Clinical significance of melatonin receptors in the human myometrium James Olcese, Ph.D., Stephen Beesley, Ph.D.  Fertility and Sterility  Volume 102, Issue 2, Pages 329-335 (August 2014) DOI: 10.1016/j.fertnstert.2014.06.020 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Circadian expression of hNFATc1 and hMNTR1b. RNA was extracted from cultured, synchronized hTERT myometrial cells over 24 hours, at the times indicated, and then converted to cDNA and subjected to real-time quantitative PCR. Both hNFATc1 (black lines, circles) and hMNTR1b (red lines, squares) transcripts showed differential expression over the circadian day, yet in opposite phases, i.e., the hNFATc1 expression peak preceded the hMNTR1b expression rise by 4–8 hours. The data represent the average of 3 independent experiments with duplicate samples. Statistical analysis was by one-way ANOVA with a Bonferroni post-hoc correction vs. circadian time 12, ∗P<.05. Fertility and Sterility 2014 102, 329-335DOI: (10.1016/j.fertnstert.2014.06.020) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 NFATc1 induction of hMTNR1b. Wild-type (WT) or constitutively active (CA) NFATc1 expression plasmids were transiently transfected into hTERT myometrial cells using the Mirus transfection reagent (Mirus Bio LLC). Forty-eight hours later, cells transfected with the WT NFATc1 showed a minor induction of hCOX2 (A; used here as a positive control) and hMNTR1b (B) as determined by quantitative PCR (normalization control was h18S RNA). However, transfection with the CA NFATc1 resulted in significant induction for both hCOX2 (A) and hMNTR1b (B). pBJ5 was used here as the control plasmid. The data represent the average of 3 independent experiments with duplicate samples. Statistical analysis was by one-way ANOVA with a Bonferroni post-hoc correction vs. pBJ5 control, ∗∗P<.01. Primer sequences are available upon request. Fertility and Sterility 2014 102, 329-335DOI: (10.1016/j.fertnstert.2014.06.020) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Time course of nocturnal melatonin secretion in 3 control (without bright light) and 15 light-exposed late-term pregnant volunteers. The arrows represent the time point when light was presented for 1 hour. At midnight, the melatonin levels were significantly lower in the light-exposed group (∗P<.05 by Student's t test). (B, C, D) Hourly uterine contractions before and after light exposure in 3 representative plots. Note the varying degrees and duration of suppression and the subsequent rebound of contractions after cessation of light treatment. Fertility and Sterility 2014 102, 329-335DOI: (10.1016/j.fertnstert.2014.06.020) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions