new directions in the evaluation of recurrent miscarriage william h. kutteh, m.d., ph.d., h.c.l.d....

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

http://www.fertilitymemphis.com/default.asp

DISCLOSURES

• None

LEARNING OBJECTIVES

At the conclusion of this presentation, participants should be able to:

1. Discuss the current trends in the diagnosis and treatment of RPL.

2. Describe the different society definitions of “pregnancy” and “RPL”.

3. Appreciate the role of genetic testing in developing a strategy for the evaluation of RPL

4. 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 historyReference Risk of 1

lossRisk 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)

63/19831.3%

Kutteh, 1995*(unselected women)

766/59,0351.3%

Kutteh, WH. Williams Obstetrics. Supp 15:1-4, 1995

*Considered a minimum estimate as many lost to follow up. Population 1/3 hispanic, 1/3 White, 1/3 African-American

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

Maternal Age Incidence of RPL

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 Age10% <35 years 30% 40 years50% 43 years100% > 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, 2012ASRM 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?

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

Frequency of abnormal tests in 1020 women with RPLEVIDENCE 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

Theoretical Incidence of RPL occurring by Chancefor Women with one, two and three miscarriages

AGES (years) 1 miscarriageBy chance

2 miscarriagesBy chance

3 miscarriages By chance

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

Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.

# of prior losses

2 (n=447)

3 (n=343)

4 or more(n=230)

P value2,3,or 4

Evidence based test results

41% 40% 42% NS

Investigative test results

20% 22% 21% NS

Total abnormal test results

61% 62% 63% NS

Frequency of abnormal tests in 1020 women with RPL

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

500gWilliams 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, 2012ASRM 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 ectopicKutteh experience over last 20 years of practiceBarnhart 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


Control # of prior losses

2 n=447

3 n=343

> 4 n=230

P value2,3,or 4

0.4% Parental genetics

2.8% 5.4% 5.2% NS

7.5% Anatomy 18.7% 18.2% 16.7% NS0.5% Lupus

anticoagulant5.0% 2.9% 1.9% NS

6.7% Anticardiolipin 15.6% 13.1% 17.1% NS3.9% TSH 8.1% 6.5% 6.2% NS6.8% Factor V 4.2% 8.1% 10.3% NS


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, 2012ASRM 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

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):2868-71Engels, Am J Med Genet A. 2008 Oct 15;146A(20):2611-6Neri, Am J Med Genet. 1983 Dec;16(4):535-61Stephenson, Hum Reprod. 2006 Apr;21(4):1076-82. Epub 2006 Jan 5Sugiura-Ogasawara, Fertil Steril. 2004 Feb;81(2):367-73.Carp, Fertil Steril. 2006 Feb;85(2):446-50

27

The karyotype results from the parents provides prognostic information for subsequent pregnancies

Karyotype of POC provides prognosis for subsequent pregnancy

Ogasawara, Fertil Steril. 2000 Feb;73(2):300-4Carp, Fertil Steril. 2001 Apr;75(4):678-82 Hassold TJ Am J Hum Genet 1980; 32: 723-730

Euploid Miscarriage

Aneuploid Miscarriage

0

10

20

30

40

50

60

70•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%

% Aneuploid in 2nd Miscarriage

Risk of Aneuploidy based on Maternal AgeSporadic (Control) vs. Recurrent miscarriage

0%

10%

20%

30%

40%

50%

60%

70%

80%

18-29* 30-35* 36-39 >40

Control

RPL

Maternal Age in years

Risk of cytogenetic abnormality in miscarriage

Stephenson et al., Hum Reprod. 2002 Feb;17(2):446-51

* P<0.05

Aneuploidy in Products of Conception

• Monni G, Ibba RM, Zoppi MA. Prenatal Genetic Diagnosis through Chorionic Villus Sampling. In: Milunsky A, Milunsky JM, eds. Genetic Disorders and the Fetus. 6 th 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.

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 0.9 %11 0.5 %5 0.4 %

19 0.2 %1 0 %

59.2%

6.6%

8% of all SAB are 45, X

Possibility exists that aneuploidies on these chromosomes survived longer and thus allowed a karyotype to be obtained from POC

Maternal Cell Contamination: Parental Support Technology vs. Traditional Karyotype

15%

25%47%

13%

46 XX MCC ANEUPLOID 46 XY

37%

49%

14%

46 XX ANEUPLOID 46 XY

600 POC cases analyzed using Parental SupportGSN data. First 600 sequential cases (448 fetal results)

1920 POC cases analyzed using cytogenetic karyotypingMenasha 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)

Ch14.9% Ch2

5.2%

Ch33.9%

Ch43.7%

Ch54.1%

Ch63.9%

Ch74.0%

Ch84.6%

Ch94.9%

Ch104.0%

Ch114.4%Ch12

4.0%

Ch134.2%

Ch144.1%

Ch154.6%

Ch165.8%

Ch174.8%

Ch184.2%

Ch193.5%

Ch204.5%

Ch214.8%

Ch224.7%

X/Y3.1%

1702 SNP microarrays obtained 1404 (82%) Cleavage Stage

298 (18%) Blastocyst Stage759 (45%) Euploid Embryos943 (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



2 n=447

3 n=343

> 4 n=230

P value 2,3,or 4


2.8% 5.4% 5.2% NS

7.5% Anatomy 18.7% 18.2% 16.7% NS0.5% Lupus




Congenital Uterine Anomalies

3-D Sonohysterograpy for the Evaluation of the Uterine Cavity

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)

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).

Jaslow and Kutteh. Fertil Steril 99: 1916-22, 2013.

Comparison of Uterine Anomalies Primary RM compared with Secondary RM

Primary RM(n = 479)

Secondary RM(n = 425) P

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)

0.2(1) ns

Septate uterus

6.3(30)

3.1(13) 0.028

T-shaped uterus

0.4(2)

0.2(1) ns

Unicornuate uterus0.8(4)

0.5(2) ns

Acquired anomalies 14.6(70)

11.8(50) ns

Adhesions 4.0(19)

4.2(18) ns

Fibroid(s) 7.3(35)

5.4(23) ns

Polyp(s) 4.0(19)

2.4(10) nsValues are % occurrence (n). Jaslow and Kutteh. 99:1916-22, 2013.

What is evidence-based?- Autoimmune



2 n=447

3 n=343

> 4 n=230

P value2,3,or 4


2.8% 5.4% 5.2% NS

7.5% Anatomy 18.7% 18.2% 16.7% NS0.5% Lupus




Pathophysiology of aPL IT’S NOT JUST ANTICOAGULATION !

Girardi,Redecha,Salmon. Nature Med 10:1222-1226, 2005

• 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

What is evidence-based?- Endocrine



2 n=447

3 n=343

> 4 n=230

P value2,3,or 4


2.8% 5.4% 5.2% NS

7.5% Anatomy 18.7% 18.2% 16.7% NS0.5% Lupus


6.7% Anticardiolipin 15.6% 13.1% 17.1% NS3.9% TSH 8.1% 6.5% 6.2% NS6.9% HgbA1c 14.1% 16.3% 17.4% NS


Thyroid Function and RPL

Schwartz et al. J Clin Endocrinol Metab 95: 44-48, 2010

• 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

Metformin Improves Pregnancy Outcomes

Zheng and Shan. J Endocrinol Invest 36:797-802, 2013

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)

• Meta analysis of metformin use in pregnant women• All women with PCO by Rotterdam Criteria• Total of 8 studies and 1,106 women

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 KaryotypeAneuploid karyotype Unbalanced chromosomal

translocation or inversion

Perform parental karyotypes and offer

preimplantation genetic diagnosis for future pregnancy attempts

No further evaluation Euploid karyotype

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 RPLEuploid POC After ≥2 Pregnancy Losses

ORAt Least 2 Consecutive Miscarriages With No POC Diagnosis

OrAt Least 3 Nonconsecutive Miscarriages With No POC Diagnosis

Anatomic Evaluation

(Ex: HSG, SHG)

Endocrinologic Evaluation

(EX: TSH, Prolactin, Hyperglycemia)

Targeted Surgical

Correction

Targeted Medical or

Surgical Correction

Add Progesterone

Support to Future

Pregnancies Until 10 Weeks

Gestation

Autoimmune Factors:

aPLLAC

β2GP 1

Start ASA, SQ heparin, Calcium,

& Vitamin D preconceptually

and continue until delivery(Follow CBC)

Evaluation of Lifestyle/Environment(Ex: Caffeine, Tobacco, Alcohol, Environmental

Exposures, Obesity)

Appropriate Alterations to

Lifestyle, Nutrition, or Environment

.

Genetics: Karyotype of Parents if no

POC karyotype Obtained

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

48

Lund et al. Obstet Gynecol 119: 37-43, 2012

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)

new directions in the evaluation of recurrent miscarriage william h. kutteh, m.d., ph.d., h.c.l.d....

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