Adaptation and Modulation of Memory and Regulatory T Cells in Pregnancy
Kieffer, Tom Eduard Christiaan
DOI:
10.33612/diss.97355536
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Publication date: 2019
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Kieffer, T. E. C. (2019). Adaptation and Modulation of Memory and Regulatory T Cells in Pregnancy. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.97355536
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9
Prednisolone treatment during early
pregnancy disrupts maternal immune
adaptation and modulates offspring
development in mice
Manuscript in preparation
Tom E.C. Kieffer
1,2Peck Y. Chin
1Ella S. Green
1Lachlan M. Moldenhauer
1Jelmer R. Prins
2Sarah A. Robertson
11Robinson Research Institute & School of Paediatrics and Reproductive
Health, University of Adelaide, Adelaide, South Australia, Australia
2Department of Obstetrics and Gynaecology, University Medical Center
ABSTRACT
Prednisolone is increasingly utilized in women undergoing In Vitro Fertilization (IVF) treatment in the hope of improving embryo implantation rates, purportedly by lowering the risk of immunological rejection through its immunosuppressive effects. However, active processes of the maternal immune system, including generation of regulatory T (Treg) cells, are essential for pregnancy success. To investigate the effects of prednisolone treatment in the peri-implantation phase on Treg cell generation, pregnancy outcome, and fetal development, female mice were administered pred-nisolone on day 0.5 until 2.5 post coitum (pc). Predpred-nisolone disrupted the normal expansion of CD4+ cells, Treg cells, and effector T cells within the para-aortic lymph nodes (PALN) at day 3.5 pc. IFNG+ Treg cell and IFNG+ effector cell counts in the PALN were lower at day 9.5 pc in mice treated with prednisolone. Pregnancy rates were unaffected, but the number of implantation sites was higher after prednisolone treatment and placental and birth weights were lower. Furthermore, body weights of offspring after prednisolone treatment were altered in a sex dependent manner into adult life. Male offspring exposed to prednisolone in utero had lower adrenal weights at week 20 after birth compared to males born after placebo treatment. We conclude that prednisolone interferes with normal maternal immune adaptation to increase litter size, but this has consequences for offspring growth and development. These results emphasize the necessity for more studies to investigate the safety of prednisolone use during early pregnancy in women.
INTRODUCTION
In recent years, prednisolone has been increasingly utilized off-label during in vitro fertilization (IVF) treatment in women with suspected implantation failure and women suffering from recurrent pregnancy loss1,2. For these indications, prednisolone is pre-scribed in the peri-implantation phase and the first trimester in the hope of improving embryo implantation rates and protecting against pregnancy loss. The underlying justification is that prednisolone acts to suppress an assumed abnormal maternal immune response and so promotes trophoblast proliferation and invasion, thereby improving the quality of implantation and likelihood of establishing pregnancy3–6. However, the contemporary consensus view is that specific and well-regulated acti-vation of innate and adaptive immune cells in the endometrium - rather than physical exclusion or broad immune suppression - is favorable for successful implantation and pregnancy success7,8. Moreover, there is no clinical proof of effectiveness and safety of this use of prednisolone, and indeed a Cochrane review and recent studies, show
lack of efficacy except for women with specific autoimmune disorders5,6,9,10. Thus, the validity of its wider use is under debate.
Prednisolone is the most commonly used corticosteroid drug in pregnancy and is well known to act through the constitutively-expressed glucocorticoid receptor and mineralocorticoid receptor inducing various anti-inflammatory and immunosuppressive responses in target cells1,11–13. Prednisolone exerts non-specific suppressive effects on natural killer (NK) cells, dendritic cells and macrophages, which all contribute to implantation and early placental development through tissue remodeling effects particularly in the uterine vasculature13–15. Importantly, the T-lymphocyte response is suppressed by prednisolone in a manner that affects all T cell subsets, regardless of their immune effector or immune regulatory function7,13,14,16,17. T-lymphocytes, and in particular regulatory T lymphocytes (Treg cells) have important roles in the peri-implan-tation phase and in pregnancy thereafter18. Treg cells, identified by expression of the transcription factor FOXP3, have critical immune-regulating and immunosuppressive properties imparting immune tolerance and anti-inflammatory activity in gestational tissues18,19, and recently have also been implicated in uterine vascular remodeling and reducing oxidative stress in the placenta20,21.
Several studies have shown effects of prednisolone on Treg cells. Some in vitro22 and in vivo23–25 studies show that glucocorticoids shift the balance of T cell subsets to favor Treg cells. However other in vivo studies show expansion of the Treg cell population only in patients or mice with auto-immune disorders16,23–25. Others have reported reduced numbers of Treg cells after glucocorticoid treatment in a dose response manner in patients without immune disorders and in naïve mice26. In asthma and multiple sclerosis mouse models, decreased numbers of Treg cells were also shown after glucocorticoid therapy27,28. These studies imply that glucocorticoids affect Treg cell generation differently depending on the tissue and disease context. However, no studies have been performed to evaluate the effects of early pregnancy glucocorticoid treatment on maternal Treg cells.
In this study, we investigate the effects of prednisolone treatment in the peri-im-plantation phase on the maternal Treg cell population in mice. We also analyze the consequences of prednisolone treatment on pregnancy outcome and postnatal development of offspring. The results show that prednisolone impairs CD4+ T cell activation and inhibits Treg cell generation in early pregnancy, causing elevated litter size associated with relatively minor but potentially significant changes in offspring growth and development.
MATERIALS AND METHODS
Mice and Treatments
CBA x C57Bl/6 F1 (CBA F1) female mice were purchased from the University of Adelaide Animal Facility and BALB/c male mice from the Animal Resource Centre (Perth, Australia). All mice were housed under specific pathogen-free conditions on a 12L:12D cycle, and food and water were administered ad libitum. All experiments were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific purposes, with approval of the University of Adelaide Animal Ethics Committee.
Vaginal smears were made daily to monitor the estrous cycle. When in pro-estrus, females were placed with a BALB/c male. The next day, presence of vaginal copu-latory plugs was checked between 0800 h and 1000 h. The day of sighting of a vaginal plug was considered day 0.5 post coitum (pc). From this day, female mice were housed separately from males, with 2-4 females per cage. Mice were randomly allocated to either the prednisolone or the placebo group.
On days 0.5 pc, 1.5 pc and 2.5 pc, plugged females were administered either prednisolone (Sigma, St Louis, USA) (20 µg in 100 µl 0.04% chloroform methanol (1:1) and 99.96% sterile phosphate buffered saline (PBS)) or carrier control (100 µl 0.04% chloroform methanol (1:1) and 99.96% sterile PBS) via intraperitoneal injection (ip) between 0800 h and 1000 h. Groups of mice were killed at estrus, and on days 3.5 and 9.5 pc for flow cytometry analysis of CD4+ cells. On day 5.5 pc mice were killed for assessment of implantation numbers. On day 18.5 pc mice were killed for analysis of late gestation fetal and placental development. Another group of mice progressed to term to determine perinatal outcomes and track post-natal development of offspring until 20 weeks after birth.
Flow Cytometry
On day 3.5 pc or 9.5 pc, estrus or pregnant CBA F1 females were sedated with 2% 2.2.2-tribromoethanol (Avertin, Sigma, St Louis, USA), after which peripheral blood was collected by cardiac puncture. Then, mice were killed by cervical dislocation and uterus-draining para-aortic lymph nodes (PALN), and spleens were harvested. Blood was diluted in Hanks buffered salt solution (HBSS) and mononuclear leukocytes were isolated by centrifugation over Lympholyte (Cedarlane Laboratories, Hornby, Canada)
according to the manufacturerOs instructions. Leukocytes were isolated from PALN by mechanical dispersion between glass microscope slides. Spleens were crushed through a 70 µm cell strainer after which red blood cells were lysed using red blood cell lysis buffer (154 mM NH4Cl, 99.9 mM KHCO3, 0.13 mM Na2EDTA diluted in 800 mL distilled H2O, pH 7.2).
Aliquots of 106 cells PALN and spleen cells were incubated (4 hours, 37°C, 5% CO2) in phorbol 12-myristate 13-acetate (PMA; Sigma; 20 ng/mL), ionomycin (Invi-trogen; 1 nM) and BD Golgiplug (Brefeldin A; BD Biosciences, Franklin Lakes; 1 µl/ mL as per the manufacturer)s instructions) in RPMI for in vitro stimulation. For live-dead staining, aliquots of cells from tissues and blood were incubated (10 min, room temperature) with 0.001% Fixable Viability Stain 620 (BD Biosciences) in PBS and thereafter washed with PBS. To reduce non-specific binding, cells were incubated (15 min, room temperature) with anti-Fc-γIIR antibody (FcBlock; BD Biosciences). Subsequently cells were incubated (25 min, 4oC) with APC-Cy7 anti-CD4 (GK1.5, BD Biosciences) in 0.1% bovine serum albumin (BSA) in 0.05% sodium azide in PBS, pH 7.4 (fluorescence-activated cell sorting [FACS] buffer) and washed with 0.04% sodium azide in PBS. After surface staining, cells were permeabilized and fixed using a FOXP3 Staining Buffer set (eBioscience, USA) according to the manufacturerCs instruc-tions. Cells were incubated (25 min, 4°C) with APC anti-FOXP3 (FJK-16s, eBioscience, USA), BV510 anti-IFNG (XMG1.2, Biolegend, USA), BV421 anti-CD304 (Neuropi-lin-1) (3E12, Biolegend, USA), and PE anti-CTLA4 (UC10-4F10-11, BD Biosciences) in permeabilization buffer (eBioscience, USA). Cells were washed once with perme-abilization buffer (eBioscience, USA) and once with 0.04% sodium azide in PBS. Cells were then resuspended in PBS and analyzed using a FACSCanto flow cytometer (BD Biosciences, USA). Analysis was performed using FlowJo v10 (LLC, USA). Cells stained with a single antibody and fluorescence-minus-one controls were used for setting compensation and gates29.
Assessment of Day 5.5 and 18.5 Pregnancy Outcomes
On day 5.5 pc or day 18.5 pc mice were killed by cervical dislocation between 1000 h and 1200 h. On day 5.5 pc, the uterus was removed and the number of implantation sites was counted. On day 18.5 pc the uterus was removed, and the number of viable, dead and resorbing implants was counted. Total implantation count was the sum of viable and non-viable implantations. Viable pregnancy was defined as the presence of at least one viable implantation site, and viability rate was the proportion of mice with a vaginal plug that progressed to viable pregnancy at day
18.5 pc. Each viable fetus was dissected from the amniotic sac and umbilical cord. Fetal and placental weights were recorded and expressed as absolute numbers and fetal:placental weight ratio.
Offspring Cohort
For outcome at term and neonatal analyses, the time of spontaneous delivery was noted to the nearest 12 h, the number of pups were counted and birth weight was recorded. Pups were weighed at 12-24 h, 8 days and 21 days after birth when pups were weaned and housed with littermates according to sex. All progeny were weighed again at 4 weeks and then every 2 weeks until 20 weeks of age.
Body Morphometry Analysis
At 20 weeks, mice were killed by cervical dislocation, weighed and autopsied for full body composition analysis. The following tissues were excised and weighed individually; brain, heart, lungs (left and right), kidneys (left and right), liver, adrenal glands (left and right), thymus, spleen, testes (males, left and right), seminal vesicle (males), epididymis (males), ovaries (females, left and right), uterus (females), qua-driceps (left and right), triceps (left and right), biceps (left and right), gastrocnemius muscle (left and right), retroperitoneal fat, peri-renal fat, epididymal fat (males, left and right) and parametrial fat (females). Weights of bilateral tissues and organs were combined for each mouse. Total muscle weight was calculated by summing the weights of quadriceps, triceps, biceps, and gastrocnemius muscles. Total central fat weight was calculated by summing the weights of retroperitoneal fat, peri-renal fat and epididymal fat (for males) or parametrial fat (for females), and the muscle:cen-tral fat ratio was determined. Total cenmuscle:cen-tral fat weight was subtracted from total body weight to calculate total lean weight.
Statistics and Data Analysis
Normality of distribution was assessed by Kolmogorov-Smirnov test. Normally distributed data were analyzed using Student T-test. Non-normally distributed data were analyzed using Mann-Whitney U test. Pregnancy viability was analyzed by χ2-test. Progeny weights, fetal and placental weights, and body composition data is expressed as estimated marginal means ± SEM and analyzed by Mixed Model Linear Repeated Measures ANOVA and post-hoc Sidak t-test, with mother as subject and litter size as a covariate. Statistical analysis was conducted using SPSS for Windows,
version 20.0 software (SPSS Inc, Chicago, IL, USA) and GraphPad Prism 5 software for Microsoft Windows (GraphPad software Inc., San Diego, CA, USA; Microsoft, Seattle, WA, USA). Data are displayed as mean ± standard error of the mean (SEM). Differences between the groups were considered statistically significant if p < 0.05.
RESULTS
Prednisolone alters CD4+ cell populations in the PALN
To examine the effects of prednisolone treatment in the peri-implantation phase on CD4+ T cell populations and Treg cells specifically, PALN from mice treated with prednisolone or placebo (control mice) were analyzed by flow cytometry at estrus (peri-ovulation), on day 3.5 pc (peri-implantation) and day 9.5 pc (mid-gestation) (Figure 1A). The mice that received placebo treatment showed dynamic changes in CD4+ T cells (Figure 1B) resulting in a progressive increase in the number of total CD4+ cells from estrus to implantation and mid-gestation (Figure 1C), consistent with the expected process of activation of the adaptive immune response in early pregnancy reported previously30–33. After prednisolone treatment, this increase failed to occur and the number of CD4+ cells remained similar across the time points (Figure 1C).
The proportion of Treg cells amongst CD4+ T cells on day 3.5 pc was 21% lower after prednisolone treatment compared to control mice (Figure 1D) and this contri-buted to a 37% reduction in the total Treg cell number (Figure 1E). By day 9.5, total Treg numbers were 53% lower after prednisolone treatment compared to controls (Figure 1E).
-To evaluate whether prednisolone differentially influences thymic Treg cells (tTreg cells) or peripheral Treg cells (pTreg cells), neuropilin-1 (NRP1) was used to distinguish these two Treg subtypes34. NRP1 is expressed on tTreg cells which emerge from the thymus as fully competent regulatory cells, whereas pTreg cells show lower or absent NRP1 and are induced in the periphery from naïve Th0 cells which are induced to express FOXP3 after priming with cognate antigen by tolerogenic DCs in the presence of TGFB and IL1034. The numbers of both tTreg and pTreg cells were reduced in early gestation in mice treated with prednisolone compared to mice treated with placebo (Figure 1F, 1G). CD4+FOXP3- effector T (Teff) cells were also seen to progressively increase in number in control mice across early gestation, but this did not occur in mice treated with prednisolone (Figure H).
Figure 1. The effects of peri-implan-tation prednisolone treatment on the proportions and total cell number of CD4+ cells, Treg cells, thymic Treg
cells, peripheral Treg cells, and ef-fector T cells in the uterus draining para-aortic lymph nodes (PALN) in early and mid-gestation. PALN from CBA F1 females at the estrus phase (est) of the cycle, on day 3.5 post coitum (pc), and on day 9.5 pc after mating with a BALB/c male, were analyzed by fl ow cyto-metry. Representative dotplots of fl ow cytometric analysis, gating of CD4+ cells, effector T cells (CD4
+-FOXP3-), Treg cells (CD4+FOXP3+)
cells, and gating of thymic Treg cells (CD4+FOXP3+NRP1+) and
periphe-ral Treg cells (CD4+FOXP3+NRP1-)
within the Treg cell population after prednisolone or placebo treatment on day 3.5 pc is shown (A). Data are proportions CD4+ cells (% of
live) (B), total CD4+ cell numbers (C),
proportions Treg cells (% of CD4+)
(D), total Treg cell numbers (E), total thymic Treg cell numbers (F), total peripheral Treg cell numbers (G), and total effector T cell numbers
(H) in the PALN of mice treated with prednisolone (open squares), place-bo (black point) or mice at the estrus phase without treatment (closed squares) (n = 5-12 mice per group, per time point, per treatment). The effect was evaluated using Mann-Whitney U-test to compare the prednisolone group with the placebo group at the same time point (mean ± SEM) (*p < 0.05, **p < 0.01 and #p < 0.1).
Figure 2. The effects of peri-implantation prednisolone treatment on CD4+ cells, Treg cells (CD4+FOXP3+),
and effector T cells (CD4+FOXP3-) in the spleen and blood in early and mid-gestation. Spleens and blood
from CBA F1 females at the estrus phase (est) of the cycle, on day 3.5 post coitum (pc), and on day 9.5 pc, after mating with a BALB/c male, were analyzed by fl ow cytometry. Data are proportions of CD4+ (% of live) (A), proportions of Treg cells (% of CD4+) (B), numbers of CD4+(C), and numbers of Treg
cells (D) in the spleen, and proportions of CD4+ cells (% of live) (E), and proportions of Treg cells (% of
CD4+) in blood of mice treated with prednisolone (open squares), placebo (open circles) or mice at the
estrus phase without treatment (closed squares) (n = 5-12 mice per group, per time point, per treatment). The effect was evaluated using Mann-Whitney U-test to compare the prednisolone group with the placebo group at the same time point (mean ± SEM).
Expression of cytotoxic T lymphocyte antigen 4 (CTLA4), a marker of immune suppressive function in Treg cells, was also analyzed. CTLA4 ligates costimulatory molecules on antigen presenting cells and thereby induces regulatory pathways35,36. CTLA4 expression on Treg cells was not affected by prednisolone treatment (not shown). Prednisolone does not affect systemic T cell proportions
To determine whether the effects of prednisolone treatment seen locally in the PALN are also present systemically, lymphocytes from spleens and blood were analyzed as well. Although the absolute number of spleen CD4+ T cells and CD4+ Treg cells appeared moderately lower at day 3.5 pc in prednisolone treated animals, this did not reach statistical significance (Figure 2A, 2B, 2C, 2D). In the blood, CD4+ T cell and Treg cell proportions did not differ between treatment groups (Figure 2E, 2F). Prednisolone alters T cell phenotype in PALN at mid gestation
IFNG secretion by Treg cells is reported to be essential for their immunosuppressive function 37. To determine the effect of prednisolone on cytokine secretion from Treg cells and T effector cells, in vitro stimulation of lymphocytes with PMA was performed and IFNG expression was analyzed. The proportion of Treg cells positive for IFNG was not affected by prednisolone treatment in PALN on day 3.5 pc, however the pro-portion of Treg cells expressing IFNG on day 9.5 pc was reduced after prednisolone (Figure 3B). This difference was only observed in the PALN and not in the spleen or blood (Figure 3D and F). In effector T cells, expression of IFNG indicates a T helper 1 (Th1) phenotype. In the PALN, Th1 cell numbers were lower on day 9.5 pc after prednisolone treatment compared to control (Figure 4C). In the spleen, no difference was observed, but the proportion of IFNG+ effector T cells in blood was lower on day 9.5 pc in the mice after prednisolone treatment compared to controls.
Prednisolone treatment increases litter size, and alters fetal and placental development
Next, we examined whether prednisolone treatment affects pregnancy viability, litter size, and fetal and placental growth parameters. Pregnancy outcomes were evaluated on day 5.5 pc, 18.5 pc, and day 1 after birth. Analysis of the proportion of mated mice progressing to viable pregnancy showed that prednisolone did not affect the likelihood of mated mice progressing to a viable pregnancy (Figure 4A). Assessment of the number of implantation sites in mice treated with prednisolone at
Figure 3. The effects of peri-implantation prednisolone treatment on the proportions of IFNG+
T regulatory (Treg) cells and effector T cells in the uterus draining para-aortic lymph nodes (PALN), spleen, and blood, in early and mid-gestation. PALN, spleens and blood from CBA F1 females at the estrus phase (est) of the cycle, on day 3.5 post coitum (pc), and on day 9.5 pc, after mating with a BALB/c male, were analyzed by fl ow cytometry. Gating of IFNG+
Treg cells (CD4+FOXP3+) cells after prednisolone or placebo treatment on day 3.5 pc is shown
(A). Data from PALN and spleens are proportions and total cell numbers of IFNG+ Treg cells
(% of CD4+FOXP3+) (B, D) and proportions and total cell numbers of IFNG+ effector T cells
(CD4+FOXP3-IFNG+) (% of CD4+FOXP3-) (C, E). Data from blood are proportions of IFNG+ Treg
cells (% of CD4+FOXP3-) (F), and IFNG+ effector T cells (% of CD4+FOXP3-) (G). All data are from
mice treated with prednisolone (open squares), placebo (open circles) or mice at the estrus pha-se without treatment (clopha-sed squares) (n = 5-12 mice per group, per time point, per treatment). The effect was evaluated using Mann-Whitney U-test to compare the prednisolone group with the placebo group at the same time point (mean ± SEM) (*p < 0.05 and **p <0.01).
each time point, revealed that on average prednisolone generated a 11.5% increase in litter size measured as total implants or 10.9% measured as viable implants, that was evident from as early as day 5.5 and sustained through pregnancy and after birth (Figure 4B, C). The rate of fetal loss identifi ed as resorptions sites or dead fetuses in late gestation was unaffected by maternal prednisolone administration (Figure 4D). Fetal weights were comparable between the prednisolone and the control group (Figure 5A), but placental weights were signifi cantly lower after prednisolone treatment Figure 4. The effects of peri-implantation prednisolone treatment on pregnancy
via-bility, total litter size, number of viable pups per litter, and pup survival. Pregnancy viability was noted as the proportion of mice with a vaginal plug that had at least one viable fetus measured on day 5.5 post coitum (pc), 18.5 pc, and day 1 after birth (A). Total litter size (B), number of viable pups per litter (C), and pup survival
(D) was counted on day 5.5 pc and 18.5 pc. Pup survival is shown as proportion of viable pups on day 5.5 pc. All data are from mice treated with prednisolone (open squares), placebo (open circles) (n = 20 mated mice per group, per time point, per treatment). The effect of prednisolone was evaluated using χ2-tests for pregnancy viability and student T test for litter size and viable pups to compare the prednisolone group with the placebo group (mean ± SEM) (*p < 0.05).
compared to controls, after statistical correction for litter size (p < 0.05) (Figure 5B). The fetal:placental weight ratio did not differ between the prednisolone and the control group (Figure 5C).
Figure 5. The effects of peri-implantation prednisolone treatment on fetal weight (A), placental weight (B), and fetal:placental weight ratio (C) recorded on day 18.5 post coitum. Numbers of mated mice are shown in parentheses. The effect of prednisolone was evaluated using Mixed Model Linear Repeated Measures ANOVA and post-hoc Sidak T-test for fetal and placental weights to compare the prednisolone group with the placebo group (mean ± SEM) (*p < 0.05).
Prednisolone treatment postpones parturition and alters offspring post-natal development in a sex-specific manner
To analyze the effects of prednisolone treatment on gestation length, post-natal growth, survival and postnatal growth of offspring, we noted timing of parturition and measured pup survival and weights of progeny on day 1 and day 8 after birth (Figure 6A, 6B). On day 21 after birth, offspring were weaned and sexed, and weights were then analyzed separately for male and female progeny until week 20 (Figure 6C, 6D).
The day of parturition of control mice was on average day 18.8 pc, whereas mice treated with prednisolone delivered on average 11.4 hours later (p < 0.01) (Figure 6A). On day 1 after birth, pups born after prednisolone treatment showed 9.4% lower weight compared to pups born after placebo treatment (p < 0.01) (Figure 6B). Lower weight was still evident on day 8 after birth in pups from pregnancies with prednisolone treatment, compared to pups from pregnancies with placebo treatment (p < 0.05) (Figure 6B).
Figure 6. The effects of peri-implantation prednisolone treatment on gestation length and progeny weights. Day of parturition was noted to the nearest 12 hours (A). Pups were weighed on day 1 and day 8 after birth (B). Progeny was sexed at 21 days postpartum and weighed every 2 weeks until week 20 (C, D). Numbers of mated mice are shown in parentheses. The effect of prednisolone was evaluated using Mixed Model Linear Repeated Measures ANOVA and post-hoc Sidak T-test to compare the prednisolone group with the placebo group (mean ± SEM) (#p <0.1, *p < 0.05 and **p <0.01).
Female progeny after prednisolone treatment had a signifi cantly higher weight compared to controls at three weeks postpartum, and a trend towards a higher weight at 4 weeks postpartum (Figure 6C). Thereafter until week 20, no differences in weight of female progeny was observed.
From three weeks until 16 weeks after birth, no effects of prednisolone treatment on male progeny weight was observed (Figure 6D). A trend towards lower body weights of male progeny after prednisolone treatment compared to placebo was observed at week 16 (Figure 6D). A signifi cantly lower weight of male progeny after prednisolone treatment was observed at week 18, with a trend towards a signifi cantly lower weight at week 20 postpartum (Figure 6D).
At week 20, body morphometry analysis was performed to evaluate effects of prednisolone on a wide range of individual organs and tissues (Table 1 and 2). In male progeny of dams exposed to prednisolone treatment, adrenal weight was reduced by 18% (Table 1). The weights of all other organs and tissues measured did not show significant alterations between the groups in male progeny (Table 1). In female progeny, no differences in body morphometry were found after predniso-lone treatment (Table 2).
DISCUSSION
In this study, we report that prednisolone treatment in the peri-implantation phase alters features of the maternal immune adaptation in early pregnancy. The normal increase of CD4+ cells and CD4+ Treg cells in early pregnancy was impaired in the uterus draining lymph nodes in mice that received prednisolone treatment. In addition, prednisolone treatment had consequences for pregnancy outcome and development of offspring. We observed a higher number of implantation sites with prednisolone treatment, but no effect on fetal viability or survival in utero or after birth. However, mice after prednisolone had lower placental weights, lower birth weights, and post-poned parturition. Progeny showed minor adverse developmental effects most evident in males, with growth impairment until weaning, then alterations in body weights into adulthood, plus lower adrenal weights evident at autopsy at 20 weeks.
Earlier human and mouse studies on glucocorticoid treatment in a non-pregnant setting showed both inducing and reducing effects on T cell populations in different immune mediated conditions16,23,24,26–28. Pregnancy is a substantial immune and inflammatory challenge to the mother and is accompanied by clear evidence of immune activation in the innate and adaptive compartments, that is required for optimal fetal and placental development30–32. In this study we show that CD4+ T cell numbers increased over the course of pregnancy in control mice, which was consistent with earlier studies33,38. With prednisolone treatment, this increase was impaired, levels of CD4+ T cells remained at non-pregnant levels, and with that lower numbers of CD4+ T cells were observed at day 9.5 pc compared to controls.
Amongst the CD4+ T cells, Treg cells were particularly affected by prednisolone. In control mice, Treg cells accumulated in the PALN during early pregnancy. This finding is in line with previously published studies showing that Treg cells accumu-late at the fetal-maternal interface in the peri-implantation phase39–41. In mice after prednisolone treatment, Treg cell accumulation was disturbed, as shown by the lower proportions and numbers of Treg cells. Reduced Treg cells after prednisolone treatment
Table 1. Body morphometry of male progeny
Absolute weight placebo
n = 26 prednisolonen = 28
Lean body weight (g) 29.23 ± 1.17 29.66 ± 1.14
Muscle:fat ratio 0.36 ± 0.06 0.40 ± 0.05
Total central fat (mg) 3596 ± 452 3159 ± 419
Epididymal fat (mg) 1090 ± 150 897 ± 140 Retroperitoneal fat (mg) 725 ± 52 701 ± 50 Peri-renal fat (mg) 417 ± 62 367 ± 58 Subcutaneous fat (mg) 1362 ± 202 1197 ± 186 Combined muscle (mg) 1129 ± 25 1127 ± 24 Gastrocnemius (mg) 339 ± 6 339 ± 6 Quadriceps (mg) 432 ± 9 438 ± 8 Biceps (mg) 83 ± 6 80 ± 6 Triceps (mg) 275 ± 10 270 ± 9 Brain (mg) 439 ± 3 442 ± 3 Heart (mg) 162 ± 4 161 ± 3 Lungs (mg) 229 ± 6 233 ± 6 Thymus (mg) 60 ± 3 58 ± 3 Kidneys (L + R) (mg) 539 ± 19 544 ± 18 Adrenals (L + R) (mg) 11.0 ± 1.0 9.0 ± 1.0* Liver (mg) 90 ± 5 84 ± 5 Spleen (mg) 399 ± 27 454 ± 26 Seminal vesicle (mg) 225 ± 4 217 ± 4 Testes (L + R) (mg) 152 ± 10 141 ± 9 Epididymis (L + R) (mg) 90 ± 5 84 ± 5
were associated with lower levels of Treg cells of peripheral and thymic origin, both of which are involved in the immune response to pregnancy.
A likely explanation for the impaired T cell response is the suppressive effect of corticosteroids on maturation and antigen presentation of dendritic cells (DCs)13,42. It is known that after corticoid treatment DCs are less competent at activating T cells and inducing the Treg cell population13. In early pregnancy, uterine DCs are critical for the events of T cell activation43,44. They act to take up paternal antigens present in seminal fluid, then traffic to draining PALN to stimulate T cell activation and prolifera-tion45. Altered functionality of DCs could also explain the differential impact on Tregs as opposed to T effector cells, since the balance of T effector cells versus Treg cells in any T cell response is largely determined by the phenotype and function of DCs46,47.
The effects of prednisolone were strongly evident when IFNG production in Tregs was assessed. Even though IFNG is commonly associated with a pro-inflammatory response, it has been shown that IFNG secretion by Treg cells is critical for exerting their immunosuppressive effects37,48. Notably, IFNG is essential for tolerance in graft versus host disease and organ transplantation48,49. A previous study showed that pred-nisolone inhibits IFNG positive Treg cells in vitro50. In line with this study, we found that IFNG positive Treg cells in the PALN from mice after prednisolone treatment are less frequent compared to control mice. To our knowledge, IFNG secretion by Treg cells has not been investigated in pregnancy. Presumably, IFNG secretion by Treg cells affects macrophages and DCs, and may be involved in endometrial vasculature remodeling, and maintenance of the decidua51–53. Also, IFNG expression by CD4+ T effector cells was reduced after prednisolone.
Increasing the chance of embryo implantation and pregnancy success has been the goal of prednisolone treatment in the peri-implantation phase in clinical use. However, the effectivity of prednisolone treatment in the implantation phase for increasing implantation rates and pregnancy success in unselected populations has not been proven, as shown in the Cochrane review by Boomsma et al.9, and several more recent studies5,6. Consistent with a fertility-boosting effect, we showed incre-ased numbers of implantation sites in mice after prednisolone treatment compared to placebo control mice. In general terms, the offspring showed similar likelihood of live birth and post-natal survival, however there were consequences for fetal-placental development, and outcomes for offspring, that are important to note.
Table 2. Body morphometry of female progeny
Absolute weight placebo
n = 34 prednisolonen = 17
Lean body weight (g) 23.31 ± 0.72 24.41 ± .057
Muscle:fat ratio 0.40 ± 0.05 0.41 ± 0.04
Total Central fat (mg) 2649 ± 403 2702 ± 326
Parametrial fat (mg) 744 ± 131 738 ± 105 Retroperitoneal fat (mg) 568 ± 46 580 ± 37 Peri-renal fat (mg) 291 ± 60 307 ± 47 Subcutaneous fat (mg) 1050 ± 174 1077 ± 143 Combined muscle (mg) 897 ± 27 919 ± 22 Gastrocnemius (mg) 275 ± 12 277 ± 10 Quadriceps (mg) 342 ± 12 355 ± 10 Biceps (mg) 66 ± 5 67 ± 4 Triceps (mg) 215 ± 5 220 ± 4 Brain (mg) 459 ± 5 456 ± 4 Heart (mg) 138 ± 4 134 ± 3 Lungs (mg) 201 ± 10 212 ± 8 Thymus (mg) 54 ± 3 53 ± 2 Kidneys (L +R) (mg) 352 ± 10 352 ± 8 Adrenals (L +R) (mg) 13 ± 0 13 ± 0 Liver (mg) 1300 ± 53 1316 ± 43 Spleen (mg) 88 ± 4 97 ± 4 Uterus (mg) 126 ± 10 122 ± 8 Ovary (L+R) (mg) 31 ± 2 34 ± 1
Mean ± SEM, Student T-test
We report lower placental weights after prednisolone treatment in early pregnancy. Lower placental weights after prednisolone could be related to the increased litter sizes, even though the effect was evident even when statistical correction for litter size was performed. Interestingly, previous studies have shown that antenatal exposure
to glucocorticoids affects placentation and gene expression in the placenta in a sex dependent manner54,55. Also, reduced regulatory T cells in early pregnancy has been linked to uterine artery dysfunction20. It seems likely that the reduced Treg cell propor-tions and suppressed immune response after prednisolone treatment has affected the placentation processes, resulting in lower placental weights at day 18.5 pc.
The increased gestational length we found, might be due to several factors. Firstly, T cells have been shown to be involved in parturition56,57, and since we showed that prednisolone in early pregnancy has a sustained impact on the immune response at least until day 9.5 pc, it seems possible that prednisolone exposure has constrained the T cell response involved in parturition. Secondly, the underdeveloped adrenal gland could also be a contributor to the postponed parturition in mice exposed to prednisolone treatment, since adequate glucocorticoid production by the fetus is known to be one of the signals to initiate parturition58.
In this study, we found evidence for late fetal growth restriction as shown by the evidence of normal fetal weights on day 18.5 pc but lower pup weights after birth following prednisolone treatment, compared to controls. Since fetal weights are known to increase substantially in the final days of gestation in wildtype mice59, the underdeveloped placenta observed in prednisolone treated mice, could have substantial effects in late gestation and might be an underlying mechanism for the late fetal growth restriction. Evidence shows that glucocorticoid treatment in human pregnancy reduces birth weights60,61, which is in line with our results. We noted that the effects of prednisolone treatment during pregnancy are sex dependent, with male offspring born from pregnancies with prednisolone treatment showing lower weights in adulthood from 16 weeks after birth, and female offspring having higher weights after prednisolone treatment at 3 weeks after birth. Altered epigenetic programing might play a role, as it has been shown that antenatal exposure to glucocorticoids affects programming of the epigenome in offspring in a sex dependent manner62–64.
Previous studies in humans report that glucocorticoid treatment in pregnancy acts to program neuroendocrine, metabolic, and cardiovascular disorders in the fetus61. In addition, cleft lip and palate and other anomalies were associated with corticos-teroid treatment in early pregnancy65–67. Our data suggest no major overt effects of peri-implantation prednisolone on pregnancy or offspring outcomes. However impor-tantly, this study was not powered to detect fetal congenital anomalies, so further studies are required to investigate the safety of use of prednisolone in early pregnancy.
Our findings do suggest that prednisolone either directly, or indirectly via dis-ruption of the maternal immune response, exerts programing effects on offspring phenotype, as shown by the altered pup weights until 20 weeks after birth and the lower adrenal weights in male progeny. The effect on adrenal gland development is in line with other studies showing effects of antenatal glucocorticoid treatment on hypothalamic-pituitary-adrenal regulation68,69. A study in rats induced higher maternal corticosterone levels through stress and also found lower adrenal weights in male offspring69. In general it is known that prednisolone can induce adrenal insufficiency because corticoid therapy partly replaces adrenal function70. In addition, adrenal embryonic development in mice has been shown to be dependent on several immune associated proteins such as GATA4, and GATA671. With disruption of the normal immune response through prednisolone treatment, development of the adrenal gland might be affected. The consequences of an underdeveloped adrenal gland might be significant, since adrenal insufficiency induced through antenatal overexposure to glucocorticoids was associated with subfertility in male rats69.
Our mouse model mimics peri-implantation prednisolone treatment used in assisted reproductive technology to prevent implantation failure9. In human studies and in rou-tine clinical care, prednisolone has been used in doses varying from 5 mg to 60 mg per day9. Calculating the dose per kg, the 20 μg administered in this mouse model resembles 60 mg prescribed in clinics. Treatment in human studies usually starts from ovulation, oocyte retrieval, or from the day of embryo transfer, for a period varying from 3 days up to months9. To reduce animal numbers and refine our experiments, prednisolone treatment in our model was administered on the day of appearance of a vaginal plug and not before mating. Prednisolone was administered on three consecutive days from the day of vaginal plug until day 3.5 pc, just before embryo implantation commences. In the clinical setting, prednisolone is commonly prescribed in combination with other medications such as aspirin, and hormones for ovarian stimulation and oocyte retrieval. This adds complexity to defining the specific effects of these individual agents, and could mask the effects of prednisolone on pregnancy success and the maternal immune response72.
To conclude, we report that prednisolone treatment in early pregnancy disrupts the normal maternal immune response in pregnancy, does not affect pregnancy viability or pup survival, but does have consequences for placental weights, body weights and development of offspring. These data raise further questions on the rationale for and safety of clinical use of prednisolone in human reproductive medicine.
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