Differential effects of the immunosuppressive calcineurin inhibitors cyclosporine-A and tacrolimus on ovulation in a murine model

Abstract

Study question: Do therapeutic levels of cyclosporine-A and tacrolimus affect ovulation in a rat gonadotrophin-induced ovulation model?

Summary answer: Cyclosporine-A, but not tacrolimus, decreases ovulation rate when administered for 5 days before induced ovulation.

What is known already: The mainstays of immunosuppression in solid organ transplantation, to prevent rejection, are the calcineurin inhibitors cyclosporine-A or tacrolimus. These drugs could potentially affect fertility in transplanted patients. Since ovulation is an inflammation-like process with pivotal roles for several immune cells and modulators, it is possible that the calcineurin inhibitors, with broad effects on the immune system, could interfere with this sensitive, biological process.

Study design size duration: Experimental design at university-based animal facilities. A total of 45 immature Sprague-Dawley rats were used. The study was carried out over 3 months.

Participants/materials setting methods: Immature Sprague-Dawley rats (n = 45) were randomly assigned to receive equivalent doses of tacrolimus (0.5 mg/kg/day; TAC), cyclosporine-A (10 mg/kg/day; CyA) or vehicle (Control). Ovarian hyperstimulation was induced with 10 IU of equine chorionic gonadotrophin, and ovulation was triggered with 10 IU of hCG. Oocytes were retrieved from the oviducts and ovulation rates were calculated. Various subpopulations of white blood cells were counted in peripheral blood and ovarian tissue samples.

Main results and the role of chance: Animals in the CyA group showed a lower ovulation rate when compared to the TAC and Control groups (CyA: mean 9 oocytes (range 0-22); TAC: 21 oocytes (8-41); Control: 22 oocytes (6-39); P = 0.03). Regarding counts of the white blood cell subpopulations and resident neutrophils in the ovary, no significant differences were observed between the groups.

Limitations reasons for caution: Although the ovulation process is highly conserved within species, the differences between rodents and humans may limit the external translatability of the study.

Wider implications of the findings: These findings suggest that tacrolimus should be the preferred calcineurin inhibitor of choice in transplanted patients who are aiming for pregnancy.

Study funding/competing interests: Swedish Research Council and ALF of Sahlgrenska Academy, Sweden. Rio Hortega Grant from the Instituto de Salud Carlos III, Spain (CM09/00063). There are no conflicts of interest.

Keywords: calcineurin; cyclosporine-A; immunosuppression; ovary; ovulation; rat; tacrolimus; transplantation.

Figure 1.

Scheme of the experimental design using a rat gonadotrophin-induced ovulation model. Sprague–Dawley rats were synchronized for ovulation by injecting 10 IU of equine chorionic gonadotrophin (eCG) s.c.; 48 h later, ovulation was induced by injecting 10 IU of hCG i.p. and 20 h after induction, the animals were euthanized by cervical dislocation. During the 7 days prior to euthanasia, the animals received tacrolimus 0.5 mg/kg/day (TAC; n = 15); cyclosporin-A 10 mg/kg/day (CyA; n = 15); or normal saline (Control; n = 15).

Figure 2.

Ovulation rate outcomes in the rat gonadotrophin-induced ovulation model. Bars represent the median (vertical line) and ranges for the ovulation rate in different groups. Significant differences between Control and CyA group are represented by * (P<0.05). MII: metaphase II.

Figure 3.

Analysis of ELANE, MPO, RUNX2 and TIMP3 mRNA expression levels in rat ovary. Columns represent fold changes (y-axis) using the control group as reference (1-fold change). Bars represent SDs of delta-Ct measurements. ACTB, actin beta; ELANE, elastase neutrophil expressed; MPO, myeloperoxidase; RUNX2, runt-related transcription factor 2; TIMP3, tissue inhibitor of metalloproteinases.

Figure 4.

Macrophage distribution within the rat ovaries. Two main patterns of CD163+ cells were identified in rat ovaries. (A) Macrophages around the vascular structures (asterisks). (B) Macrophages within the newly formed corpus luteum. The rectangles delineated by dotted lines are shown at three different magnifications. These images illustrated ovaries from animals in the tacrolimus group.