1. Infertile women with isolated polycystic ovaries are deficient in endometrial expression of osteopontin but not αvβ3 integrin during the implantation window 
Renée DuQuesnay, MRCOG, Corrina Wright, M.B., B.Ch., Anita Abdul Aziz, Ph.D., Gordon W.H. Stamp, FRCPath., Geoffrey H. Trew, MRCOG, Raul A. Margara, M.D., John O. White, Ph.D. 
Fertility and Sterility 
Volume 91, Issue 2, Pages (February 2009)
DOI: /j.fertnstert
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Cycle-dependent expression of Integrin β3 in human endometrium. (A) Endometrial samples obtained throughout the menstrual cycle in a cohort of fertile women and women with infertility secondary to male factors were analyzed using RT-QPCR for the abundance of integrin β3 transcripts. L19-specific primers were included to serve as an internal control and normalize the RNA input used in the RT-QPCR. β3 mRNA appeared constant throughout the proliferative and early secretory phase with an abrupt fall in the midsecretory phase (P=.01). This low level of β3 transcripts continued in the late secretory phase (P=.001) of the menstrual cycle. Immunohistochemical staining for β3 integrin in the endometrium of fertile women (B) confirmed its temporal expression. Endometrial specimens, histologically dated, from fertile women <40 years were obtained during the proliferative phase (a), early secretory phase (b), midsecretory phase (c), and late secretory phase (d) of the menstrual cycle (original magnification ×20). Integrin αvβ3 is strongly expressed in the stroma during the proliferative (a—day 8) and early secretory (b—day 17) endometrium, whereas the glandular and luminal epithelium show little or no expression (a and b). During the midsecretory phase (c—day 20) an increase in staining was observed around the circumference of the glandular and luminal epithelium. The epithelial staining continued to intensify during the late-secretory phase (d—day 25), see arrows.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Cycle-dependent expression of osteopontin (OPN) in human endometrium. (A) The RT-QPCR analysis of osteopontin mRNA expression in control endometrium throughout the menstrual cycle reveals a marked increase in transcript levels starting in the midsecretory phase and continuing throughout the late secretory phase of the menstrual cycle (P=.04). The temporal expression of the protein reflects this on immunohistochemical staining (B). Staining for osteopontin was negative in the proliferative phase (a—day 8) and increased markedly from day 17 (b) into the midsecretory (c—day 21) and late secretory phases (d—day 24). Staining was exclusively glandular and luminal epithelial cells and the stroma remained negative throughout the cycle. In the late secretory phase immunoreactivity also appeared within glandular secretions (original magnification ×20).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Integrin β3 expression during the “window of implantation” in the endometrium of women with ovulatory PCO. Endometrial samples taken during the “window of implantation” on day LH +7 from infertile women with PCO morphology were compared with a control group of fertile women. All samples were confirmed to be in date according to Noye's criteria. There is no significant difference in integrin β3 mRNA levels (A) between the two groups (P=.1). The intensity of immunostaining for integrin β3 in the two groups was assessed using the semiquantitative HSCORE (B). This also showed no significant difference in β3 protein expression in either the luminal epithelium (P=.06) or the glandular epithelium (P=.41) between these two groups of women.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Osteopontin expression during the “window of implantation” in the endometrium of women with ovulatory PCO. Endometrial samples as described previously in Figure 3 were assessed using RT-QPCR for osteopontin transcripts on day LH +7. Infertile women with isolated PCO showed no difference in message from control fertile women (A). Immunostaining of specimens obtained at the same stage of the menstrual cycle (B), however, demonstrates a significant reduction in osteopontin expression in both the luminal and glandular epithelial compartments.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

6. Figure 5
Analysis of the regulation and expression of osteopontin in Ishikawa cells in response to treatment with sex steroid hormones. Ishikawa cells were treated with no hormones or in the presence of E2 (10−8 mol/L), progesterone (10−6 mol/L), and the synthetic androgen, R1881 (10−9 mol/L), alone or in combination for a period of 24–48 hours. The RT-QPCR was performed as described. Treatment with E2 resulted in an increase in gene transcription (A, P=<.0001) in comparison to treatment with progesterone at 48 hours. This increase was not enhanced by the addition of progesterone, and the addition of R1881 did not attenuate the response. Treatment with R1881 alone had no effect (not shown). Western analysis (B) of 100 μg whole-cell lysate from Ishikawa cells treated for 48 hours were probed using LF166 and LF123 (rabbit polyclonal antibodies raised against recombinant human osteopontin). An increase in osteopontin (∼41 kDa) was confirmed on treatment with both E2 and progesterone, but was more pronounced with E2. The combination of E2 + progesterone did not have an additive effect. The increase in osteopontin seen with E2 + progesterone was not affected by the addition of R1881. Again, R1881 alone had no effect on OPN levels.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions