Clomiphene Citrate Administered in Periconception Phase Causes Fetal Loss and Developmental Impairment in Mice.

Clomiphene citrate is a common treatment for ovulation induction in subfertile women, but its use is associated with elevated risk of adverse perinatal outcomes and birth defects. To investigate the biological plausibility of a causal relationship, this study investigated the consequences in mice for fetal development and pregnancy outcome of periconception clomiphene citrate administration at doses approximating human exposures. A dose-dependent adverse effect of clomiphene citrate given twice in the 36 hours after mating was seen, with a moderate dose of 0.75 mg/kg sufficient to cause altered reproductive outcomes in 3 independent cohorts. Viable pregnancy was reduced by 30%, late gestation fetal weight was reduced by 16%, and ∼30% of fetuses exhibited delayed development and/or congenital abnormalities not seen in control dams, including defects of the lung, kidney, liver, eye, skin, limbs, and umbilicus. Clomiphene citrate also caused a 30-hour average delay in time of birth, and elevated rate of pup death in the early postnatal phase. In surviving offspring, growth trajectory tracking and body morphometry analysis at 20 weeks of age showed postweaning growth and development similar to controls. A dysregulated inflammatory response in the endometrium was observed and may contribute to the underlying pathophysiological mechanism. These results demonstrate that in utero exposure to clomiphene citrate during early pregnancy can compromise implantation and impact fetal growth and development, causing adverse perinatal outcomes. The findings raise the prospect of similar iatrogenic effects in women where clomiphene citrate may be present in the periconception phase unless its use is well-supervised.

Differences in Immune phenotype in decidual tissue from multigravid women compared to primigravid women.

PROBLEM: Women with a previous uncomplicated pregnancy have lower risks of immune-associated pregnancy disorders in a subsequent pregnancy. This could indicate a different maternal immune response in multigravid women compared to primigravid women. In a previous study, we showed persistent higher memory T cell proportions with higher CD69 expression after uncomplicated pregnancies. To our knowledge no studies have reported on immune cells in general, and immune memory cells and macrophages specifically in multigravid and primigravid women. METHOD OF STUDY: T cells and macrophages were isolated from term decidua parietalis and decidua basalis tissue from healthy primigravid women (n = 12) and multigravid women (n = 12). Using flow cytometry, different T cell populations including memory T cells and macrophages were analyzed. To analyze whether a different immune phenotype is already present in early pregnancy, decidual tissue from uncomplicated ongoing pregnancies between 9 and 12 weeks of gestation from multigravida and primigravid women was investigated using qRT-PCR. RESULTS: Nearly all T cell subsets analyzed in the decidua parietalis had significantly higher CD69+ proportions in multigravid women compared to primigravid women. A higher proportion of decidual (CD50- ) M2-like macrophages was found in the decidua parietalis in multigravid women compared to primigravid women. In first trimester decidual tissue higher FOXP3 mRNA expression was found in multigravid women compared to primigravid women. CONCLUSIONS: This study shows that decidual tissue from multigravid women has a more activated and immunoregulatory phenotype compared to decidual tissue from primigravid women in early pregnancy and at term which could suggest a more balanced immune adaptation towards pregnancy after earlier uncomplicated pregnancies.

Decidual memory T-cell subsets and memory T-cell stimulatory cytokines in early- and late-onset preeclampsia.

PROBLEM: Preeclampsia is a major cause of fetal and maternal mortality and morbidity. Disturbed fetal-maternal immune tolerance, and therewith memory T cells, might be involved in its etiology. This study aims to give insight into memory T-cell populations and its associated cytokines in the decidual layers in early-onset preeclampsia (EO-PE) and late-onset preeclampsia (LO-PE). METHOD OF STUDY: Lymphocytes were isolated from the decidua parietalis and basalis from EO-PE (n = 6), LO-PE (n = 8) and healthy (n = 15) pregnancies. CD4+ and CD8+ central- (CCR7+ ), effector- (CCR7- ), tissue resident- (CD103+ ), and regulatory- (Foxp3+ ) memory cell (CD45RO+ ) populations and their activation status (CD69+ ) were analyzed using flow cytometry. qRT-PCR analysis was performed on decidua parietalis and basalis biopsies to detect mRNA expression of interferon-gamma, interleukin-1B, IL2, IL6, IL7, IL8, IL10, IL15, and IL23. RESULTS: CD4+ central-memory (CM) cell proportions were lower in the decidua parietalis in LO-PE (P < .0001) and EO-PE (P < .01) compared to healthy pregnancies. CD8+ memory (P < .05) and CD8+ CM (P < .01) cell proportions were also lower in the decidua parietalis in EO-PE compared to healthy pregnancies. This was accompanied by higher IL15 (P < .05) and IL23 (P < .05) and lower IL7 (P < .05) mRNA expression in decidua basalis biopsies from EO-PE compared to healthy pregnancies, analyzed by qPCR. CONCLUSION: In conclusion, decidual memory T-cell proportions, their activation status, and associated cytokines are altered in preeclampsia and might therefore be involved in fetal-maternal immune tolerance and the pathophysiology of preeclampsia.

Lower activation of CD4+ memory T cells in preeclampsia compared to healthy pregnancies persists postpartum.

Insufficient adaptations of the maternal immune system are associated with pregnancy complications such as preeclampsia. Memory T cells might be implicated in the pathophysiology of preeclampsia and its recurrence risk. Therefore, peripheral blood samples were taken from healthy pregnant women (n = 15), preeclamptic pregnant women (n = 15), formerly healthy pregnant women (n = 16), and formerly preeclamptic women (n = 15). CD4+ and CD8+ memory cells (CD45RO+), central-memory cells (CM, CD45RO+CCR7+), effector-memory cells (EM, CD45RO+CCR7-), and their activated (CD69+) proportions were analyzed using flow cytometry. A magnetic Luminex assay was performed on plasma samples from all groups to analyze 16 cytokines associated with memory T cells homeostasis and T cell differentiation. Proportions of CD4+ and CD8+ memory cell populations did not differ between preeclamptic and healthy pregnant women or between formerly preeclamptic and formerly healthy pregnant women. However, activated proportions of the general CD4+ memory, CD4+ EM, and CD4+ CM population in the peripheral blood were lower during preeclampsia compared to healthy pregnancies and were also lower postpartum in formerly preeclamptic compared to formerly healthy pregnant women. This was accompanied by lower IL2 concentrations in plasma from formerly preeclamptic compared to formerly healthy pregnant women. The lower activated proportions of memory T cells after a preeclamptic pregnancy were not associated with altered memory T cell associated cytokine plasma concentrations. These findings showed lower activation of memory CD4+ T cell subsets in and after preeclampsia as compared with healthy pregnancies, which makes their implication in the preeclampsia recurrence risk likely.

Memory T Cells in Pregnancy.

Adaptations of the maternal immune response are necessary for pregnancy success. Insufficient immune adaption is associated with pregnancy pathologies such as infertility, recurrent miscarriage, fetal growth restriction, spontaneous preterm birth, and preeclampsia. The maternal immune system is continuously exposed to paternal-fetal antigens; through semen exposure from before pregnancy, through fetal cell exposure in pregnancy, and through microchimerism after pregnancy. This results in the generation of paternal-fetal antigen specific memory T cells. Memory T cells have the ability to remember previously encountered antigens to elicit a quicker, more substantial and focused immune response upon antigen reencounter. Such fetal antigen specific memory T cells could be unfavorable in pregnancy as they could potentially drive fetal rejection. However, knowledge on memory T cells in pregnancy has shown that these cells might play a favorable role in fetal-maternal tolerance rather than rejection of the fetus. In recent years, various aspects of immunologic memory in pregnancy have been elucidated and the relevance and working mechanisms of paternal-fetal antigen specific memory T cells in pregnancy have been evaluated. The data indicate that a delicate balance of memory T cells seems necessary for reproductive success and that immunologic memory in reproduction might not be harmful for pregnancy. This review provides an overview of the different memory T cell subtypes and their function in the physiology and in complications of pregnancy. Current findings in the field and possible therapeutic targets are discussed. The findings of our review raise new research questions for further studies regarding the role of memory T cells in immune-associated pregnancy complications. These studies are needed for the identification of possible targets related to memory mechanisms for studies on preventive therapies.

Lower FOXP3 mRNA Expression in First-Trimester Decidual Tissue from Uncomplicated Term Pregnancies with a Male Fetus.

Pregnancies with a male fetus are associated with higher risks of pregnancy complications through maladaptation of the maternal immune system. The pathophysiology of this phenomenon is unknown. A possible pathway could be a fetal sex-dependent maternal immune response, since males have a Y chromosome encoding specific allogenic proteins, possibly contributing to a different response and higher complication risks. To analyze whether fetal sex affects mRNA expression of maternal immune genes in early pregnancy, real-time PCR quantification was performed in the decidual tissue from primigravid pregnancies (n = 20) between 10 and 12 weeks with uncomplicated term outcomes. Early-pregnancy decidual mRNA expression of the regulatory T-cell marker, FOXP3, was sixfold lower (p < 0.01) in pregnancies with a male fetus compared to pregnancies with a female fetus. Additionally, mRNA expression of IFNγ was sixfold (p < 0.05) lower in pregnancies with a male fetus. The present data imply maternal immunologic differences between pregnancies with male and female fetuses which could be involved in different pregnancy pathophysiologic outcomes. Moreover, this study indicates that researchers in reproductive immunology should always consider fetal sex bias.

Pregnancy persistently affects memory T cell populations.

Pregnancy is an immune challenge to the maternal immune system. The effects of pregnancy on maternal immunity and particularly on memory T cells during and after pregnancy are not fully known. This observational study aims to show the short term and the long term effects of pregnancy on the constitution, size and activation status of peripheral human memory T-lymphocyte populations. Effector memory (EM) and central memory (CM) T-lymphocytes were analyzed using flow cytometry of peripheral blood from 14 nulligravid, 12 primigravid and 15 parous women that were on average 18 months postpartum. The short term effects were shown by the significantly higher CD4+ EM cell and activated CD4+ memory cell proportions in primigravid women compared to nulligravid women. The persistent effects found in this study were the significantly higher proportions of CD4+ EM, CD4+ CM and activated memory T cells in parous women compared to nulligravid women. In contrast to CD4+ cells, activation status of CD8+ memory cells did not differ between the groups. This study shows that pregnancy persistently affects the pre-pregnancy CD4+ memory cell pool in human peripheral blood. During pregnancy, CD4+ T-lymphocytes might differentiate into EM cells followed by persistent higher proportions of CD4+ CM and EM cells postpartum. The persistent effects of pregnancy on memory T cells found in this study support the hypothesis that memory T cells are generated during pregnancy and that these cells could be involved in the lower complication risks in multiparous pregnancies in humans.

Immunomodulators to treat recurrent miscarriage.

Recurrent miscarriage is a reproductive disorder affecting many couples. Although several factors are associated with recurrent miscarriage, in more than 50% of the cases the cause is unknown. Maladaptation of the maternal immune system is associated with recurrent miscarriage and could explain part of its pathophysiology. Modulating the maternal immune system toward pregnancy tolerance could benefit pregnancy outcome. Although there is a clear scientific rationale that modulating the maternal immune system could benefit recurrent miscarriage, only a few studies suggest possible beneficial effects of immune modulators as a therapy for recurrent miscarriage. Therapies skewing the maternal immune response to a tolerating regulatory T cell rich environment seem especially promising; however, more research is needed to find effective and safe maternal immune modulators for reproductive pathologies as recurrent miscarriage. Moreover, the possible side effects on maternal, fetal, and neonatal immune function are essentially unknown, and its elucidation is crucial before any possible therapeutic strategies could be clinically implemented.