Decoding the molecular cascade of embryonic-uterine modulators in pregnancy loss of PCOS mother- an "in vivo" study

Abstract

Background: Polycystic ovary syndrome is associated with an increased rate of spontaneous abortion/early pregnancy loss and pups delivered to PCOS animals were abnormal. Currently, assisted reproductive technology has been used to help numerous infertile couples to have their babies. However, there is a low implantation rate after the transfer of embryos. Till now, it could not be concluded whether the reduced pregnancy rates observed were due to abnormal embryos or endometrial modification. Further, transgenic mouse models have been used to find out the molecular deficits behind early pregnancy complications. But, the deletion of crucial genes could lead to systemic deficiencies/embryonic lethality. Also, pregnancy is a complex process with overlapping expression patterns making it challenging to mimic their stage-specific role. Therefore, the motive of the current study was to investigate the probable molecular cascade to decipher the early pregnancy loss in the letrozole-induced PCOS mouse model.

Methods: PCOS was induced in mice by oral administration of letrozole daily for 21 days. Following, the pregnancy was established and animals were sacrificed on the day 6th of pregnancy. Animals were assessed for early pregnancy loss, hormonal profile, mRNA expression of steroid receptors (Ar, Pr, Esr1/2), decidualization markers (Hox10/11a), adhesion markers (Itgavb3, Itga4b1), matrix metalloproteinases and their endogenous inhibitor (Mmp2/9, Timp1/2) and key mediators of LIF/STAT pathway (Lif, Lifr, gp130, stat3) were analyzed in the embryo implanted region of the uterus. Morphological changes in ovaries and implanted regions of the uterus were assessed.

Results: Mice treated with letrozole demonstrated significant increases in testosterone levels along with a decline in progesterone levels as compared to control animals. PCOS animals also exhibited decreased fertility index and disrupted ovarian and embryo-containing uterus histopathology. Altered gene expression of the steroid receptors and reduced expression of Hox10a, integrins, Mmp9, Timp1/3, Gp130 & Stat3 was observed in the implanted region of the uterus of PCOS animals.

Conclusion: Our results reveal that majority of the molecular markers alteration in the establishment of early pregnancy could be due to the aberrant progesterone signaling in the embryonic-uterine tissue of PCOS animals, which further translates into poor fetal outcomes as observed in the current study and in several IVF patients.

Keywords: Letrozole; Mice; Polycystic ovary syndrome; Pregnancy loss; Progesterone.

Fig. 1

Plan of work for evaluating the pregnancy loss of letrozole-induced PCOS mouse model

Fig. 2

Hematoxylin and eosin-stained sections of the ovary and implanted region of the uterus. a. Control group b. PCOS group. CL: corpus luteum; CF: cystic follicle; GF- Graafian follicle, magnification 4X. Pictorial representation of a number of implanted sites. c. Control group d. PCOS group. Arrows indicate the implanting embryo. Hematoxylin and eosin-stained sections of embryo implanted region of the uterus e. Control group, black arrow indicates embryo f. PCOS group, black arrows indicate accumulation of erythrocytes. 1. Mesometrium. 2. Mesometrial endometrium. 3. Myometrium. 4. Anti-mesometrial decidua. 5. Embryo. Scale bar = 100 μm

Fig. 3

Steroid hormone receptors in the implanted region of the uterus. Values are mean fold changes in gene expression, a. Androgen receptor b. Progesterone receptor c. Estrogen receptor-α d. Estrogen receptor-β in the letrozole-induced PCOS mice model. Error bars represent SEM; N = 6 per group. * P < 0.05, **P < 0.01 ***P < 0.001 as compared to control group

Fig. 4

Integrin gene expression in the implanted region of the uterus. Values are mean fold changes in gene expression, a. Integrin- αv b. Integrin- β3 c. Integrin- α4 d. Integrin- β1 in the letrozole induced PCOS mice model. Error bars represent SEM; N = 6 per group. **P < 0.01, ***P < 0.001 as compared to the control group

Fig. 5

Transcription factor gene expression in the implanted region of the uterus. Values are mean fold changes in gene expression, a. Homeobox-10A b. Homeobox-11A in the letrozole induced PCOS mice model. Error bars represent SEM; N = 6 per group. **P < 0.01 and ns-non-significant as compared to the control group

Fig. 6

Matrix metalloproteinases and their inhibitors in the implanted region of the uterus. Values are mean fold changes in gene expression, a. MMP-2, and b. MMP-9. c. Gelatin gel zymograms showing pro-MMP2, active MMP2, and active MMP9 activity (arrows) in the letrozole induced PCOS mice model (represented gel picture was cropped from the same gel- uncropped/original gel picture is given in supplementary fig. S3) d. Quantification of total (Pro and active) MMP9 and MMP2 by computer-based densitometry analysis. e. & f. Values are mean fold change in gene expression of tissue inhibitor of metalloproteinase TIMP-1 & TIMP-3 respectively. Error bars represent mean ± SEM; N = 6 per group. * P < 0.05, **P < 0.01, ***P < 0.001, ns-non-significant as compared to the control group

Fig. 7

Key mediators of LIF-STAT3 related genes in the implanted region of the uterus. Values are mean fold changes in gene expression, a. Leukemia inhibitory factor b. Leukemia inhibitory factor receptor c. Glycoprotein 130 d. Signal transducer and activator of transcription 3 in the letrozole-induced PCOS mice model. Error bars represent SEM; N = 6 per group. **P < 0.01, ***P < 0.001, ns-non-significant as compared to control group

Fig. 8

Diagrammatic summary of the current study. Ar androgen receptor, Pgr progesterone receptor, Hox10a homeobox transcription factor 10a, MMP Matrix metalloproteinase, Timp tissue inhibitor of metalloproteinase, Lifr leukemia inhibitory factor receptor, Gp130 glycoprotein 130, Stat3 signal transducer and activator of transcription 3