From bench to bedside in reproduction

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From bench to bedside in reproduction

• Siobhan Quenby
• Professor Of Obstetrics
• University of Warwick

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What is recurrent miscarriage?

• 3 consecutive miscarriages
• Very distressing
• Occurs over 1-2 years
• Increasingly desperate for baby
• 3 couples trying for a baby
• 600,000 births in UK per year
• 18,000 couple in UK per year
• 50 cases no known cause in 30 blood tests

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Historical perspective

• Definition RM 3 consecutive pregnancy losses
  before the 20th weeks
• Sporadic miscarriage rate is 15
• RM rate 0.153 0.3-0.4.
• The actual prevalence of RM is 1-3

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Karyotypical abnormality

• High (29-57) in RM population
• Stern et al., 1996,
• Ogasawara et al., 2000,
• Carp et al., 2001,
• Stephenson et al., 2002
• Same rate recurrent and spontaneous miscarriage

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Three treatments

• Anti-thrombotic
• Aspirin
• Heparin
• Immunotherapy
• -IVIG
• Hormones
• Progesterone

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One treatable cause APS

• ACA
• IgG, IgM
• Lupus anticoagulant
• DRVVT
• Platelet neutralisation
• 2 ve tests six weeks apart

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Antithrombotic

• APS
• Cochrane review Empson et al., 2010

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Pathophysiology APS

• First trimester
• Placental histology
• not thrombosis or infarcts
• (Sebier et al., 2003)
• Lack of trophoblast invasion
• (Sebier et al., 2002)
• Second trimester
• Placental thrombosis is identified

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Idiopathic first trimester miscarriage

• Canada, Laskin et al 2008 N88
• included APS, thrombophilia, antinuclear
  antibodies
• Asp 78 V aspLMWH 79
• Metanalysis Cochrane -2009 N189, Idiopathic
• Asp 81 v placebo 81
• Asp 82 v LMWH 84
• Netherlands ALIFE N299, Kaandorp et al 2010
• Included idiopathic, thrombophilia, excluded APS,
  2 miscarriages
• Asp 62 V asp LMWH 69 v placebo 67
• Scotland, SPIN, N294, Clark et al., 2010
• Included idiopathic, thrombophilia, excluded APS,
  2 miscarriages
• Standard care 80V aspLMWH 78
• HABENOX N207, Visser et al., 2010 n207
• Idiopathic, 3 miscarriages
• LMWH Placebo 71 V LMWH and aspirin 65 V
  Aspirin 61

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Aspirin

• Netherlands study
• Placebo v aspirin
• Absolute risk that causes miscarriage is 5.2
• CI (-6.1-16.6)
• Kaandorp et al 2010

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Mechanism miscarriage ?

• Excessive oxidative stress
• (Burton and Jauniaux J Soc Gynecol Investig
  2004113425)

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Remaining questions

• Heparin and aspirin work second trimester loss?
• Type and dose?
• Recurrent IVF failure?

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Progesterone
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Sub clinical hyopthyroidismReid et al., 2010
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Immunotherapy

• Porter et al 2010 Cochrane

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Cochrane Porter 2010

• A specific assay to diagnose immune-mediated
  early pregnancy loss and a reliable method to
  determine which women might benefit from
  manipulation of the maternal immune system are
  urgently needed

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Secondary RMStephenson et al., 2010
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Uterine Natural Killer cells in womb lining
patient with two normal deliveries
Patient who had ten miscarriages
uNK cells more numerous in RM Quenby et al,
1999,2005 Clifford et al, 1999, Tuckerman et
al., 2007
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Case History

• 17 consecutive miscarriages
• No Cause found
• Most NK cells in study n40
• Preconceptual prednisolone 5mgs
• Two further miscarriages
• Higher dose (prednisolone 20mg)
• Live Birth aged 42
• (IUGR 32/40)
• Alive and well age 4 years
• Quenby et al., 2004

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Quenby et al., 2005 Fert Steril
uNK cells
P0.0009
Normal range
Before Prednisolone
After prednisolone
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Uterine NK cells studies finding more cells in RM
Study N Inclusion criteria Method Results
Lachapelle et al., 1996 20 3 or more idiopathic Flow cytometry D18-25 ?CD16 D56dim ? of CD16-CD56bright cells
Quenby et al., 1999 22 3 or more idiopathic Immunohistochemistry D19-22 Manual counting ? of CD16 D56 cells c.f. controls
Clifford et al., 1999 29 3 or more idiopathic Immunohistochemistry D20-23 Manual counting ? CD56 cells in RM lt13 wk c.f. controls
Emmer et al., 2002 9 2 or more idiopathic Immunohistochemistry Tissue collected after miscarriage Manual counting ? expression of CD56 CD16
Dosiou and Giudice 2005
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Uterine NK cells studies finding no difference
in RM
Study N Inclusion criteria Method Results
Shimada et al., 2004 17 2 or more idiopathic Flow cytometry Midluteal phase No difference in of CD56, CD56CD16- or CD56CD16 cells
Michimata et al., 2002 20 2 or more idiopathic Immunohistochemistry D18-21 Manual counting No difference in numbers of CD56or CD16 cells
In summary, there is likely no difference in the
percentage of total uNK cells in endometrial
leukocytes of women with RPL compared with women
without PL. Dosiou and Giudice
2005 histological evaluation of endometrium is
limited
Kim-Kwak and Gilman-Sachs 2008
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Predict outcome?
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Predict outcome?
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Cell counting

• Manual hand counting under microscope
• Manual counting of digital images
• Digital Image analysis

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Manual counting

• Time consuming
• Different fields analysed
• Retrospective checking of counts
• Observer dependent fatigue, distractions
• Poor intra and inter observer agreement
• Risk to observer RSI

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Digital image analysis

• Semi- automated
• Manual thresholding
• PC Freeware
• Constants size, circularity parameters

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Results
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New developments -
Study N Inclusion criteria Method Results
Quenby et al., 1999 22 3 or more idiopathic Immunohistochemistry D19-22 Manual , Frozen Liverpool patients ? of CD16 D56 cells c.f. controls
Quenby et al., 2005 85 3 or more idiopathic Immunohistochemistry D19-23 Manual, Frozen UK wide ? CD56 , CD16 -, CD3 cells c.f. controls
Drury et al., 2007 120 3 or more idiopathic Immunohistochemistry LH 7 Paraffin, Image analysis UK wide ? CD56 , CD16-, CD3- cells
Tuckerman et al., 87 3 or more idiopathic Immunohistochemistry LH 7 Paraffin ? CD56 ,
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NATURE MEDICINE 2006
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More blood UNK cells more blood flow
Recurrent miscarriage Recurrent IVF failure
Quenby et al., 2009
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Prednisolone treatment reduces endometrial
angiogenic growth factor expression at LH7
Lash 2008
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Figure 2
A
B
C
D
E
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Observational studyPrednisolone in early
pregnancy n22

• Included
• gt5 miscarriages,
• high uNK cells preconceptually
• Age gt 35
• Expect 40 live birth rate
• Outcome to date
• 16 term deliveries
• 6 miscarried
• 73 live birth rate

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A randomised controlled trial of prednisolone for
women with recurrent miscarriage and high levels
of uNK cells in the endometrium.

• Funded by The Moulton Charitable Foundation

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Inclusion

• gt2 consecutive idiopathic first trimester
  miscarriages
• gt5 of endometrial cells CD56,
• Age 20 - 40.

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Exclusion

• Known cause for pregnancy losses
• APS
• parental balanced translocation,
• uterine anomaly
• subseptate uterus, cervical weakness
• known thrombophilia
• Contraindications to steroid therapy
• hypertension, diabetes, mental health problems,
  obesity BMIgt230

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Outcome measures

• Primary
• Live birth rate
• Secondary
• Miscarriages
• First/second trimester losses
• Karyotype of miscarried pregnancies
• Still births
• Obstetric complications
• IUGR, Pre-eclampsia, abruption, gestation at
  delivery
• Fetal abnormality
• Side effects of steroids

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Feasibility trial

• Aims
• See if recruitment possible
• Test trial procedures
• Recruit 40 women

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Demographics
Placebo Prednisone
Age (mean) 33 34
BMI (mean) 26 26
No who had LB 3 4
NO Miscarriages (median) 4 4
uNK (median) 5.7 7.2
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Screened 160 women gt2 miscarriages lt40 Had timed
biopsy
68 (43) Screen ve
40 women conceived randomized
Prednisolone 20 women
Placebo 20 women
miscarriages 6 fetus, 3 sac 3
ectopic Karyotype 1/2 trisomy 22 1/2 normal
Live Birth 8 women 40
Live birth 12 women 60
Miscarriages 4 fetus, 3 sacs 1 molar Karyotype 1/3
trisomy 22 2/3 normal
Relative risk 1.5 95 CI 0.8-2.9 absolute risk
difference 20 95 CI 11-48
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Power calculation 90 power 5 a
Screened 850 women gt2 miscarriages lt40 timed
biopsy
340 (40) Screen ve
214 women conceived randomized
Prednisolone 107 women
Placebo 107 women
Live Birth 43 women 40
Live birth 64 women 60
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Success in control group
Author date patients control Live birth rate
Kutteh 1996 APS aspirin 44
Rai 1997 APS aspirin 42
Farquharson 2002 APS aspirin 72
Laskin 2009 RM -all aspirin 78
Cochrane heparin 2009 idiopathic Aspirin placebo 82 81
Kaandorp 2010 idiopathic Aspirin placebo 67 62
Clark 2010 idiopathic Intensive care 80
El-Zibdeh 2005 idiopathic placebo 70
Cochrane IVIG 2010 Idiopathic control 60
Stephenson 2010 secondary placebo 62
Visser 2010 idiopthic Aspirin 61
Quenby 2010 Endometrial raised NK cell placebo 40
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Live birth rates
weeks general pop 0 previous miscar- riages 1 previous miscar- riage 2 previous miscar- riages 3 previous miscar- riages 4 Previous miscar- riages previous live birth
Biochem- ical lt5 75
Clinical 5-10 88 94 86 77 72 58 95
First trimester 10-12 97
reference Bottomley 2009 Bhattacharya et al., 2010 Bhattacharya et al., 2010 Bhattacharya et al., 2010 Bhattacharya et al., 2010 Bhattacharya et al., 2010 Bhattacharya et al., 2008
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What next

• EME grant trial with clinical and science
  outcomes?
• Warwick CTU
• Clinical live birth rate
• Science
• Protien damage- Thornalley
• Endometrial differentiation Brosens
• Angiogensis- Bulmer
• Geneotyping ? Correlated outcome