1. with a history of infertility or miscarriages
Elevated levels of Tc17 and NK17 cells in early pregnancy are associated with spontaneous abortion in women
with a history of infertility or miscarriages
Nadera Mansouri-Attia, PhD
13th Congress of ISIR-ESRI 2016
June 22 – , Erfurt, Germany

2. No conflict of interest
Disclosure statement
No conflict of interest

3. Monitoring of immunologic reactions at the maternal-fetal interface during early pregnancy
Fetus is a semi-allograft
Maternal immune tolerance towards the fetus
Degree of immunosuppression to allow fetal growth
Sufficient immune function to fight infections
Th1/Th2 paradigm during pregnancy:
Th1/Th2: shift towards Th1 bias in threatened miscarriage (Calleja-Agius et al. Gynecol Endocrinol. 2011)
Th1/Th2: use to determine therapies efficiency (Lee et al. Am J Reprod Immunol. 2016)
Helpful but overly simplistic (Chaouat et al. Int Arch Allergy Immunol. 2004)
Treg (Figueiredo and Schumacher. Immunology. 2016):
Maintain homeostasis
Promote fetal survival by inhibiting effector T cells and secreting anti-inflammatory factors such as IL-10 and TGF-β
Fetus
Decidua
Adapted from Weiss et al. Reproductive Sciences. 2009

4. Aims of the Study
Retrospective cross-sectional study to determine association of peripheral blood levels of various immune cell types in relation to pregnancy outcome
Does Th1/Th2 balance correlate with pregnancy outcome in our study?
Use these parameters to monitor pregnancy and detect women “at risk”
Study the effects of immunomodulatory agents such as Neupogen:
- on intracellular cytokine levels
- to counteract these deleterious effects
- to improve the pregnancy outcome

5. Methods
Women with a history of infertility or spontaneous abortion, undergoing an IVF cycle in our center were included in the study
All women who conceived (positive β-hCG) were included regardless of the therapy used
Blood was drawn and levels of CD4+ T cells, CD8+ T cells, NK cells, and NKT cells positive for intracellular cytokines (IL-10, IL-4, TNF-α, IFNγ, IL-17) were measured, as well as levels of Treg cells, at various time points:
T0=pre-pregnancy; T1=4-5 weeks; T2=6-9 weeks; T3=10-12 weeks; T4=13-16; T5=17-20 weeks).
Treg cells were identified as CD3+CD4+CD25hiCD127loFoxP3+ (immunosuppressive)

6. Patient demography Characteristics 278 Age in Y (SEM) 34.65 (0.33)
Infertility
primary
27%
secondary
73%
Previous miscarriage
None
75 (27%) 1
50 (18%) 2
51 (18.3)
3 or more
102 (36.7%)
Previous deliveries
24.82%
Average of previous losses (SEM)
2.10 (0.12)
Duration of infertility (Y)
2.42 (0.23)

7. CD3+CD8+T cells (Tc cells)

8. CD3+CD4+ T cells (Th cells)

9. CD3+CD56+ T (NKT) cells

10. CD3-CD56+ (NK) cells

11. Th1/Th2 ratio does not correlate with pregnancy outcome

12. IL-17-producing cells during early pregnancy
IL-17 is a proinflammatory cytokine that plays a role in host defense against bacteria and fungi but also in the pathogenesis of autoimmune diseases
There are many IL-17 producing cells including CD4+ T cells (Th17), CD8+ T cells (Tc17), NK cells (NK17), and NKT cells (NKT17)
IL-17 producing CD8+ T cells may also be γδ T cells or MAIT cells (Kim and Jordan. Cell Mol Life Sci. 2013)
Tc17 cells are key mediators of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (Gartlan et al. Transplantation. 2015)
While Th17 were previously associated with a negative pregnancy outcome (Figueiredo and Schumacher. Immunology. 2016), nothing is known so far on Tc17 or NK17 in relation to pregnancy outcome

13. CD3+CD8+ IL-17 (Tc17) cells *
T- test:
*, p<.05
**, p<.01
***, p<.001
Miscarriages are associated with a spike in CD3+CD8+ IL-17 production

14. CD3-CD56+ IL-17 (NK/IL-17) cells*
4-5
10-12
13-16
17-20
Gestational age
Miscarriages are associated with a spike in CD3-CD56+ IL-17 production

15. CD3+CD4+ IL-17 (Th17) cells
No correlation between CD3+CD4+ IL-17 production and pregnancy loss.

17. Treg cell trafficking during early pregnancy
Treg cells are enriched in the fetal-maternal interface during early pregnancy
(Mjosberg et al. Biol Reprod 2010)
Treg cells were shown to migrate from the peripheral blood to the decidua in pregnant subjects (Tilburgs et al. Immunol 2008):
This correlates with decreasing Treg cell levels in peripheral blood of pregnant women (Wegienka et al. J Reprod Immunol 2011)
Treg cells might be recruited and expanded by the recognition to fetal antigens (Aluvihare et al. Nat Immunol 2004)
Lower proportion of decidual Treg cells was found in the POC of spontaneous abortion cases vs terminated early pregnancy (Sasaki et al. Mol Hum Reprod 2004)
RM patients have a lower ability to induce and maintain immune
tolerance towards fetal alloantigens

19. Effect on Neupogen (G-CSF) on levels of immune cells positive for intra cellular cytokines in peripheral blood
CONTROL GROUP
NEUPOGEN GROUP
p-value
Characteristics
N=136
N=142
Age (Y)
32 (+/- 1.14)
35.32 (+/-0.44)
 **, p= 0.006
Infertility
primary
44 (32.35%)
31 (21.83%)
ns, p=0.08
secondary
92 (67.65%)
111 (78.17%)
ns, p=0.33
Previous deliveries
41 (30.14%)
28 (19.7%)
Previous miscarriage
None
ns, p=0.32 1
24 (17.64%)
26 (18.31%)
ns, p=0.95 2
25 (18.38%)
ns, p=0.99
3 or more
43 (31.61%)
59 (41.58%)
ns, p=0.30
Duration of infertility (Y)
2.37 (+/- 0.28)
2.48 (+/-0.19)
ns, p=0.7

20. Decrease in levels of peripheral blood Treg cells is enhanced by Neupogen at critical stage of implantation
*** **
T- test:
*, p<.05
**, p<.01
***, p<.001
Reduction of 60% in Neupogen treated patients vs. 20% in control patients at the time of implantation.

21. Neupogen reduced CD3+CD8+ IL-17 in peripheral blood at critical stage of implantation** *
1.8-fold decrease in levels of IL-17 producing CD8+ T cells in Neupogen treated patients at time of implantation.

22. Tc17 and NK IL-17: covariant in correlation with loss
But are they co-dependant?
CD3+CD8+ IL-17 and CD3-CD56+ IL-17 are both required to correlate with a loss in our patient population

23. Conclusions
Th1/Th2 ratio: our data do not support its role a biomarker of pregnancy outcome (supported largely by the literature)
CD3+CD8+ IL-17 and CD3-CD56+ IL-17 increase is associated with a miscarriage during early pregnancy
Higher levels of Treg cells in the peripheral blood reflect a lack of recruitment to the decidua and this is associated with miscarriage
Correlation with the use of Neupogen and the:
- accentuation in Treg cells proliferation and recruitment to the decidua
- inhibition CD4+CD8+ IL-17 production
This could significantly and positively impact the pregnancy outcome

24. Braverman Reproductive Immunology Thanks to all our patients!
Acknowledgements
Braverman Reproductive Immunology
Jeffrey Braverman, MD, FACOG
Darren Ritsick, PhD
Andrea Vidali, MD
Meredith Campbell
Katie Carlson
Medical staff
Reprosource
Benjamin Leader, MD, PhD
Kevin Arnold, PhD
Meenakshi Khare, PhD
Thanks to all our patients!

26. Effect of Neupogen (G-CSF) on IL-17 producing cells

27. Effect of Neupogen (G-CSF) on IL-17 producing cells

28. Effect of Neupogen (G-CSF) on IL-17 producing cells

29. Losses on Neupogen could not be attributed to a spike in Tc17 cells

30. Losses in the control groups could be attributed to a spike in Tc17 cells

31. Successful patients on Neupogen have lower Tc17 levels than successful Controls** *

32. *

33. *
***
***