NEW DIRECTIONS IN THE EVALUATION OF RECURRENT MISCARRIAGE William H. Kutteh, M.D., Ph.D., H.C.L.D. Professor of Obstetrics and Gynecology Vanderbilt University Medical Center

DISCLOSURES None

LEARNING OBJECTIVES At the conclusion of this presentation, participants should be able to: Discuss the current trends in the diagnosis and treatment of RPL. Describe the different society definitions of “pregnancy” and “RPL”. Appreciate the role of genetic testing in developing a strategy for the evaluation of RPL Understand the effect of maternal age and number of prior losses on predicting future live births

CONTROVERSIES in RPL How many losses diagnose RPL? What counts as a pregnancy loss? Should we get karyotypes on parents? Should we get karyotypes on POC? What is the prognosis for a live birth?

Natural miscarriage history Reference Risk of 1 loss Risk of 2 Losses Risk of 3 Losses Alberman, 1988 (study of female MD) 10.4% 2.3% 0.34% Wilcox et al., 1988 (preclinical + clinical) 63/198 31.3% Kutteh, 1995* (unselected women) 766/59,035 1.3% *Considered a minimum estimate as many lost to follow up. Population 1/3 hispanic, 1/3 White, 1/3 African-American Kutteh, WH. Williams Obstetrics. Supp 15:1-4, 1995

# Miscarriages to Define RPL Theoretical Incidence of RPL Based on Number of Miscarriages Used to Define # Miscarriages to Define RPL Incidence of RPL Two 1/45 Three 1/300 Four 1/2000 Five 1/13,000 Six 1/90,000 Seven 1/600,000 Eight 1/4,000,000 Incidence based on mean sporadic miscarriage rate of 15% (=μ). Incidence=μnumber of miscarriages (μ = sporadic miscarriage rate of 15%). Saravelos SH, Regan LR. Obstet Gynecol Clinics N Am. 2014.

Spontaneous Pregnancy Loss: Role of Maternal Age American Society for Reproductive Medicine: Patient’s Fact Sheet: RPL. 2005. Hassold T et al 1980 Ann Hum Genet 44:151-178 7

Theoretical Incidence of RPL Based on Maternal Age 20 1/85 25 1/70 30 1/45 35 1/16 40 1/4 45 1/2 Incidence based on mean sporadic miscarriage rate according to age. Incidence=μ2 (μ = sporadic miscarriage rate for age). Saravelos SH, Regan LR. Obstet Gynecol Clinics N Am. 2014.

Maternal Age is Related to Aneuploidy in oocytes Aneuploidy Risk Maternal Age 10% <35 years 30% 40 years 50% 43 years 100% > 45 years Pellester F, Andreo B,Arnal F, Humeau C, Demaille J Maternal aging and chromosomal anormalities:new data drawn from in vitro unfertilized human oocytes, Hum Genet 112 : 195, 2003

How many losses? -Traditional Three or more spontaneous, consecutive pregnancy losses (fathered by the same partner) Williams OB 21st Edition “most generally accepted definition”

How many losses? - ACOG “RPL is typically defined as two or three or more consecutive pregnancy losses” “Patients with two or more consecutive, spontaneous losses are candidates for an evaluation to determine the etiology” ACOG Practice Bulletin No. 24, February 2001 (withdrawn)

How many losses? - ASRM “RPL is a disease distinct from infertility defined by two or more failed consecutive failed pregnancies” “Clinical evaluation may proceed following two first trimester pregnancy losses” ASRM Committee Opinion Fertil Steril. 99:63, 2012 ASRM Practice Committee Fertil Steril 98:1103-1101, 2012

How many losses? - Insurance After two consecutive losses, most insurance companies will pay for a complete evaluation of recurrent pregnancy loss Kutteh experience over last 20 years of clinical practice.

Does the Number of losses affect the frequency of abnormal findings in women with RPL? Frequency of abnormal tests in 1020 women with RPL EVIDENCE BASED TESTS INVESTIGATIVE TESTS Karyotpe parents Prolactin Evaluate uterine anatomy Antiphosphatidyl serine Lupus anticoagulant Midluteal progesterone Anticardiolipin antibodies Mycoplasma/ureaplasma Thyroid stimulating hormone Factor II (prothrombin) DNA Factor V Leiden DNA MTHFR/Homocysteine Christiansen et al. Semin Reprod Med 24;5-16,2006. Jauniaux etal. Hum Reprod 21:2216-2222, 2006. Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010

Theoretical Incidence of RPL occurring by Chance for Women with one, two and three miscarriages AGES (years) 1 miscarriage By chance 2 miscarriages 3 miscarriages 20-24 11% 1.21% 0.13% 25-29 12% 1.44% 0.17% 30-34 15% 2.25% 0.34% 35-39 25% 6.25% 1.56% Saravelos SH and LiTC. Human Reprod. 27:1882-1886, 2012

Possible RPL Etiologies based on Number of Losses Frequency of abnormal tests in 1020 women with RPL # of prior losses 2 (n=447) 3 (n=343) 4 or more (n=230) P value 2,3,or 4 Evidence based test results 41% 40% 42% NS Investigative test results 20% 22% 21% Total abnormal test results 61% 62% 63% Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.

Spectrum of Pregnancy Loss Pregnancy of Unknown Location (PUL) Early embryonic (< 6 wks) Embryonic (> 6 to 9 wks) Fetal loss (> 9 to 20 wks) Miscarriage (< 20 wks) Stillbirth (> 20 wks) Silver et al. Obstet Gynecol 118: 1402-1408, 2011.

What counts as a Loss? -Traditional Miscarriage is the loss of a pregnancy before 20 weeks of gestation or less than 500g Williams OB 21st Edition “most generally accepted definition”

What counts as a loss? - ACOG “Loss of a recognized pregnancy in the first or early second trimester <15 wks)” “Most are evident by the 12th week and the demise precedes clinical features of pregnancy loss by one or more weeks” ACOG Practice Bulletin No. 24, February 2001 (withdrawn)

What counts as a loss? - ASRM “Pregnancy is defined as a clinical pregnancy documented by ultrasonography or histopathologic examination” ASRM Committee Opinion Fertil Steril. 99:63, 2012 ASRM Practice Committee Fertil Steril 98:1103-1101, 2012

What counts as a loss? - Patient A positive pregnancy test from home (or their doctors office) that does not result in a baby Kutteh experience over last 20 years of clinical practice

What counts as a loss?- My Opinion A pregnancy that is documented by an appropriately rising quantitative hCG that fails Using this definition there is < 7% chance of being an ectopic Kutteh experience over last 20 years of practice Barnhart KT. Obstet Gynecol 104:50-55, 2004

What is a complete workup? - ACOG Karyotypes on both partners Uterine cavity evaluation Glucose level LAC, aCL, β2-glycoprotein (No inherited thrombophilias) ACOG Bulletin No. 24, Feb 2001 (withdrawn) ACOG Bulletin No. 124, September 2011

What is evidence-based?- Genetics Frequency of abnormal tests in 1020 women with RPL Control # of prior losses 2 n=447 3 n=343 > 4 n=230 P value 2,3,or 4 0.4% Parental genetics 2.8% 5.4% 5.2% NS 7.5% Anatomy 18.7% 18.2% 16.7% 0.5% Lupus anticoagulant 5.0% 2.9% 1.9% 6.7% Anticardiolipin 15.6% 13.1% 17.1% 3.9% TSH 8.1% 6.5% 6.2% 6.8% Factor V 4.2% 10.3% Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.

What is a complete workup? - ASRM Karyotypes on both partners Uterine cavity evaluation Prog, PCOS, HgbA1c LAC, aCL, antiβ2 GP1 (No inherited thrombophilias) ASRM Committee Opinion Fertil Steril. 99:63, 2012 ASRM Practice Committee Fertil Steril 98:1103-1101, 2012

Parental Genetic Abnormalities (found in 3-5% of couples with RPL) Reciprocal translocation 59% Robertsonian translocation 27% Inversions 9% Sex chromosome aneuploidy 4% Supernumerary chromosome 1% Balanced translocation

Prognosis based on parental karyotypes The karyotype results from the parents provides prognostic information for subsequent pregnancies Parents Karyotype Subsequent Miscarriage Reciprocal translocation 50-70% Robertsonian translocation 30-50% (Exception is translocation to same chromosome) Inversions 30% Normal 30% Brigham, Hum Reprod. 1999 Nov;14(11):2868-71 Engels, Am J Med Genet A. 2008 Oct 15;146A(20):2611-6 Neri, Am J Med Genet. 1983 Dec;16(4):535-61 Stephenson, Hum Reprod. 2006 Apr;21(4):1076-82. Epub 2006 Jan 5 Sugiura-Ogasawara, Fertil Steril. 2004 Feb;81(2):367-73. Carp, Fertil Steril. 2006 Feb;85(2):446-50 Unexplained: Hum Reprod. 1999 Nov;14(11):2868-71. A longitudinal study of pregnancy outcome following idiopathic recurrent miscarriage. Brigham SA, Conlon C, Farquharson RG. Robertsonian: Am J Med Genet A. 2008 Oct 15;146A(20):2611-6. Genetic counseling in Robertsonian translocations der(13;14): frequencies of reproductive outcomes and infertility in 101 pedigrees. Engels H, Eggermann T, Caliebe A, Jelska A, Schubert R, Schüler HM, Panasiuk B, Zaremba J, Latos-Bieleńska A, Jakubowski L, Zerres KP, Schwanitz G, Midro AT. Reciprocal and Robertsonian: Am J Med Genet. 1983 Dec;16(4):535-61. Reproductive risks for translocation carriers: cytogenetic study and analysis of pregnancy outcome in 58 families. Neri G, Serra A, Campana M, Tedeschi B. Reciprocal, inversions, robertsonian: Hum Reprod. 2006 Apr;21(4):1076-82. Epub 2006 Jan 5. Reproductive outcomes in recurrent pregnancy loss associated with a parental carrier of a structural chromosome rearrangement. Stephenson MD, Sierra S. Tranlsocations: Fertil Steril. 2004 Feb;81(2):367-73. Poor prognosis of recurrent aborters with either maternal or paternal reciprocal translocations. Sugiura-Ogasawara M, Ozaki Y, Sato T, Suzumori N, Suzumori K. Offspring: Fertil Steril. 2006 Feb;85(2):446-50. Embryonic karyotype in recurrent miscarriage with parental karyotypic aberrations. Carp H, Guetta E, Dorf H, Soriano D, Barkai G, Schiff E. 27

Karyotype of POC provides prognosis for subsequent pregnancy % Aneuploid in 2nd Miscarriage If the POC of the first miscarriage are normal, the second miscarriage will be aneuploid in 35% If the POC of the first miscarriage are aneuploid, the second miscarriage will be aneuploid in 65% Fertil Steril. 2000 Feb;73(2):300-4. Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Ogasawara M, Aoki K, Okada S, Suzumori K. Fertil Steril. 2001 Apr;75(4):678-82. Karyotype of the abortus in recurrent miscarriage. Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G. Ogasawara, Fertil Steril. 2000 Feb;73(2):300-4 Carp, Fertil Steril. 2001 Apr;75(4):678-82 Hassold TJ Am J Hum Genet 1980; 32: 723-730

Risk of Aneuploidy based on Maternal Age Sporadic (Control) vs Risk of Aneuploidy based on Maternal Age Sporadic (Control) vs. Recurrent miscarriage Risk of cytogenetic abnormality in miscarriage Hum Reprod. 2002 Feb;17(2):446-51. Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Stephenson MD, Awartani KA, Robinson WP. * P<0.05 Maternal Age in years Stephenson et al., Hum Reprod. 2002 Feb;17(2):446-51

Aneuploidy in Products of Conception Chromosome Number % of All Trisomies 16 24.7 % 22 13.9 % 21 12.3 % 15 8.3 % 13 6.8 % 18 4.8 % 14 4.4 % 7 3.4 % 2 3.2 % 8 3.0 % 9 2.9 % 4 2.8 % 20 2.7 % 10 1.5 % 12 1.2 % 6 1.0 % 3 0.9 % 17 11 0.5 % 5 0.4 % 19 0.2 % 1 0 % 59.2% Possibility exists that aneuploidies on these chromosomes survived longer and thus allowed a karyotype to be obtained from POC 8% of all SAB are 45, X 6.6% Monni G, Ibba RM, Zoppi MA. Prenatal Genetic Diagnosis through Chorionic Villus Sampling. In: Milunsky A, Milunsky JM, eds. Genetic Disorders and the Fetus. 6th ed. Oxford, UK: Wiley-Blackwell. 2010. Kearns WG, er.al Preimplantation genetic diagnosis and screening. Semin Reprod Med. 2005 Nov;23(4):336-47. Review.

Maternal Cell Contamination: Parental Support Technology vs Maternal Cell Contamination: Parental Support Technology vs. Traditional Karyotype [Data pull 5/18/11; GSN aneuploidy rate 51%, Menasha aneuploidy rate 55%] Male: 79/600 (13%); Female: 89/600 (15%); MCC: 152/600 (25%); Aneuploidy: 280/600 (47%) NOTE: Overall, 46,XX makes up 40%(241/600)– of these 63% (152/241) are MCC. Currently, we have an 8% conversion rate. 600 POC cases analyzed using Parental Support GSN data. First 600 sequential cases (448 fetal results) 1920 POC cases analyzed using cytogenetic karyotyping Menasha et al. Genetics in Medicine 2005; 7(4): 251-264 31

Aneuploidy Exists in all Chromosomes Aneuploidy in the developing embryo exists at significant rates in all 23 pairs of chromosomes at both the cleavage and blastocyst stage as identified by SNP microarray PGS preformed on couples with > 2 previous pregnancy losses Brezina, Kearns, Kutteh. J Assist Reprod Genetics. In Press, 2014.

Results: % Aneuploidy by Chromosome in RPL (After PGS on 1702 embryos from RPL patients) 1702 SNP microarrays obtained 1404 (82%) Cleavage Stage 298 (18%) Blastocyst Stage 759 (45%) Euploid Embryos 943 (55%) Aneuploid Embryo Significant levels of aneuploidy occurs in all chromosomes during early human Embryogenesis Range of aneuploidy was From 3.1% to 5.8% Brezina, Kearns, Kutteh. J Assist Reprod Genetics. In Press, 2014.

What is evidence-based?- Anatomy Frequency of abnormal tests in 1020 women with RPL Control # of prior losses 2 n=447 3 n=343 > 4 n=230 P value 2,3,or 4 0.4% Parental genetics 2.8% 5.4% 5.2% NS 7.5% Anatomy 18.7% 18.2% 16.7% 0.5% Lupus anticoagulant 5.0% 2.9% 1.9% 6.7% Anticardiolipin 15.6% 13.1% 17.1% 3.9% TSH 8.1% 6.5% 6.2% 6.8% Factor V 4.2% 10.3% Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.

Congenital Uterine Anomalies

3-D Sonohysterograpy for the Evaluation of the Uterine Cavity

consecutive patients with RM. Prevalence of uterine anomalies among 904 consecutive patients with RM.   % occurrence (n) Total frequency of anomaliesa 19.5 (176) Congenital anomalies 6.7 (61) Bicornuate uterus 0.8 (7) Didelphic uterus 0.2 (2) Septate uterus 4.8 (43) T-shaped uterus 0.3 (3) Unicornuate uterus 0.7 (6) Acquired anomalies 13.3 (120) Adhesions 4.1 (37) Fibroid(s) 6.4 (58) Polyp(s) 3.2 (29) Jaslow and Kutteh. Fertil Steril 99: 1916-22, 2013. aFive patients (0.6%) had both congenital and acquired anomalies. The combinations were septum and adhesions, septum and fibroid(s), septum and polyp(s), bicornuate uterus and fibroid(s), and unicornuate uterus and polyp(s). 

Comparison of Uterine Anomalies Primary RM compared with Secondary RM All uterine anomalies 22.8 (109) 15.8 (67) 0.009 Congenital anomalies 8.8 (42) 4.5 (19) 0.011 Bicornuate uterus 1.0 (5) 0.5 (2) ns Didelphic uterus 0.2 (1) Septate uterus 6.3 (30) 3.1 (13) 0.028 T-shaped uterus 0.4 (2) Unicornuate uterus 0.8 (4) Acquired anomalies 14.6 (70) 11.8 (50) Adhesions 4.0 (19) 4.2 (18) Fibroid(s) 7.3 (35) 5.4 (23) Polyp(s) 2.4 (10) Values are % occurrence (n). Jaslow and Kutteh. 99:1916-22, 2013.

What is evidence-based?- Autoimmune Frequency of abnormal tests in 1020 women with RPL Control # of prior losses 2 n=447 3 n=343 > 4 n=230 P value 2,3,or 4 0.4% Parental genetics 2.8% 5.4% 5.2% NS 7.5% Anatomy 18.7% 18.2% 16.7% 0.5% Lupus anticoagulant 5.0% 2.9% 1.9% 6.7% Anticardiolipin 15.6% 13.1% 17.1% 3.9% TSH 8.1% 6.5% 6.2% 6.8% Factor V 4.2% 10.3% Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.

Pathophysiology of aPL IT’S NOT JUST ANTICOAGULATION ! Inhibit hCG release from placental explants Block of in vitro trophoblast migration &invasion Inhibit formation of giant, multinucleated cell Inhibit of trophoblast cell adhesion molecules (alpha 1 and 5 integrins, E and VE cadherins) Activate complement on the trophoblast surface inducing an inflammatory response Girardi,Redecha,Salmon. Nature Med 10:1222-1226, 2005

What is evidence-based?- Endocrine Frequency of abnormal tests in 1020 women with RPL Control # of prior losses 2 n=447 3 n=343 > 4 n=230 P value 2,3,or 4 0.4% Parental genetics 2.8% 5.4% 5.2% NS 7.5% Anatomy 18.7% 18.2% 16.7% 0.5% Lupus anticoagulant 5.0% 2.9% 1.9% 6.7% Anticardiolipin 15.6% 13.1% 17.1% 3.9% TSH 8.1% 6.5% 6.2% 6.9% HgbA1c 14.1% 16.3% 17.4% Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.

Thyroid Function and RPL Increased pregnancy loss rate in thyroid antibody negative women with TSH levels between 2.5 and 5.0 in the first trimester 4,123 women prospectively evaluated Miscarriage rate doubled in group with TSH 2.5 to 5.0 compared to below 2.5 group Endocrine Society Guidelines advise TSH between 1.0 and 2.5 for pregnancy Schwartz et al. J Clin Endocrinol Metab 95: 44-48, 2010

Metformin Improves Pregnancy Outcomes Meta analysis of metformin use in pregnant women All women with PCO by Rotterdam Criteria Total of 8 studies and 1,106 women OUTCOME Odds Ratio (95% CI) Miscarriage 0.32 (0.19-0.55) Gestational DM 0.37 (0.25-0.56) Preeclampsia 0.53 (0.30-0.95) Preterm delivery 0.30 (0.13-0.68) Zheng and Shan. J Endocrinol Invest 36:797-802, 2013

What about Lifestyle Factors. Risks of miscarriage increase 1 What about Lifestyle Factors? Risks of miscarriage increase 1.5 -2 fold Tobacco (>15/day) Ethanol (> 5/week) Obesity (BMI > 30) Caffeine (> 2-3 cups/day)

Initial Evaluation for Early RPL Miscarriage #1 (No action unless clinically indicated 2nd Miscarriage Obtain Miscarriage Karyotype Unbalanced chromosomal translocation or inversion Aneuploid karyotype No further evaluation Euploid karyotype Perform parental karyotypes and offer preimplantation genetic diagnosis for future pregnancy attempts RPL Workup Brezina and Kutteh. Clin Reprod Med Surg. 2nd Ed.pp197-208,2013. Modified from Bernardi et al. Fertil Steril 98:156-161,2012

Workup for Early RPL Euploid POC After ≥2 Pregnancy Losses OR At Least 2 Consecutive Miscarriages With No POC Diagnosis Or At Least 3 Nonconsecutive Miscarriages With No POC Diagnosis Anatomic Evaluation (Ex: HSG, SHG) Endocrinologic Evaluation (EX: TSH, Prolactin, Hyperglycemia) Add Progesterone Support to Future Pregnancies Until 10 Weeks Gestation Autoimmune Factors: aPL LAC β2GP 1 Evaluation of Lifestyle/Environment (Ex: Caffeine, Tobacco, Alcohol, Environmental Exposures, Obesity) Genetics: Karyotype of Parents if no POC karyotype Obtained Targeted Surgical Correction Targeted Medical or Surgical Correction Start ASA, SQ heparin, Calcium, & Vitamin D preconceptually and continue until delivery (Follow CBC) Appropriate Alterations to Lifestyle, Nutrition, or Environment Preimplantation Genetic Testing if Appropriate and Desired: PGS/PGD Brezina and Kutteh. Clin Reprod Med Surg. 2nd Ed.pp197-208,2013 .

What about True Unexplained RPL? Current evaluation completed Test results all return as normal Chromosomes on POC are normal Subsequent live birth is 40% to 80% Depends on maternal age Depends on number of prior losses J Assist Reprod Genet. 2002 Nov;19(11):536-8. Tissue sampling technique affects accuracy of karyotype from missed abortions. Lathi RB, Milki AA. 48

Chance of Live Birth based on # Prior Losses Current Diagnostic and Treatment Strategies (n-665) Lund et al. Obstet Gynecol 119: 37-43, 2012

Chance of Live Birth based on Maternal Age Current Diagnostic and Treatment Strategies (n=665) Lund et al. Obstet Gynecol 119: 37-43, 2012