Optimized Biobanking Procedures for Preservation of RNA in Tissue: Comparison of Snap-Freezing and RNAlater-Fixation Methods.

Introduction: Personalized treatment, supported by biomarkers, would improve survival of ovarian cancer patients. RNA molecules are potentially important biomarkers. The Danish CancerBiobank provides an infrastructure for handling and storage of biological material, including RNA, from Danish cancer patients. The aim of this study was to investigate the effects of handling-time and fresh-freezing versus RNAlater® fixation on RNA degradation in solid tissue from pelvic mass samples. Materials and Methods: We evaluated RNA quality in surgical tissue from patients with a pelvic mass. Corresponding samples were either fresh-frozen or fixed in RNAlater, at eight different time points after the surgery. Integrity was measured using a bioanalyzer, and the amount and quality were further investigated by quantitative reverse transcription-polymerase chain reaction measuring the expression of housekeeping genes B2M and HPRT1. Results: Our results show that tissue RNA is stable up to at least 180 minutes after the surgery, as the quality was comparable to the quality of RNA handled immediately. Likewise, patient RNA was of acceptable quality after both fresh-frezing and RNAlater fixation, but RNAlater fixation was slightly more effective for RNA preservation. Discussion and Conclusion: Our data suggest that RNA in pelvic mass samples is relatively stable. Knowledge about RNA stability is an important prerequisite for research in RNA biomarkers, where the challenge is to balance the need for careful RNA handling and storage with the need for effective large-scale biobanking in a busy clinical setting where patient treatment is the main priority.

Glycodelin in endometrial flushing fluid and endometrial biopsies from infertile and fertile women.

OBJECTIVE: To investigate in the natural cycle just before IVF, whether glycodelin levels in endometrial flushing fluid obtained days LH+1 and LH+7 can be used in predicting pregnancy in the following IVF cycle, and whether there are differences in women with tubal factor infertility compared to women with unexplained infertility and fertile controls. STUDY DESIGN: A prospective observational multicentre study of 21 fertile and 75 infertile women (25 showed abnormal tubes with no signs of hydrosalpinges, 18 had uni- or bi-lateral hydrosalpinges, 17 were salpingectomised because of hydrosalpinges, and 15 women had unexplained infertility). Endometrial flushing at days LH+1 and LH+7, endometrial biopsy, and blood sampling at day LH+7 were performed before down-regulation for IVF. Glycodelin levels in endometrial flushing fluids (EFF), biopsies, and plasma samples were related to tubal pathology, endometrial dating and IVF outcome. Furthermore, total protein concentration was measured in EFF to investigate the influence of normal endometrial maturation on protein concentrations from days LH+1 and LH+7. RESULTS: At day LH+1, EFF glycodelin levels were higher in infertile women with abnormal tubes compared to fertile women, particularly in women conceiving after the following IVF. For women with unexplained infertility, a higher level at day LH+1 was present only in women not conceiving after the following IVF. ROC curve analysis showed that at day LH+1 EFF glycodelin levels had no predictive value for IVF outcome. At day LH+7, glycodelin levels in endometrial flushing fluids and biopsies depended on endometrial dating. CONCLUSIONS: At day LH+1, glycodelin concentration is increased in endometrial flushing fluid from infertile women with abnormal tubes compared to fertile controls without being a valuable predictor of subsequent pregnancy. At day LH+7 the glycodelin level depends on endometrial dating.

Ultrasound characteristics and histological dating of the endometrium in a natural cycle in infertile women compared with fertile controls.

OBJECTIVE: To compare endometrial dating and ultrasound texture in a natural cycle before IVF and relate these to outcome and to fertile references. DESIGN: Prospective study with a fertile reference group. SETTING: Four university IVF clinics. PATIENT(S): Seventy-five IVF patients and 21 fertile women. INTERVENTION(S): Ultrasound, biopsy, blood sampling. In vitro fertilization-ET in the following cycle. MAIN OUTCOME MEASURE(S): Endometrial evaluation, P, IVF outcome. RESULT(S): At day LH+7, 42% infertile vs. 67% fertile women demonstrated endometria that were in phase (statistically nonsignificant difference). Nine percent had an accelerated endometrium, and 47% (infertile) vs. 24% (fertile) had a delayed endometrium (statistically nonsignificant difference). Statistically significantly fewer women with tubal factor and no hydrosalpinges had an endometrium in phase (20%) than was the case in fertile women (67%). Dating could not predict outcome. Statistically significantly more women in the hydrosalpinx group had a visible midline at day LH+7 compared with the case in other infertile groups. Forty-three percent conceived after IVF-ET. Plasma P was statistically significantly lower in nonpregnant women compared with in women with ongoing pregnancies and with fertile controls. A periovulatory hyperechogenic endometrium resulted in no ongoing pregnancies. CONCLUSION(S): In a natural cycle preceding IVF, a low midluteal P level predicts a low implantation rate. A periovulatory hyperechogenic endometrium or hydrosalpinges visible at ultrasound may have some predictive value. Endometrial dating was of no help.

The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interactions in vitro.

OBJECTIVE: To investigate the effect of the anti-P Org 31710 on human blastocyst attachment to cultured endometrial epithelial cells. DESIGN: Experimental in vitro study. SETTING: University hospital. PATIENT(S): Eleven fertile endometrial donors. INTERVENTION(S): Timed endometrial biopsy for cell cultures. MAIN OUTCOME MEASURE(S): Blastocyst attachment rate on endometrial cell cultures; secretion of glycodelin and leukemia inhibitory factor into the culture medium measured by RIA and ELISA techniques; and expression of progesterone receptors, interleukin-1 receptor type-1, and integrin subunit beta(3) on endometrial epithelial cells examined by immunohistochemistry. Endometrial pinopodes visualized by scanning electron microscopy. RESULT(S): Eleven of 16 human blastocysts attached to control cultures, whereas none of 10 blastocysts attached when Org 31710 was added to the culture medium (P=.0007). Immunohistochemical studies demonstrated no significant differences between groups. Biochemical analyses displayed a trend toward higher glycodelin secretions and, by scanning electron microscopy, a tendency toward less pinopode formation in the Org 31710 group, but the results did not reach statistical significance. The presence of swollen microvilli, precursors of endometrial pinopodes, was significantly reduced on cultures with Org 31710 (P=.03). CONCLUSION(S): The study presents a model for human blastocyst-endometrial interactions responding to an anti-P drug. The exact mechanism for the anti-attachment properties of Org 31710 on the cultured endometrial cells and the blastocysts needs further evaluations.

Biochemical evaluation of endometrial function at the time of implantation.

OBJECTIVE: To review the literature on various endometrial factors assumed to be of importance to implantation and to evaluate their potential clinical value in the assessment of endometrial function at the time of implantation in infertile women in natural and stimulated cycles. DESIGN: Literature review. RESULT(S): Cytokines such as leukemia inhibitory factor, colony-stimulating factor-1, and interleukin-1 have all been shown to play important roles in the cascade of events that leads to implantation. They participate in a synchronized cooperation between the endometrium and the preimplanting embryo under the influence of steroid hormones. The same applies to the integrin alpha(v)beta(3), glycodelin, and the polymorphic mucin 1. The usefulness of these factors to assess endometrial receptivity and to estimate the prognosis for pregnancy in natural and artificial cycles remains to be proven. CONCLUSION(S): The studies performed to date have mostly included only small groups of patients with a lack of fertile controls, and only a few prospective, controlled trials have been carried out. Therefore, definite conclusions about the clinical value of these factors in the assessment of endometrial function and prognosis for pregnancy after artificial reproductive therapy cannot be drawn at present. Further evaluation of their importance for and function during implantation is needed.