Prospective assessment of midsecretory endometrial leukemia inhibitor factor expression versus ανβ3 testing in women with unexplained infertility

In the population studied, healthy controls were younger than the patients with UI ( P <.001; Table 1 ). The overall mean LIF HSCORE was not different between patients and control subjects, whereas the average HSCORE for the β 3 integrin subunit was reduced in patients with UI ( P <.01). Endometrial LIF protein by immunostaining was positive in most (17/20; 85%) samples from healthy controls (example of positive immunostaining shown in Fig. 1A ), but was reduced (HSCORE < 1) in 19/55 (65%) of the endometrial samples from women with UI (example shown in Fig. 1B ). This difference did not achieve predetermined statistical significance ( P =.14). Positive immunostaining was present predominantly in the epithelial glands and lumen, with distinct immunostaining on the uterodome (pinopode) projections on the luminal surface ( Fig. 1A , inset). The reverse transcriptase–PCR results for integrin expression in the UI group were arbitrarily divided into two groups: those with normal integrin expression and those with low integrin expression. We compared LIF mRNA expression with β 3 integrin subunit expression in women with UI and control subjects. As expected endometrial LIF was low in the proliferative phase and increased 24-fold in the midsecretory phase ( P =.004). In UI patients low LIF expression was largely confined to the group of women who also had low β 3 integrin subunit mRNA expression ( Fig. 1C ). In subjects with low β 3 integrin subunit expression there was a 4.4-fold reduction in LIF mRNA abundance ( P =.03) compared with the group with normal ανβ 3 integrin expression. Using multivariant logistic regression analysis, with pregnancy as the dependent variable and adjusting for age, β 3 integrin subunit, and LIF HSCOREs, LIF was the only variable to provide an association with pregnancy ( P =.014). We examined the time to pregnancy in women based on either LIF or β 3 integrin subunit expression in women undergoing monitored cycles. Monitored cycles were conducted by clinicians based on patient preferences and were not included in the conduct of the present study. Cycle treatments including oral ovulatory agents, injectable gonadotropins, and IVF and frozen embryo transfer and the proportions of each were similar between women with normal or abnormal LIF expression ( Table 2 ). In monitored cycles, women with normal integrin expression were found to have a significant advantage with a reduced time to pregnancy compared with those with absent integrin expression ( P <.01). Still, many women who lacked ανβ 3 integrin expression conceived successfully ( Fig. 2A ), possibly due to the high proportion with histologic delay. When we compared LIF immunohistochemistry results to β 3 integrin subunit expression, women with absent integrins, but positive LIF staining, exhibited a significantly higher pregnancy rate (PR) than women who lacked both LIF and ανβ 3 integrin expression. Women with UI, low LIF, and low integrin expression rarely conceived within the 12-month time period of observation ( Fig. 2B ). Two conceptions occurred in this group—one in the month of biopsy and the other after 12 months of monitored cycles.

Numerous proteins have been shown to be biomarkers for the assessment of endometrial receptivity based on their temporal and spatial relation to the attachment phase of the embryo to the endometrium or through the use of mouse gene knockout models ( 44 ). The use of ανβ 3 integrin for determination of endometrial receptivity is a well-established test for endometrial receptivity ( http://Etegritytest.com ) but its use is limited by relative poor performance of most anti-integrin antibodies in formalin-fixed tissues ( 45 ), as well as the uniform absence of staining in biopsies exhibiting subnuclear vacuoles consistent with histologic delay ( 31 , 46 ). In the present study, we report for the first time that LIF and ανβ 3 integrin expression patterns are related, with simultaneous loss of both biomarkers in a subset of women with UI. We also showed that reduced LIF expression was more specific to poor reproductive outcomes and has an advantage versus integrin expression alone, because it is expressed earlier in the cycle compared with the abrupt initiation of ανβ 3 integrin expression on cycle day 20 (LH + 6). We found that histologically “in phase” samples that were missing ανβ 3 integrin usually lacked LIF expression, whereas “out of phase” samples lacking ανβ 3 integrin expression usually expressed LIF normally. In those patients where LIF was missing (in cases with delayed histology), reproductive outcomes appear to be compromised, similar to cases where ανβ 3 integrin expression was lacking in “in phase” histology (type II defects). These data suggest that the presence of normal LIF expression in cases where β 3 integrin subunit is absent due to histologic delay, is reassuring regarding endometrial receptivity. When absent, LIF provides a clear advantage versus integrin testing alone, as ανβ 3 integrin expression is always negative and lacks any predictive value for pregnancy outcome. As a well-established endometrial biomarker critical for implantation in rodents as well as primates ( 10 – 12 , 47 – 50 ), LIF may have advantages versus integrin testing alone for the assessment of endometrial receptivity. In this prospective case-controlled comparison of UI with control subjects, endometrial LIF expression was elevated only during the secretory phase compared with the proliferative phase, consistent with previous reports ( 15 ). We and other investigators have reported reduced LIF expression in the endometrium of women with infertility ( 17 , 22 , 51 – 53 ) and such defects in LIF expression have been previously associated with endometriosis and adenomyosis ( 54 – 57 ) and hydrosalpinx ( 33 ), similar to reports on integrin expression ( 3 ). These similarities in patterns of expression may reflect common regulatory controls such as HOXA10 ( 7 ). The synchronous loss of both LIF and ανβ 3 integrin expression argues for a common cause in their dysfunctional expression. Future studies are needed to determine what those factors are that lead to this particular type of endometrial dysfunction. Reduced endometrial LIF expression in women with UI may be associated with an endometrial P resistance, abrogating the anti-inflammatory actions of P ( 58 , 59 ). Endometrial LIF is positively regulated by estrogen (E) and P, heparin-binding EGF-like growth factor ( 41 , 60 ), and hCG ( 61 ), whereas ανβ 3 integrin is negatively impacted by E ( 8 ), but stimulated by heparin-binding EGF (reference value). Increased local production of E by aromatase expression has been linked to inflammatory changes associated with endometriosis ( 62 , 63 ). In unpublished studies, we find that E does not inhibit LIF expression, as it does ανβ 3 integrin expression in endometrial epithelium. This may account for the early onset of LIF expression during the secretory phase. Endometrial LIF expression is indirectly regulated by multiple factors including cytokines, including stimulation by interleukin-1, TNF- α , platelet-derived growth factor, EGF, and transforming growth factor β (TGF- β ), as well as down-regulation by INF-γ ( 40 ). An inhibitory role of INF-γ on LIF was recently reported in diabetic NOD mice, a murine model of impaired implantation and fertility ( 50 ). The INF-γ is elevated in the serum and PF of women with endometriosis ( 64 , 65 ) and produced by endometriotic monocytes ( 66 ). The INF-γ induces other proinflammatory cytokines associated with endometriosis and poor reproductive performance, such as interleukin 6 ( 67 – 69 ), and is a possible mechanism by which LIF expression is decreased in eutopic endometrium of women with endometriosis. Inflammatory cytokines have only been studied in the context of the uterine ανβ 3 integrin by intraperitoneal (IP) injection of PF from women with endometriosis and reduced fertility. In those studies we reported a coordinated reduction in not only ανβ 3 integrin expression but also LIF and HOXA10 in the uteri of injected female mice ( 26 ). The strengths of the study include the prospective collection of endometrial biopsies in women with UI. In addition, we had expert assistance from a gynecological pathologist (D.P.S.) and an experienced reader of HSCOREs (B.A.L.). Weaknesses include the nonuniform treatment protocols of women after the biopsy was performed. Because many women with UI and their physicians choose to do some form of ovulation induction other than IVF, we examined the time to pregnancy is this “real life” exposure to different types of monitored cycles. The types of medications used did not differ between groups, therefore we believe that this did not alter the outcomes or the conclusions of the study. In conclusion, ανβ 3 integrin expression has been used as a biological marker of endometrial receptivity but is limited by the lack of expression in samples with histologic delay. The LIF and ανβ 3 integrin expression occur during the window of implantation in healthy controls and the absence of both biomarkers is associated with poor reproductive outcomes. Like the ανβ 3 integrin, LIF expression was reduced in a subset of women with UI, but the presence of LIF was reassuring when present in samples exhibiting histologic delay. A reduction in normal expression of secretory phase endometrial LIF predicted poor reproductive outcomes and may reflect an inflammatory basis for infertility. More research is required to identify the factor(s) involved in implantation defects and how to best treat such problems before undergoing expensive or invasive therapies for infertility.

All tissues were obtained in accordance with the Committee for the Protection of Human Subjects at the University of North Carolina and Greenville Hospital System under approved Institutional Review Board protocols. We recruited ovulatory women with healthy male partners with at least 1 year of UI for inclusion in this study. Each signed an informed consent for an Institutional Review Board-approved protocol (GHS #00015759) to obtain a urinary LH-timed endometrial biopsy. To be included, each woman was required to have regular cyclic menses (25–32 days apart), partners with normal sperm parameters according to the World Health Organization, and at least one patent fallopian tube. Women with PCOS or known uterine fibroids were excluded. A total of 66 women were initially recruited, but after exclusion, 55 were included. Exclusion included the discovery of fibroids, male factor infertility, ovulatory dysfunction, or lack of adequate tissue for analysis. As controls, 20 paid volunteer female subjects were recruited who regular cyclic menses using a separate Institutional Review Board protocol at UNC (05-1757). None of these subjects had signs or symptoms of endometriosis or a history of infertility and all were in good health. There was no attempt to match controls to the cases in terms of age, body mass index (BMI), or gravidity. All subjects underwent an endometrial biopsy, timed to the midsecretory phase using urinary LH testing. Additional endometrial biopsies were obtained from the proliferative phase in healthy volunteers to verify the cycle dependency of LIF expression. Endometrial biopsies were performed using a pipelle suction curettage on LH + 7–10; cycle day 21–24 in all subjects. The menstrual cycle stage was determined by a single pathologist (D.P.S.) using the dating criteria of Noyes et al. ( 42 ). Portions of endometrial biopsies were snap frozen in liquid nitrogen in the clinic and transported to the laboratory where they were stored at −80°C until further use, whereas the remainder of the samples was placed in 10% buffered formalin for paraffin embedding and sectioning. Immunohistochemistry was performed on sections of endometrium and stained for LIF as well as the ανβ 3 integrin. For LIF immunostaining, primary antibody (N-18; Santa Cruz) was serially diluted in a solution of phosphate-buffered saline (PBS) containing 1% normal goat serum and 0.1% sodium azide to optimize the appropriate concentrations to achieve maximum sensitivity and specificity. Monoclonal antibodies to the β 3 integrin subunit (SSA6; provided by Sepal, Inc.) were used at concentrations determined by limiting dilution on cryopreserved sections. After initial incubation in blocking solution (4% normal goat serum for 30 minutes at room temperature), primary antibody was applied and further incubated at 4°C overnight. Tissue sections were deparaffinized in xylene and rehydrated then incubated with primary antibody at 4°C overnight. Negative control sections were treated with nonimmune serum diluted in the same manner. The semiquantitative assessment of expression was made using the HSCORE (0–4), by a single blinded observer (B.A.L.) and calculated using the following equation: HSCORE = Σ Pi (i + 1)/100, where i is the intensity of staining with a value of 1, 2, or 3, (weak, moderate or strong, respectively) and Pi is the percentage of stained epithelial cells for each intensity, varying from 0–100%. The use of HSCORE has previously been validated as a semiquantitative assay for immunohistochemical staining ( 43 ). To evaluate and compare LIF expression with integrin expression, we first examined messenger RNA (mRNA) derived from endometrium from healthy controls during the proliferative or midsecretory phase using quantitative real-time reverse transcriptase–polymerase chain reaction (PCR), performed an MX3000 real-time thermocycler (Stratagene) with the conditions listed here. We also studied the mRNA expression pattern during the midsecretory phase in women with UI for comparison with control subjects. Total RNA was extracted from frozen endometrial biopsies using Trizol Reagent (Ambion) according to the manufacturer's suggested conditions. The RNA quantification was performed using RiboGreen (Invitrogen) with a ribosomal RNA standard curve. First-strand complementary DNA (cDNA) was synthesized from 1,000 ng of total RNA using AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies). A no-template control was used as a negative control. In the no-template control the primer, probe, and master mix were included without cDNA. Quantitative real-time PCR was performed using primers specific for LIF and β 3 integrin subunit. Relative quantitation was obtained using the delta-delta Ct method with peptidylprolyl isomerase A (PPIA, cyclophilin A) as a constitutive housekeeping control gene. The total reaction volume for all real-time PCR experiments was 20 μ L contained cDNA from 90 ng total RNA, 1 μ L 20 × TaqMan Mix of primer and probe (Applied Biosystems), 10 μ L 2 × Brilliant II QPCR Master Mix (Agilent Technologies). Reactions were performed in 96-well plates on a Stratagene MX3000 device. Thermal cycler conditions were one cycle at 50°C for 5 minutes, and one cycle at 95°C for 10 minutes, followed by 40 cycles of 95°C for 25 seconds, 60°C for 1 minute. The PCR primers and fluorogenic probes included Hs04194521-s1 (PPIA), HS01001469 (ITGb3; β 3 integrin subunit), and Hs01055668-m1 (LIF) (Applied Biosystems). These probe-primer sets are designed across an exon–exon junction, and therefore, are expected to provide signal only from mRNA of these genes and not from similar sequence. The PPIA was chosen because previous work suggested that PPIA exhibits little variation across the menstrual cycle (Steven L. Young, unpublished data). The reverse transcriptase–PCR data were grouped by cycle phase and analyzed by one-way analysis of variance (ANOVA) using Tukey's multiple comparison test for post hoc analysis. The demographic data and HSCOREs for LIF and the β 3 integrin subunit were compared by Student's t-test using 95% confidence ( P <.05) for significance ( Table 1 ). Fisher's exact test was used for comparisons of categorical data. Multiple logistic regression was used to compare age, BMI, LIF, and β 3 integrin subunit expression with pregnancy outcomes. Time to pregnancy and pregnancy outcomes were compared between UI cases based on LIF and β 3 integrin subunit staining (HSCORE) using Kaplan Meier survival analysis and Prism statistical software in monitored cycles (GraphPad). Monitored cycles included natural cycles, and ovulation induction with oral or gonadotropin therapies, and included IVF and frozen embryo transfer. Cycles that occurred after laparoscopy were excluded.