronic hypertension following hypertensive pregnancy

Complication Rate HR CI p No hypertensive disorder 13.5 1 (reference) Gestational hypertension 82.7 5.72 (5.28-6.20) <0.001 Mild preeclampsia 52.2 4.00 (3.80-4.21) <0.001 Severe preeclampsia 92.4 6.73 (6.04-7.49) <0.001

Lykke JA, Langhoff-Roos, Sibai BM, Funai E, Triche EW, Paidas MJ. Hypertensive pregnancy disorders and subsequent cardiovascular morbidity and type 2 diabetes mellitus in the mother. Hypertension. 2009 Jun;53(6):944-51.

4 Subsequent maternal death from non- cardiovascular causes following placenta mediated complications

Combinations Rate HR CI p

Preterm delivery only 6.4 1.39 (1.23-1.58) <0.001

Small-for-gestational-age 8.1 1.65 (1.50-1.82) <0.001

Preterm + SGA 10.4 1.93 (1.55-2.41) <0.001

Lykke JA, Jens Langhoff-Ross J, Lockwood CJ, Triche EW, Paidas MJ. Maternal mortality from cardiovascular and non-cardiovascular causes following pregnancy complications in first delivery. Paediatr Perinat Epidemiol 2010; Jul 1;24(4): 323-30. 5 Recurrent Pregnancy Loss (RPL): Overview Inherited Thrombophilias Antiphospholipid antibodies (APA) Genetic Abnormalities Alloimmune Losses Mullerian Tract Anomalies Infectious Diseases Endocrine Causes Work-up and Treatment Implantation & Immunity: Implications

Inherited Thrombophilias • Deficiencies of the natural anticoagulants: Antithrombin, protein C, protein S

• Factor V Leiden: Caused by base nucleotide change in factor V gene, resulting in a arginine  glutamine switch at position 506, which impairs APC inactivation

• Prothrombin gene mutation G20210A: Mutation in promoter region causes increased gene transcription 2- to 3-fold, increasing protein levels

• MTHFR leads to hyperhomocysteinemia: Increased procoagulants and platelet aggregation; decreased anticoagulant activity, and decreased fibrinolysis

Paidas MJ et al. Semin Perinatol. 2005;29(3):150-163. Lockwood CJ. Obstet Gynecol. 2002;99(2):333-341.

Venous thromboembolism

Bourjeily G, Paidas MJ, Khalil H, Rosene-Montella K, Rodger M. Lancet. 2010 Feb 6;375(9713):500-12 Link Between Thrombophilias & SAB < 10 weeks Large European retrospective cohort study involving 571 women with thrombophilia having 1524 pregnancies vs. 395 controls having 1019 pregnancies found a significant association between any inherited thrombophilia and stillbirth (OR 3.6; 95% CI: 1.4-9.4) but not SAB (OR 1.3; 0.9-1.7). Preston et al. Lancet. 1996; 348:913-6. Link Between Thrombophilias & SAB < 10 weeks

Large case-control study nested in a 32,700 cohort revealed an association between FVL and pregnancy loss after 10 weeks (OR 3.46; 95% CI 2.53-4.72) but not for losses occurring between 3 and 9 weeks.

Lissalde-Lavigneey al. J Thromb Haemost. 2005; 3:2178-84. U.S. Prospective Studies have found no link between Inherited Thrombophilias and Adverse Pregnancy Outcomes

• Maternal FVL not associated with increased pregnancy loss, preeclampsia, placental abruption, or SGA in a low-risk, prospective cohort. (Obstet Gynecol. 2005;106(3):517-24)

• No association between PGM and pregnancy loss, preeclampsia, abruption, or SGA in a low-risk, prospective cohort. (Obstet Gynecol. 2010;115(1):2-4)

FVL/PGM & Pregnancy Loss Systematic Review and Meta-analysis Rodger MA et al. PLoS Medicine 2010

FVL: OR 1.52 (CI 1.06, 2.19) 7 Studies n= 16158 PGM: OR 1.13 (CI 0.64, 2.01) 4 Studies ns n= 8954 Absolute Event Rate 4.2% vs 3.2% (“Odds 52% higher with FVL”)

Rodger MA, Betancourt MT, Clark P, Lindqvist PG, Dizon-Townson D, Said J, Seligsohn U, Carrier M, Salomon O, Greer IA. The association of factor V leiden and prothrombin gene mutation and placenta-mediated pregnancy complications: a systematic review and meta-analysis of prospective cohort studies. PLoS Med. 2010 Jun 15;7(6):e1000292. FVL/PGM & composite placenta mediated complications Systematic Review and Meta-analysis Rodger MA et al. PLoS Medicine 2010

FVL: OR 1.08 (CI 0.87, 1.35) 4 Studies ns n= 11405 PGM: OR 1.27 (CI 0.94, 1.71) 4 Studies ns n= 8,954

Gris: 160 patients, open-label, quasi-random • Hx loss >10 wks with FVL, PGM, or PS def • Tx: Enox 40 mg or ASA 100 mg from 8 wks • LMWH vs ASA: RR 10.00 (1.56-64.20) • LMWH improves live birth rate Gris JC et al. Blood. 2004;103(10):3695-3699. Tulppala: 82 patients, RCT, placebo • Three or more unexplained consecutive losses • Tx: ASA 50 mg vs placebo from positive urine preg • No difference in live birth rate: RR 1.00 (0.78-1.29) Tulppala M et al. Hum Reprod. 1997;12(7):1567-1572.

Empirical Treatment of Recurrent Loss Patients with Heparin and Aspirin of No Benefit •Methods: RCT 364 with unexplained recurrent miscarriage Tx with placebo, vs. LDA vs. LDA and LMWH.

•Results: Live-birth occurred in 54.5% in LDA/LMWH group, 50.8% in LDA-only group, and 57.0% in placebo group

No effect: Inherited Thrombophilia, previous LB, age, #SAB 2 Kaandorp S. et al . N Engl J Med. 2010 Mar 24.

SPIN (Scottish Pregnancy Intervention) study: a multicenter, randomized controlled trial of low- molecular-weight heparin and low-dose aspirin in women with recurrent miscarriage.

Clark P, Walker ID, Langhorne P, Crichton L, Thomson A, Greaves M, Whyte S, Greer IA; Scottish Pregnancy Intervention Study (SPIN) collaborators. Blood. 2010 May 27;115(21):4162-7.

United Kingdom & New Zealand n=294 2 or more unexplained losses ≤ 24 wks No benefit to LDA & LMWH 16

Heparin & LDA induce an anti-angiogenic state in first trimester trophoblast in vitro (HTR8 cell line): Decreasing VEGF Increasing sFLT1 Increasing sEndoglin

Han CS, Mulla MJ, Brosens JJ, Chamley LW, Paidas MJ, Lockwood CJ, Abrahams VM. Aspirin and heparin effect on basal and antiphospholipd antibody modulation of trophoblast function. Obstet Gynecol 2011. In press Antiphospholipid Antibodies

Reported ORs for LAC-associated fetal loss range from 3.0 to 4.8

Anti-cardiolipin antibodies display a wider range of reported OR’s of 0.86 to 20.0

Galli et al Hematol J. 2003; 4:180-6; Greaves Lancet. 1999; 353:1348-53.

Antiphospholipid Antibodies There is conflicting evidence whether APA are associated with RPL (> 3) in the absence of stillbirth:

•At least 50% of pregnancy losses in APA pts occur after the 10th wk (Lupus 1996; 5:409-13).

•Compared to pts having unexplained early SAB without APA, those with APA more often display fetal cardiac activity (86% vs. 43%; P < 0.01) (Hum Reprod 1995; 10:3301-4).

•At least one RCT showed no benefit to treatment with heparin (Farquharson . Obstet Gynecol. 2002; 100:408-13).

Antiphospholipid Antibodies

There is no evidence that APA are associated with IVF failure:

•Meta-analysis shows no association between APA & either IVF clinical pregnancy (OR 0.99; 0.64–1.53) or live birth rates (OR 1.07; 0.66–1.75) (Fertil Steril. 2000; 73:330–3).

•Treatment of APA does not improve IVF success rates (Fertil Steril. 2003; 80:376-83). Recurrent Pregnancy Loss (RPL): Overview Inherited Thrombophilias Antiphospholipid antibodies (APA) Genetic Abnormalities Alloimmune Losses Mullerian Tract Anomalies Infectious Diseases Endocrine Causes Work-up and Treatment Implantation & Immunity: Implications

Genetic Causes of Recurrent Pregnancy Loss Etiology of Loss Example Aneuploidy 64-88% isolated loss Parental genetic factors Fragile sites, bal trans, SYCP3 gene mut, inver Mat metabolic factors Low folate levels, elevated homocysteine Mat gamete aging Decr telomerase act, decr embryonic poly- A-bind Mat Mendelian single gene defects Homozygosity of VEGF -1154 A/A gene), p53 Fetal Mendelian single gene Lethal multiple pterygium syn, defects Incontinentia pigmenti, homozygousity for AD polycystic kid

2% of pregnant women experience two consecutive pregnancy losses 0.4 experience 3 consecutive losses

Toth B, et al.J Reprod Immunol. 2011 Jun;90(1):117-23. Lancet 1990; 336:673-5. Reprod Nutr Dev 1988; 28:1555-68. Array CGH on abortus tissue (n=27) Findings: 11 unique copy number variants in 13 losses in 8 couples (imprinted genes) Chromosomal Abnormalities

Potential Treatments: • IVF with PGD – conflicting but promising results with both AMA-associated RAB and those related to parental balanced trans- locations. Treff NR et al. Mol Hum Reprod. 2010 Aug;16(8):583-9 Treff NR et al. Fertil Steril. 2011 Apr;95(5):1606-12.e1-2 Rubio et al. Am J Reprod Immunol. 2005; 53:159-6; Platteau et al. Fertil Steril. 2005; 83:393-5; Sugiura-Ogasawara et al. Hum Reprod. 2005; 20:3267-70.

• Folate and B6 supplementation Alloimmune losses

Maternal tolerance of fetal allograft is mediated by trophoblast expression of  non-immunogenic HLA-G  Fas ligand  immunosuppressive agents: hCG, PAPP-A, progesterone, and cortisol

Am J Reprod Immunol 1998; 40: 136-44. J Clin Endocrinol Metab 1996; 81:3119-22.

Alloimmune losses

Theories and Diagnostic tests • Absence of maternal-anti-paternal leukocyte “blocking” antibodies. • Excess circulating activated Natural Killer cells

J Soc Gynecol Invest 1997; 4: 267-73; Hum Reprod 1996; 11:1569-74; Am J Reprod Immunol 1999; 41:91-8.

No Benefit to IVIG in RCT & Meta- analysis Alloimmune losses

Design: Double-blind, multi-centered RCT of Paternal WBC immunization (n=91) vs. saline placebo (n=92).

Outcome: Success rate was 31/86 (36%) in treatment group and 41/85 (48%) in the control group (OR 0.60, 95% Cl: 0.33-1.12; p=0.108).

Lancet 1999; 354: 365-9. Recurrent Early Pregnancy Loss Recommendations

1) Parental karyotypes and/or assessment of abortus’ karyotype. If abortus has normal karyotype, perform Array CGH.

2) If parental chromosomal abnormalities (e.g., translocation) consider IVF ± preimplantation screening.

3) If recurrent aneuploid POCs in older couples consider donor gametes; if occur in younger couples continue spontaneous conceptions +/- folate. Recurrent Early Pregnancy Loss Recommendations 4) When karyotypes of abortus’ specimens are euploid and/or the patients losses are intermittent & occur at the same gestational age, analyze placentas of prior losses for trophoblast inclusions. (J Theor Biol. 2003; 225:143-5)

5) If fetal Mendelian disorders detected or suspected (e.g., intermittent secondary euploid RPL), consider further attempts at spontaneous conception or donor gametes.

6) If maternal endometrial implantation disorders are suspected (e.g., recurrent primary losses in a young patient) consider surrogacy (gestational carrier). Recurrent Early Pregnancy Loss Recommendations

7) Evaluate uterus with sonohysterography and 3-D US and with hysteroscopic repair of remediable defects prior to conception.

8) Rule-out APA (LAC, ACA, anti-ß2-glycoprotein-I). If positive x 2 at least 12 weeks apart, treat with LMWH, and LDA in subsequent pregnancy.

Thrombophilia Screening & Treatment 2011 message: Limit screening Wait for trials to conclude

What to order: When to order: 1) PC (activity) 1) Personal history VTE 2) PS (Free antigen) 2) Strong family hx VTE 3) ATIII (activity) 3) RAB: APA, HC or give folate 4) FVL(PCR) 4) Placenta-mediated 5) PGM 20210A (PCR) complications: APA 6) HC, fasting 7) Platelet Count 8) LAC, ACA, B2 GP1 Conclusions 1) The majority of early pregnancy losses are a result of genetically abnormal conceptuses (useful role of PGD in IVF) 2) Application of sophisticated genetic testing (SNP, comparative genomic hybridization) to abortus specimens will likely decrease the percentage of ‘unexplained fetal loss’. 3) Empiric use of heparin and aspirin does not improve pregnancy outcome in patients with recurrent pregnancy loss and genetic thrombophlilia. 4) Implantation events require further investigation. Erlacher A. , NYU Pathology Embryonic day 10.5 36 BACKGROUND IMPLANTATION Mammalian embryo/allograft = the “perfect transplant”

IMPLANTATION

Increased Endometrial receptivity Proinflammation Priming of endometrial stromal cells by ovarian steroids Preimplantation Factor (PIF) 15 Amino Acids , 1612 Daltons (MVRIKPGSANKPSDD)

• Initial Discovery of PIF’s Presence : Roussev 1996, Barnea 1999 • Identification, Sequence, Purification Source: Viable Mouse Embryo Culture Media , Barnea 2007 • Synthetic version with same properties as native peptide , >95% pure (Applied Biosystems) • Isolated from conditioned media by affinity chromatography & HPLC • Sequence by mass spec Fmoc method (US Patent 7, 723,708, B2, Sep 25, 2007) : Preimplantation Factor (PIF) a

Circumsporozoite protein (Plasmodium falciparum) Repeat Region I region Region II

PIF (15) c 352 362

QVRIKPGSANKPKDELDYAN [H]-MVRIKPGSANKPSDD-[OH] * PIF (15) b Active [H]-MVRIKPGSANKPSD-[OH] PIF (14) PIF [H]-MVRIKPGSANKPS-[OH] PIF (13) Region [H]-MVRIKPGSA-[OH] PIF (9) HYPOTHESIS PIF creates a favorable environment for implantation

PREIMPLANTATION Modulate Systemic Immunity PIF

IMPLANTATION Autocrine Survival factor

Antipathogen& Embryo Implantation Endometrial Receptivity

PIF is produced by embryo and binds to immune cells to create tolerance. PIF also binds to maternal endometrium to enhance receptivity and maintain pregnancy 41 Apoptotic Pathway MA HESC: Bcl-2 DL BCL2 P 1 A1 D PBEP1 K C BI R D R T 8 C 8 6

CASP8 TNF BIF4B SP10 HRK

PTC RA SS ZNF COL4 148 RY A2 TR Bcl- PAWR BP 2 2 GA TA3 MD ATF2 M2 F A Bcl-2 Gene S TP7 HESC -21.1 fold 3L FTDC-27.2-fold ERB2IP DST HESC Protein: PIF increased: CAPNS1 (4) cofilin (1.7); Green: Down ZNF BAG2 (4 ) & UCHL1 (2.7) 211 Red: Up prevents ubiquitin degradation. Results • Over 500 genes significantly changed upon exposure to PIF in HESC and FTDC • Over 50 proteins significantly changed upon exposure to PIF in HESC • 21 identical genes and proteins significantly changed; related pathways and networks also affected • Principal pathways affected consist of: Immunity Adhesion Remodelling/apoptosis

RESULTS Alexa-PIF interaction with human proteome array

• Top protein-protein interaction candidate with PIF was insulin-degrading enzyme (IDE), followed by two transcript variants (1 and 3) of K+ voltage-gated channel, shaker- subfamily, beta1 (KCNAB1) gene. PIF EXERTS AUTOCRINE EFFECT ON EMBRYO FITC-PIF uptake by cow embryos Stamatkin CW et al. Reprod Biol Endocrinol. 2011 May 15;9(1):63.

Embryos cultured with FITC-PIF 30 min, washed off non specific Binding, DAPI- nuclear vital stain, FITC-PIFscrambled=control

Mouse PIF: mean +/- SEM *p .05 ** p .01 PIF: SECRETED ONLY BY VIABLE EMBRYOS Single IVF Embryos (cow) PIF levels increase in VIABLE embryos

25 * Blastocyst 20 *

15 Control Day 3

10 Day 7 PIF Level ng/ml 5

0 Control Day 3 Day 7

From Day 3 to 7 culture ANOVA p = .000009 (N= 14/control N=7 matched day 3-7) media at day 3 and 7. PIF not detected in NON-VIABLE embryos

14.00

12.00 Non-cleaving embryo

10.00

8.00

6.00 Level ng/ml

PIF 4.00

2.00

0.00 Control Day 3 Day 7

Similar to Control (IVF) Degenerating embryos (N=5/matched) vs. non- cleaving (N=15) Proprietary Stamatkin CW et al. Reprod Biol Endocrinol. 2011 May 15;9(1):63. Cow: PIF CORRELATES with PREGNANCY OUTCOME Chemiluminescent ELISA

100 93.5% 88.5 % 90 80.6 % 86 % 80

% Delivery % 40

0 Day 9 Day 14 Day 19 U/S Day 28 Delivery

PIF ELISA PIF ELISA PIF ELISA N= 25/31 23/26 29/31 live birth live birth live birth PIF DETECTION – EARLY PREGNANCY Human Maternal Serum

(n=19 patients, IVF pregnancies, normal outcomes)

ng/ml

Moschendrianu, 13th Wld Cong Hum Repr 2009

PIF Mechanism of Action

• FITC - PIF binds all monocytes & macrophages • FITC - PIF binds T, B and NK cells @ lower affinity • FITC - PIF binds most mitogen-induced T and B cells

• PIF blocks stimulated PBMC proliferation & modulates cytokines (mRNA, secretion)

• PIF effect is intracellular - K+ channel-dependent • PIF effect is intracellular – insulin degrading enzyme dependent (IDE) • Inhibition of K+ channel and IDE blocks PIF’s effects

• Dual PIF MoA: maintain basal immunity while modulating activated immunity (Th2 bias)

51 Weiss L, Bernstein S, Jones R, Amunugama R, Krizman D, Jebailey L, Hazan O, Yachtin J, Shiner R, Reibstein I, Triche E, Slavin S, Or R, Barnea ER. Preimplantation factor (PIF) analog prevents type I diabetes mellitus (TIDM) development by preserving pancreatic function in NOD mice. Endocrine. 2011 Mar 22

PIF PREVENTS DIABETES LONG-TERM Adoptive Transfer NOD Model

100

90 6/7 control 80 PIF 0.83 Control

PIF 2.73 70

60 PIF Rx 50 Stop Rx 40 PIF Rx

% Diabetic % 30 PIF 20 1/7

10 0/5 0 10 14 20 30 40 50 60 70 Days Male NOD mice (650 rads) injected with splenocytes from NOD diabetic mice PIF Rx (Alzet pump) for 28 di f/u 40 di, glucose, and IPGTT PIF Rx 0/5, and 1/7, vs. PBS 6/7, P=0.001 Weiss L, et al . Endocrine. 2011 Mar 22

PIF PREVENTS PARALYSIS LONG-TERM: EAE MODEL Spinal Cord Inflammation Reduced

CONTROL INFLAMMATION PIF Rx NORMAL

PIF Rx 0.1 or 0.5mg/kg for 28 di. Alzet pump implanted SQ. Area under the curve p<0.002

Weiss L, Or R, Jones RC, Amunugama R,JeBailey J, Ramu S, Bernstein SA, Yekhtkin Y, Almogi-Hazan O, Shainer R, Vortmeyer A, Paidas MJ, Zeira M, Slavin S, Barnea ER. Preimplantation Factor (PIF) reverses neuroinflammation while Proprietary promoting neural repair in EAE model. J of Neurological Sciences, In Press, 2011. PIF PREVENTS PARALYSIS LONG-TERM: EAE MODEL PIF Effectiveness Lasts Post -Therapy

4 * * * * * * *

3.5

3 Control 2.5 PIF 0.1 mg/ml 2 PIF 0.5 mg/ml 1.5 PIF Rx

Paralysis Paralysis Score 1

0.5

0 0 10 20 30 40 Days

SJL mice injected PLP/Tuberculin/Pertussis, P<0.002 Mann-Whitney Paralysis score 1= tail weakness, 4=paraplegia Proprietary Weiss L, e t al. J of Neurological Sciences, In Press, 2011. Conclusion Preimplantation Factor 1) Is a Unique Embryo Derived Protein 2) Plays a key role in critical steps surrounding implantation 3) Is Conserved in mammals 4) Appears to have a role in pregnancy diagnostics 5) Synthetic form is being developed as a therapeutic agent (with prevention/treatment applications) for a range of immune related diseases, and pregnancy complications. 6) Mandatory Preclinical toxicology studies have conclude 7) Progress is needed concerning its gene discovery and mechanism of action. 8) Applications for human clinical trials will begin once safety is established.

Acknowledgements

Yale Reproductive Biology Unit, Dept Obstet, Gyn, & Reprod Sci: Christina. Duzyj , Graciela Krikun, S. Min Li, Joseph Huang Yale Women and Children’s Center for Blood Disorders: Michael Romano, Jack Annunziato , Salley Pels Yale Fertility Center: Pasquale Patrizio Yale Department of Comparative Anatomy: Carmen Booth, Susan Compton Yale Department of Pathology Alexander Vortmeyer Dept Obstet &Gyn, New Jersey College of Medicine and Dentistry Eytan Barnea Funding: BioIncept (MJP) Talecris (MJP) NIH 5R01HD056123-02 (SJH) NICHD Yale Child Health Research Center training grant (SP, Co- mentors MJP, SJH)

ER Barnea, SIEP PIF Collaboration Discovered PIF PIF Cellular Immunology: Endometrial Biology MJ Paidas, G. Krikun, S. J Huang, P. Patrizio, C. F. Vilmos, B. Kotlan - NCI, Budapest, Hungary Duzyj, Yale U T. Barder - Eprogen, Inc. IL Richard Jones , Next Gene Sci D. Kirk – MDSPS, WA Chandrakant Tayade, Queens U. B. Rivnay - Repromedix, MA L. JeBailey, GeneGo Inc., MI T. Harada - Tottori U, Japan

PIF Dx: IVF & serum human, cow, PIF Therapeutics: horse, pig GVHD / NeuroInflam JDM / Thyroiditis / AutoImm Hepatitis / S. Ramu, C. Stamatkin, R. Roussev, C.B. Coulam Atherosclerosis / TB CARI Chicago, IL. R. Or, S. Slavin, O. A-Hazan, Y. Azar, R. Shainer, L. Weiss - M. Varla-L., M. Promponas, T. Vasilou - Venizileo, Hebrew U BMT. Israel Greece C. O’Brien, A.Tzakis, – Liver U. Miami, FL R.G. Godke, G. Gentry – LSU, Baton Rouge, LO J. Miller, D. Weisdorf, B. Oran – BMT U. Minn MN D. Nash J. Kimber- Aberystwyth U.,UK K. Hadjioanu, P. Limbery - Pasteur Inst- Athens, Greece B. Seguin - U. Minnesota Sch. Agric USA M. Kiocea - Batts, Inc, Tox Los Angeles CA M. Ruble- Iowa State U; A. Cooper, PhD – Trudeau Institute, NY R. Gilbert , V. Absalon - Cornell Vet. Med. F. Ria, Pathology Cattolica U. Rome F. Vailard, P. Rozenberg, Poissy St Ger, France P. KarlHeintz- Baker Inst, Monash U, Melbourne H. Matsubayashi - Osaka, IVF Japan MJ Paidas, Yale, New Haven, CT P. Patrizio- Yale IVF, New Haven, CT MJ Paidas Yale, New Haven, CT 94 96 01 03 06 08 09 10-11

PIF Embryo PIF AA Collaborator Animal trials PIF Patents Pre clin & Bioassay origin sequence agreements completed ELISA clin trial Prep Timeline Thank you Work-up of repetitive implantation failures

A Window into the Reproductive Era Research Congress Center Humanitas Clinical Institute, Via Manzoni 56, 20156 Rozzzano, Milan Oct 1, 2011. Session 4: 10.20-10.40AM