1. 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

2. DISCLOSURES
None

3. 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

4. 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?

5. 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

6. # 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

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

8. Theoretical Incidence of RPL Based on Maternal Age20
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

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

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

11. 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)

12. 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: , 2012

13. 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.

14. 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: , 2006.
Jaslow & Kutteh. Fertil Steril 93: , 2010

15. 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: , 2012

16. 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: , 2010.

17. 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: , 2011.

18. 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”

19. 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)

20. 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: , 2012

21. 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

22. 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

23. 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

24. 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: , 2010.

25. 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: , 2012

26. 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

27. Prognosis based on parental karyotypes
The karyotype results from the parents provides
prognostic information for subsequent pregnancies
Parents Karyotype Subsequent Miscarriage
Reciprocal translocation %
Robertsonian translocation %
(Exception is translocation to same chromosome)
Inversions %
Normal %
Brigham, Hum Reprod Nov;14(11):
Engels, Am J Med Genet A Oct 15;146A(20):2611-6
Neri, Am J Med Genet Dec;16(4):535-61
Stephenson, Hum Reprod Apr;21(4): Epub 2006 Jan 5
Sugiura-Ogasawara, Fertil Steril Feb;81(2):
Carp, Fertil Steril Feb;85(2):446-50
Unexplained: Hum Reprod Nov;14(11):
A longitudinal study of pregnancy outcome following idiopathic recurrent miscarriage.
Brigham SA, Conlon C, Farquharson RG.
Robertsonian: Am J Med Genet A Oct 15;146A(20):
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 Dec;16(4):
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 Apr;21(4): 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 Feb;81(2):
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 Feb;85(2):
Embryonic karyotype in recurrent miscarriage with parental karyotypic aberrations.
Carp H, Guetta E, Dorf H, Soriano D, Barkai G, Schiff E. 27

28. 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 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 Apr;75(4):
Karyotype of the abortus in recurrent miscarriage.
Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G.
Ogasawara, Fertil Steril Feb;73(2):300-4
Carp, Fertil Steril Apr;75(4):678-82
Hassold TJ Am J Hum Genet 1980; 32:

29. 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 Feb;17(2):
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 Feb;17(2):446-51

30. 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
Kearns WG, er.al Preimplantation genetic diagnosis and screening. Semin Reprod Med Nov;23(4): Review.

31. 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): 31

32. 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.

33. 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.

34. 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: , 2010.

35. Congenital Uterine Anomalies

36. 3-D Sonohysterograpy for the Evaluation of the Uterine Cavity

37. 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: , 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). 

38. 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: , 2013.

39. 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: , 2010.

41. 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: , 2005

42. 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: , 2010.

43. 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

44. 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 ( )
Gestational DM ( )
Preeclampsia ( )
Preterm delivery ( )
Zheng and Shan. J Endocrinol Invest 36: , 2013

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

46. 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.pp ,2013.
Modified from Bernardi et al. Fertil Steril 98: ,2012

47. 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.pp ,2013 .

48. 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 Nov;19(11):536-8.
Tissue sampling technique affects accuracy of karyotype from missed abortions.
Lathi RB, Milki AA. 48

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

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