Regulatory T cells are paramount effectors in progesterone regulation of embryo implantation and fetal growth

Progesterone (P4) is essential for embryo implantation, but the extent to which the pro-gestational effects of P4 depend on the maternal immune compartment is unknown. Here, we investigate whether regulatory T cells (Treg cells) act to mediate luteal phase P4 effects on uterine receptivity in mice. P4 antagonist RU486 administered to mice on days 1.5 and 3.5 postcoitum to model luteal phase P4 deficiency caused fewer CD4+Foxp3+ Treg cells and impaired Treg functional competence, along with dysfunctional uterine vascular remodeling and perturbed placental development in midgestation. These effects were linked with fetal loss and fetal growth restriction, accompanied by a Th1/CD8-skewed T cell profile. Adoptive transfer at implantation of Treg cells — but not conventional T cells — alleviated fetal loss and fetal growth restriction by mitigating adverse effects of reduced P4 signaling on uterine blood vessel remodeling and placental structure and by restoring maternal T cell imbalance. These findings demonstrate an essential role for Treg cells in mediating P4 effects at implantation and indicate that Treg cells are a sensitive and critical effector mechanism through which P4 drives uterine receptivity to support robust placental development and fetal growth.

sodium azide) and resuspended in 50 μL α-Fc-cIIR antibody (Fc block; BD Biosciences; 1/100 dilution) in FACS buffer at RT for 15 min in the dark. A master-mix of fluorophore-conjugated antibodies against surface markers (Supplemental Table 1) diluted in Brilliant Stain Buffer per well (BD Biosciences) was added to cells to reach the final antibody concentrations specified. Samples were incubated for 25 min in the dark at 4°C, washed 2x then fixed and permeabilized using the Foxp3 Staining Buffer Set (eBiosciences, Thermo Fisher Scientific, Waltham MA, USA). For intracellular staining, cells were washed in 1 x permeabilization wash and stained with fluorophore-conjugated antibodies against intracellular markers (Supplemental Table 1) diluted in permeabilization wash. Samples were incubated in the dark at 4˚C for 30 min, washed 2x in FACS buffer and resuspended in PBS for analysis, or resuspended in PBS containing 1% paraformaldehyde for analysis up to 5 days post-staining. Data was acquired on a BD Canto II, BD Aria III or BD LSR Fortessa X-20, using FACS Diva Software. Single stain, unstimulated and fluorescence minus one (FMO) controls were included in each experiment. Data was analyzed using FlowJo software (Treestar, Ashland, OR, USA) with a standardized gating strategy (Supplemental Figure 10).

T cell isolation and adoptive transfer
CD4 + CD25 + (Treg cells) or CD4 + CD25 -(Tconv) T cells were isolated from spleen and lymph nodes (LNs) of BALB/c mated donor B6 or CD45.1 females on 11.5-14.5 dpc using EasySep TM mouse regulatory T cell isolation kit, according to the manufacturer's instructions (Stemcell Technologies Cat# 18783, Vancouver, Canada). The purity of isolated cell suspensions was determined by flow cytometry staining (between 80-95%). Treg and Tconv cell suspensions were adoptively transferred to RU486treated B6 mice ~8 h following the final RU486 injection on day 3.5 pc. Treg or non-Treg cells were suspended in sterile PBS at 2 x 10 6 cells/mL and 2 x 10 5 cells (100 μL) were administered intravenously via tail vein injection. In experiments using CD45.1 donor females, the presence of donor Treg or Tconv cells was confirmed in the udLN of recipients 72 h post-transfer by flow cytometry.

Placental and decidual histology
Implantation sites on 9.5 dpc and placentas on 18.5 dpc were fixed in 10% neutral-buffered formalin (Australian Biostain, Australia) for 24 h followed by two 1 x PBS washes for 24 h, and stored in 70% ethanol until processing. Tissues were dehydrated in a graded ethanol series and embedded in paraffin using a Leica TP1020 Tissue Processor (Leica Microsystems, Wetzlar, Germany). Tissues stored at -20 o C were sectioned to 6 µm on a Leica Rotary Microtome (Leica Microsystems) and mounted on slides before rehydration and staining. Midsagittal cross-sections of placental tissue (2 placentas/dam) and cross-sections of implantation sites were stained with Masson trichrome using standard protocols, imaged on a Nanozoomer-XR Digital Slide Scanner (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed using NDP.view2 Viewing software (Hamamatsu Photonics). Cross-sectional areas of placental junctional zone (JZ) and labyrinth zone (LZ) were measured on digital images of complete midsagittal sections of placentas on 18.5 dpc. The proportion (%) of each region in the placenta was estimated by dividing the cross-sectional area (mm 2 ) of that region by the total midsagittal crosssectional area of the placenta. Glycogen trophoblast cells (GlyT) of these placentae were identified and quantified as % JZ region based on their distinctive appearance (4).
Several viable and abnormal implantation sites were assessed at 9.5 dpc for the presence of a viable embryo or signs of fetal loss, characterized by absence of a fetus and degenerating or necrotic fetal tissue. Decidual vessels were analyzed morphometrically on 9.5 dpc in one viable implantation site (exhibiting normal fetal development)/dam. Vessels within the middle two quadrants of the decidua, closest to the central plane in reference to embryo location, were analyzed as described (5). Freehand region tracing and ruler functions in NDP.view2 software were used to measure total vessel circumference and area (including the lumen and wall) and vessel lumen circumference and area. The total vessel area: lumen area ratio and vessel diameter (of the internal lumen) were then calculated.

Treg cell Suppression Assay
Mated female mice received RU486 (1 mg/kg) or vehicle control on 1.5 and 3.5 dpc, before the udLN were harvested from pregnant mice on 8.5-9.5 dpc, and prepared into a single cell suspension. CD4 + CD25 + Treg cells were isolated from udLN using EasySep TM mouse regulatory T cell isolation kit, as above. CD4 + CD25 + cells were pooled from mice if required to obtain the cell number necessary. CD4 + CD25 + cells were placed into wells of a 96 well cell culture plate (Corning Inc.; Cat#3799) and serially diluted 2 in 1, from 20,000 cells down to 312.5 cells per well, plus control wells containing no CD4 + CD25 + cells, in a total final volume of 40 µl / well of suppression assay media [RMPI 1640 (Gibco) + 1/100 dilution of anti-anti antibiotic-antimycotic (Gibco) + 50 μM β-mercaptoethanol (Sigma-Aldrich) + 10% FBS (Gibco). Spleens were harvested from unmated B6 female mice, a single cell suspension generated and RBCs lysed, as described above. Responder Tconv (CD4 + CD25 -) cells were isolated from spleens using the EasySep TM mouse regulatory T cell isolation kit, as per the manufacturer's instructions. Responder Tconv cells were counted and labelled with carboxyfluorescein succinimidyl ester using the CellTrace TM CFSE Kit (ThermoFisher Scientific; Cat#C34554), as per the manufacturer's instructions. Responder Tconv cells were seeded at 10,000 cells / well in suppression assay media containing Mouse T-Activator CD3/CD28 Dynabeads TM (Gibco), following manufacturer's instructions, to activate responder CD4 + CD25 -Tconv cells, with the final volume made up to 50 µl / well. Viable CD4 + CFSE + responder cells were assessed for proliferation 96 h later by analysing CFSE proliferation peaks by flow cytometry and the extent of proliferation determined by calculating the Proliferation Index using FlowJo software for each sample. In order to normalise the extent of proliferation and suppression in each mouse, every experiment had control wells which contained activated responder cells and lacked Treg cells (responder:Treg ratio of 1:0). These control wells were given the value of 100% proliferation, to which all other responder:Treg ratios from that mouse were compared to determine the extent of suppression. The mean and standard deviation at each ratio was plotted and t-test was used to compare groups at each ratio.

Progesterone Assay
Serum was recovered from blood collected from terminally anaesthetized control, RU486-, Treg-and Tconv-treated mice and stored at -80 o C. Serum P4 concentration was measured using ALPCO Mouse/Rat Progesterone ELISA kit (ALPCO, Salem NH, USA) according to manufacturers' instructions. Assay sensitivity was 0.4 ng/mL. This kit has a detection range of 0.4-100 ng/mL and reports minimal cross-reactivity with other steroid hormones. Serum was diluted in kit diluent and samples were run in duplicate.

Statistics
Statistical analysis was performed using GraphPad Prism 8 for Windows (GraphPad software, San Diego CA, USA). For data comparing two groups, unpaired T-tests were performed. Data comparing four groups was analyzed by one-way ANOVA with Sidak post-hoc t-test. ROUT's test was used to identify outliers greater than two standard deviations from the mean, which were excluded from analysis. To analyze categorical data (e.g. pregnant vs non-pregnant) Chi-squared test was performed. Fetal/placental weight data on 18.5 dpc was analyzed using SPSS Statistics 25 (SPSS Inc., Chicago IL, USA) using Linear Mixed Model ANOVA, with dam as the subject, and data are presented as violin plots to depict median and quartile values from individual dams. All other data are presented as mean ± SEM (standard error of mean) with scatter plots using symbols to depict data from individual mice. Statistical significance was set at P ≤ 0.05.

Study Approval
All animal experiments were approved by the University of Adelaide Animal Ethics Committee (approval #31874) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 8th edition 2013.
Supplemental Figure 1. Impaired luteal phase progesterone signaling elicits a dose-dependent effect on implantation. Female C57Bl/6 (B6) mice were mated to BALB/c males and administered RU486 (0.5 -8 mg/kg) or vehicle (control) on 1.5 and 3.5 dpc, and pregnancy was assessed on 9.5 dpc. (A) Appearance of uteri from RU486-treated mice. Large arrows indicate viable implantation sites, small arrowheads indicate abnormal implantation sites, identified as small (<2 mm) and/or avascular. (B) Pregnancy rate (%mated mice with ≥1 viable implantation site), total number of implantation sites per mated female, and number of abnormal implantation sites per pregnant dam. Data from Figure 1 is duplicated here for 0 and 1 mg/kg groups. Pregnancy rate was analysed by Chi-squared test. n = 14-18 mated females per group. Number of total and abnormal implantations is shown as mean ± SEM, with individual mice indicated by symbols, and was analyzed by unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #P < 0.1.