Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos  Hung-Ching Liu, Ph.D., Zhi-Ying He, M.S., Carol A Mele, B.A., Lucinda L Veeck, Owen Davis, M.D., Zev Rosenwaks, M.D.  Fertility and Sterility  Volume 71, Issue 2, Pages 361-367 (February 1999) DOI: 10.1016/S0015-0282(98)00451-8

FIGURE 1 Correlation of amplified product with amount of template used. Known amounts of rabbit globin messenger RNA (mRNA) templates were used for amplification by RT-PCR as described in the text to show the linear relation between the amount of template and the amplified product. Top: Identification of the products by sizing on a 3% agarose gel. The amounts of rabbit globin mRNAs used for amplification in lanes 1, 2, 3, 4, 5, and 6 were 0.125, 0.25, 0.5, 1, 2, and 4 pg per tube, respectively. Lane M represents the markers. Bottom: Linear relation between the amplified products and rabbit globin mRNA up to 2 pg per tube. The dotted line is the exploration of the linear line. bp = base pairs. Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)

FIGURE 2 Method for simultaneous detection of multiple gene expression in individual embryos. The messenger RNAs (mRNAs) from individual embryos were extracted by detecting the expression of multiple genes as described in the text. To ensure the consistency of the assays, external controls with fixed amounts of rabbit globin mRNA (rGmRNA (1 pg per tube) were run simultaneously with the samples. To monitor the inter- and recovery rates, some of the controls were run with exact sequential steps for extraction and RT-PCR and some controls were run with RT-PCR directly. IGF-I = insulin-like growth factor 1; IGF-II = insulin-like growth factor 2; IGF-IR = insulin-like growth factor 1 receptor; IGF-IIR = insulin-like growth factor 2 receptor; insulin-R = insulin receptor. Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)

FIGURE 3 Frequency distribution of day 3 embryos according to their embryonic stage. Forty-nine donated day 3 human preembryos were assigned randomly to culture alone (group A, □) or to coculture with human endometrial stromal cells isolated from endometrial biopsy samples (Group B, ■) in Ham’s F-10 medium plus 10% human serum in vitro. Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)

FIGURE 4 Frequency distribution of day 6 embryos according to their embryonic stage. All embryos in group A (□) and group B (■) were cocultured for an additional 3 days in vitro. Their day 6 embryonic stages then were recorded and analyzed by Pearson’s χ2 test. Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)

FIGURE 5 Frequency distribution of embryos according to their growth potential in vitro. The embryonic growth potential in vitro, defined in the text, of group A (□) and group B (■) also was recorded and analyzed by Pearson’s χ2 test. Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)

FIGURE 6 Gene expression of embryos after culture alone or coculture with endometrial stromal cells. The expression of multiple genes was detected in embryos after culture alone (□) or coculture with endometrial stromal cells (■). The data are expressed as means ± SD. The expression of insulin-like growth factor 1 (IGF-I), and insulin-like growth factor 2 (IGF-II), insulin-like growth factor 1 receptor (IGF-IR), and insulin receptor (Insulin-R) was significantly higher in the cocultured embryos. ∗ P <.05 when the specific gene expression of embryos cultured alone and embryos cocultured was compared. IGF-IIR = insulin-like growth factor 2 receptor. Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)

FIGURE 7 Gene expression of embryos that reached the blastocyst stage after culture. The expression of insulin-like growth factor 1 (IGF-I), insulin-like growth factor 2 (IGF-II), insulin-like growth factor 1 receptor (IGF-IR), insulin-like growth factor 2 receptor (IGF-IIR), and Insulin receptor (Insulin-R) in embryos that reached the blastocyst stage was determined and compared. The expression of IGF-I and IGF-IR was significantly higher in the cocultured embryos (■) than in the embryos cultured alone (□). Fertility and Sterility 1999 71, 361-367DOI: (10.1016/S0015-0282(98)00451-8)