1 Genomic Medicine of the Future UTERINE LAVAGE IN VIVO FERTILIZATION PGD/PGS WITHOUT IVF John E. Buster, M.D., Professor Obstetrics and Gynecology, Warren Alpert Medical School of Brown University, Women and Infants Hospital, Providence, RI

2 Disclosure The speaker is Founder and Chief Medical Officer for Previvo Genetics, LLC

3 PGD/PGS WITHOUT IVF PGD (Affected child, parents, or carrier) PGS (Aneuploidy) Recurrent miscarriage Some infertility Method of embryo donation-No IVF Fertility preservation

5 5 Historical Overview of Uterine Lavage In 1984 A UCLA research team performed the world’s first ever donor human embryo transfer using “uterine lavage” for recovery of an in vivo blastocyst

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16 Data from Initial Studies Morphology of human ova recovered by uterine lavage from fertile women with single spontaneous ovulations and timed inseminations abstracted from five reports from two independent centers InsemTotal OvaUnidvidFrag2-18MorulaBlast UCLA (1- 3) Pavia (4,5) Total148*67* Buster JE et al: Am J Obstet. Gynecol. 1985, 153: Sauer MV et al: Fertil Steril. 1987, 47: Sauer MV et al: Obstet Gynecol. 1988, 71: Formigli L et al: Fertil Steril. 1987, 47: Formigli L et al: Personal Communication, * Recovery efficiency for all ova was 67/148 or 45%.

17 Data from Initial Studies Uterine embryo morphology and pregnancy outcome after transfer of a single in vivo embryo from donor to recipient women. A total of 14 intrauterine pregnancies were produced in recipient women with 10 deliveries and 4 spontaneous abortions. MorphologyUCLA (1-3) Pavia (4,5) Total 2-180/172/62/23 (9%)* Morula0/23/63/8 (37.5)* Blastocysts4/85/79/15 (60%) 1. Buster JE et al: Am J Obstet. Gynecol. 1985, 153: Sauer MV et al: Fertil Steril. 1987, 47: Sauer MV et al: Obstet Gynecol. 1988, 71: Formigli L et al: Fertil Steril. 1987, 47: Formigli L et al: Personal Communication, * Recovery efficiency for all ova was 67/148 or 45%. * There were at least two additional pregnancies that were retained in the donors. In addition, five embryos were transferred in the Formigli series from lavages that were done between days 2 and 4. All of these (total 7 embryos) would have been blastocyst if recovered on day 5.

’s vs 2015 Uterine Lavage was 30 years ahead of its time 1980’s2015 Crude devices 45% recoveryAdvanced Catheter/controller unit: 90% + Superovulation not safeSuperovulation safe Retained/ectopic pregnancy riskAntagonist shut down minimal pregnancy risk Crude devices, lavage trauma, large fluid volumes, limited flow control 2 nd generation devices/controller, atraumatic, small flluid volumes, precise fluid control PGD/PGS Genomic technology severely limitedPGD/PGS Genomic technology highly evolved Negative Public opinion Supportive public opinion

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Uterine Lavage: A Novel Portal To The Human Embryonic Genome “In Vivo PGD And PGS Without IVF”

IN VIVO PGD & PGS WITHOUT IVF Antagonist FSH. Partner IUI Blastocysts Recovered PGS: Euploid blasts vitrified PGD: Unaffected blasts identified SET: Unaffected blasts. - Cryopreservation Trophectoderm Bx and Dx Lavage Insemination Superovulation Single Embryo Transfer

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In Vivo Insemination Fertilization

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Uterine Lavage/Recovery

CRYOPRESERVATION Using established technology... To be returned in the next cycle... Or many years later...

Transfer

Advantages of the Procedure Uterine Lavage Office procedure No operating room No anesthesia Highly automated Economical Recovers in vivo blastocysts 30

Advantages of the Procedure High Transfer efficiencies Multiple trophectoderm cells for diagnosis Efficient cryopreservation and thawing In vivo fertilization, no IVF SET reduction/elimination of multiples Day 5 In Vivo Blastocysts 31

32 Why in vivo Blasts Matter 1.Conception in vivo: Vitality uncompromised by IVF culture 2.In vivo blastocyst implantation/pregnancy rates > IVF 3.Trophectoderm for diagnosis: a.Shipped for analysis anywhere b.Blastocyst bx safer/more informative than cleavage 5.Vitrification with return to patient after Dx 6.Positive Public Perception: Moral ethical genetic plan for pregnancy-not an established pregnancy. Can end need for pregnancy termination in practice of genetic medicine. The Single Most Important Rationale for Uterine Lavage is Access to the In Vivo Blastocyst

33 Comparative Trials In Animal Models: In Vivo Embryos Outperform IVF IVF embryos lower implantation/development/births IVF embryos developmentally delayed IVF blasts fewer cells per embryo IVF embryos improve when transferred to in vivo IVF culture induces aberrant gene expression Limited human trials (1980’s): Single in vivo blast transfers 60% ongoing pregnancies

34 IVF Embryos Lower Implantation/Development/Births Bovine: In vivo increased implantation Farin PW et. al Porcine: In vivo higher transfer efficiencies Stokes PJ et al Mouse: In vivo blastocysts formed fetuses at higher rates than IVF blastocysts Thouas et al, Reproduction 2003

35 IVF Embryos Are Developmentally Delayed Ovine : In vivo blasts increased hatching rates Garcia-Garcia et al, Anim Reprod Sci 2007 Mouse: IVF reduced blast formation rates Jurisicova et al, Mol Hum Reprod 1998

36 IVF Blasts Have Fewer Cells Per Embryo Mice: In vitro cultured blastocysts have fewer cells at comparable stage than in vivo Colver et al, Fertil Steril 1991

37 IVF Embryos Improve When Transferred To An In Vivo Environment Ovine: In vitro blastocysts have higher survival when transferred to an in vivo environment Garcia-Garcia et al, Theriogenology 2005 Mouse: Low viability in vitro embryos have restored viability after transfer to an in vivo enviroment Evsikov, et al, J Exp Zool 1996

38 IVF Culture Induces Aberrant Gene Expression Porcine: In vitro embryos have aberrant expression of transcriptional regulators in excess of in vivo Hyttel et al, Hum Reprod 2000

39 Data from Initial Studies Uterine embryo morphology and pregnancy outcome after transfer of a single in vivo embryo from donor to recipient women. A total of 14 intrauterine pregnancies were produced in recipient women with 10 deliveries and 4 spontaneous abortions. MorphologyUCLA (1-3) Pavia (4,5) Total 2-180/172/62/23 (9%)* Morula0/23/63/8 (37.5)* Blastocysts4/85/79/15 (60%) 1. Buster JE et al: Am J Obstet. Gynecol. 1985, 153: Sauer MV et al: Fertil Steril. 1987, 47: Sauer MV et al: Obstet Gynecol. 1988, 71: Formigli L et al: Fertil Steril. 1987, 47: Formigli L et al: Personal Communication, * Recovery efficiency for all ova was 67/148 or 45%. * There were at least two additional pregnancies that were retained in the donors. In addition, five embryos were transferred in the Formigli series from lavages that were done between days 2 and 4. All of these (total 7 embryos) would have been blastocyst if recovered on day 5.

40 Clinical Applications PGD (Affected child, parents, or carrier) PGS (Aneuploidy disorders) Recurrent miscarriage Some infertility Method of embryo donation-No IVF Fertility preservation

Future Clinical Impact Medical device is the only portal to the human embryonic genome in vivo. Embryonic diagnosis in vivo will become more widely available First pregnancies and births should immediately follow blast recoveries expected in 2015 Will facilitate study of human in vivo implantation and embryonic development never before possible

2015 Full Validation of Uterine Lavage System Lavage Cycle Effectiveness Effective Blastocyst Recovery Limited Market Release First Pregnancies Publications Further Platform Validations

43 Thank You John E. Buster, M.D.