Neutrophil depletion in the pre-implantation phase impairs pregnancy index, placenta and fetus development

Abstract

Maternal neutrophils cells are players in gestational tolerance and fetus delivery. Nonetheless, their actions in each phase of the pregnancy are unknown. We here investigated the role of maternal neutrophil depletion before the blastocyst implantation phase and outcomes in the pregnancy index, placenta, and fetus development. Neutrophils were pharmacologically depleted by i.p. injection of anti-Gr1 (anti-neutrophils; 200 µg) 24 hours after plug visualization in allogeneic-mated C57BL/6/BALB/c mice. Depletion of peripheral neutrophils lasted until 48 hours after anti-Gr1 injection (gestational day 1.5-3.5). On gestational day 5.5, neutrophil depletion impaired the blastocyst implantation, as 50% of pregnant mice presented reduced implantation sites. On gestational day 18.5, neutrophil depletion reduced the pregnancy rate and index, altered the placenta disposition in the uterine horns, and modified the structure of the placenta, detected by reduced junctional zone, associated with decreased numbers of giant trophoblast cells, spongiotrophoblast. Reduced number of placenta cells labeled for vascular endothelial growth factor (VEGF), platelet-endothelial cell adhesion molecule (PECAM-1), and intercellular cell adhesion molecule (ICAM-1), important markers of angiogenesis and adhesiveness, were detected in neutrophil depleted mice. Furthermore, neutrophil depletion promoted a higher frequency of monocytes, natural killers, and T regulatory cells, and lower frequency of cytotoxic T cells in the blood, and abnormal development of offspring. Associated data obtained herein highlight the pivotal role of neutrophils actions in the early stages of pregnancy, and address further investigations on the imbricating signaling evoked by neutrophils in the trophoblastic interaction with uterine epithelium.

Keywords: abnormal fetal development; anti-granulocytes; maternal immune system; placenta morphology; pregnancy index; window of implantation.

Figure 1

Neutrophil depletion before the implantation phase of pregnancy reduces pregnancy index and reproductive outcomes. Anti-Gr1/isotype injection and the subsequent endpoints were illustrated in the experimental workflow (A). The total number of mononuclear cells (MN) and polymorphonuclear (PMN) was monitored before and after 48h of anti-Gr1/isotype i.p. injection by manual count (B) and flow cytometry (b’), respectively. On gestational day 5.5, mice were euthanized for implantation points count (C). Representative images of implantation points (D - arrows) in the isotype (d’), and in the Anti-Gr1 (d’’) groups are shown. The lack of implantation points observed in anti-Gr1 treated mice is also depicted (d’’’). On gestational day 18.5, pregnant mice were submitted for cesarean and the number of resorption points (E), fetuses (G), and disposition of fetuses in the uterine horns (H) was analyzed. Representative images of resorption sites (F) in the isotype (f’) and Anti-Gr1 (f’’) are shown. Schematic diagram, isotype, and Anti-Gr1 placenta distribution into the uterine horns are demonstrated (H). Each number indicates a fetus inside of the placenta. Arrow indicates cervix location. Data were statistically analyzed using One-Way ANOVA followed by Tukey’s post-test (n= 5 – 9 animals/group) and p < 0.05 was considered statistically significant. * p < 0.05 versus respective control before Anti-Gr1 injection (B). No significant difference was observed (C, E, G).

Figure 2

Neutrophil depletion altered placenta morphology and reduced the number of specialized cells in the junctional zone. On gestational day 18.5, placentas were removed for gross analyses (A-C) and then submitted for histology evaluation with H&E staining (D-F). Morphometrical analysis was employed to measure the total area (G), the Lb- labyrinth (H), Dc - decidua (I), and JZ - junctional zone (J) using PAS staining. The count of the types of cells in the junctional zone and the presence of glycogen trophoblast-positive cells was performed by analyzing five fields (M). The trophoblast subtypes were quantified and indicated in the sections as glycogen-trophoblast cells (N), giant trophoblast cells (O), and spongiotrophoblast (P). * < 0.05 in comparison to control group; #p < 0.05; ##p < 0.01 in comparison to isotype control. Data were statistically analyzed using One-Way ANOVA followed by Tukey’s post-test (n = 6 – 9). Results were expressed as mean ± SEM. Arrowheads in PAS staining (K-M) indicate spongiotrophoblast (SpTs); arrows indicate glycogen trophoblasts (GlyTCs), and asterisks indicate giant trophoblast cells (TGCs). Embedding paraffin; sections thickness 4 µm. Bars 50 µm.

Figure 3

Neutrophil depletion altered angiogenesis and adhesiveness markers in the placenta. Expression and distribution of VEGF (A-C), PECAM-1 (D-F), and ICAM-1 (G-I) in the junctional zone of placenta were evaluated by immunohistochemistry. Negative control sections labeled with anti-rabbit (J) and anti-goat (K) HRP-conjugated antibodies. Count of VEGF (L), PECAM-1 (M) and ICAM-1 (N) positive cells. *, **p < 0.05; 0.01 in comparison to control group; #, ##p <0.05, 0.01 in comparison to isotype. Data were statistically analyzed using One-Way ANOVA followed by Tukey’s post-test (n= 4 – 6 placentas). Results were expressed as mean ± SEM. Immunoreactive cells for adhesiveness markers are indicated as arrowheads. Asterisks - giant trophoblast cells; TGCs. Arrows - spongiotrophoblast – SpTs. Counterstaining with hematoxylin; embedding paraffin; sections thickness 4 µm. Bars 50 µm.

Figure 4

Neutrophil depletion altered the peripheral leukocyte profile and offspring development. On gestational day 18.5, peripheral leukocytes from mice were collected for immunophenotyping of granulocytes (Gr-1), monocytes (F4/80), natural killers cells (NK1.1), and mature B cells (B220) (A). T lymphocyte subtypes were divided into CD3+ CD4/CD8 positive cells (B) and T regulatory cells, characterized as CD3+CD4+FoxP3+ positive cells (C). The fetuses were carefully removed for gross analyses (D). *p < 0.05 in comparison to the control group. Data were statistically analyzed using One-Way ANOVA followed by Tukey’s post-test (n= 3 – 4 animals/group). Results were expressed as mean ± SEM.