Objective
Components of the insulin-like growth factor (IGF) system enhance in vitro embryo quality and implantation rates in both animal models and human in vitro fertilization (IVF). We evaluated whether differences in serum levels of these components in women prior to initiation of an IVF cycle would be predictive of subsequent outcome.
Study Design
In this retrospective study sera from women obtained at day 2 of their IVF cycle (at baseline before stimulation) were assayed for IGF-I, IGF-II, and IGF binding protein (BP)-1 by enzyme-linked immunosorbent assay. Samples from 54 women with a live birth, 38 with a transient biochemical pregnancy, 45 with a spontaneous abortion, 54 who did not become pregnant, and 35 who had an ectopic pregnancy were available for analysis. Associations between the assays and outcome were evaluated by the Kruskal-Wallis test and receiver operating characteristic analysis.
Results
There were no differences in the number of oocytes retrieved, oocyte quality, fertilization rates, or embryo grade between groups. Median concentrations of IGF-I were elevated in women with a live birth (29.1 ng/mL) as compared to women with a biochemical pregnancy (25.6 ng/mL), with spontaneous abortion (21.2 ng/mL), who were not pregnant (18.7 pg/mL), or who had an ectopic pregnancy (4.2 pg/mL) ( P < .001). Conversely, median levels of IGF-II were reduced in women with a live birth (294.5 ng/mL) as opposed to 357.5, 393.6, 407.2, and 426.9 ng/mL in women with a biochemical pregnancy, with ectopic pregnancy, with spontaneous abortion, or who were not pregnant, respectively ( P < .001). Median IGFBP-1 concentrations were markedly elevated in women with a live birth (23.6 ng/mL) compared to 18.3, 14.1, 13.8, and 9.5 ng/mL in women with a biochemical pregnancy, with spontaneous abortion, who were not pregnant, or with an ectopic pregnancy ( P < .001). The combination of IGF-I and IGFBP-1 best predicted the occurrence of a live birth with an area under the curve of 0.892.
Conclusion
Maternal serum levels of IGF-I, IGF-II, and IGFBP-1 prior to initiation of an IVF cycle are correlated with the likelihood of a live birth. Alterations in maternal IGF system components may influence oocyte quality or the success of early postfertilization events and embryo implantation.
Growth and development of the preimplantation embryo is regulated by cytokines and growth factors, including the insulin-like growth factor (IGF) system. The human IGF system consists of IGF-I, IGF-II, and their binding proteins (BP), IGFBP 1-5. The IGF system plays a major role in growth, apoptosis, maturation, and development of the human embryo.
IGF-I has multiple roles in follicular and embryonal development, through its autocrine and/or paracrine interaction with specific receptors. Embryos express receptors for IGF-I, indicating a role for this growth factor in development. When IGF-I was added to porcine embryos cultured in vitro, there was increased blastocyst formation and number of cells in the inner cell mass. Supplemental IGF-I also has been shown to decrease the number of apoptotic nuclei in preimplantation in vitro fertilized (IVF) porcine and human embryos. Following addition of IGF-I, expression of the antiapoptotic Bcl-2 gene was increased, while transcription of the proapoptotic Bax gene was reduced. In a prospective study of human embryos cocultured with autologous endometrial cells or with conventional medium, higher IGF-I levels (>2 ng/mL) correlated with improved embryo quality. Furthermore, higher IGF-I levels were associated with progression to a subsequent clinical pregnancy rather than to adverse outcomes.
IGF-II is expressed by trophoblasts and placental villi and is considered a key mediator of trophoblast invasion in human beings. IGF-II messenger RNA levels are highest in the first trimester, indicating a role in early cytotrophoblastic differentiation. IGF-II has been shown to interact with IGFBP-1 in the decidua to modulate trophoblast proliferation.
Aside from IGF’s role in promoting embryonal growth and development, the IGF system is also intimately involved in follicle growth and oocyte maturation. The addition of IGF-I to an in vitro maturation culture medium had beneficial effects on porcine oocyte maturation and IVF outcome. In a study of patients with polycystic ovary syndrome and controls, IGF-I levels increased in both groups during oocyte stimulation. A higher mean number of immature oocytes were retrieved in women with polycystic ovary syndrome whose IGF-I level decreased from days 3-28, suggesting a role for IGF-I in oocyte maturation. Likewise, studies have shown that women undergoing IVF who had elevated levels of IGF-I in their follicular fluid had a higher rate of successful outcomes than did women with reduced follicular IGF-I levels. Similarly, levels of IGF-I were higher in sera from women with conception cycles compared to those with nonconception cycles. The observation that IGF-I plays a role in folliculogenesis suggests that expression of IGF-I in maternal serum, before initiation of IVF stimulation, can contribute to the success or failure of IVF.
In the present study, we evaluated whether differences in serum levels of IGF system components in women prior to initiation of an IVF cycle would be predictive of subsequent outcome.
Materials and Methods
Subjects
In this retrospective study, sera from 226 women obtained at day 2 of their IVF cycle (at baseline before stimulation) were collected. The only basis of study participation was the availability of a serum sample obtained at the time of interest from women who underwent an IVF cycle. The subjects in the study were seen from January 2010 through November 2011. The women were divided into cohorts based on pregnancy outcome: 54 with a live birth (delivery of a liveborn infant), 38 with a biochemical pregnancy (defined as transient rise and fall of human chorionic gonadotropin [hCG] without documentation of a gestational sac or ectopic pregnancy), 45 with a spontaneous abortion (nonviable fetus following observation of a uterine gestational sac), 54 who did not become pregnant (negative hCG on day 28), and 35 with an ectopic pregnancy. The ectopic pregnancy group included those women with ultrasound documentation of a gestational sac or fetal pole in the adnexa, or surgical/pathological confirmation. This study was approved by the institutional review board at Weill Cornell Medical College and all subjects gave informed written consent.
IVF protocol
Patients were treated with standard ovulation induction protocols and underwent IVF-embryo transfer. Specific protocols were selected on a case-by-case basis as determined by the woman’s attending physician. In general, ovarian stimulation began on day 3 of their treatment cycle and consisted of a combination of gonadotropins (human menopausal gonadotropin) and/or follicle-stimulating hormone. Some women received estrogen priming or a leuprolide acetate microflare and gonadotropin-releasing hormone antagonists were administered when appropriate. hCG was administered (3300-10,000 IU) when at least 2 follicles reached or exceeded 17 mm mean diameter as measured by transvaginal ultrasound. Oocyte retrieval was conducted by transvaginal ultrasound-guided follicular puncture 35-36 hours after hCG administration.
Conventional oocyte insemination or micromanipulation was performed as indicated. Morphologically normal-appearing embryos were transferred into the uterine cavity approximately 72-120 hours after retrieval. Patients underwent day 3 or day 5 transfers as clinically indicated. The number of embryos transferred was dependent on maternal age and history, but mostly 1-3 embryos were transferred. Progesterone supplementation (50 mg intramuscular/d) was initiated on the third day after hCG administration and was continued until the sonographic documentation of a fetal heart beat at approximately 7 weeks of gestation. A review of the Weill Cornell protocols was recently published.
IGF system assays
All sera were stored at –80°C until assayed. The sera were assayed for IGF-I, IGF-II, and IGFBP-1 by commercially available enzyme-linked immunosorbent assay kits (IGF-I and IGFBP-1 from R&D Systems, Minneapolis, MN, and IGF-II from Alpco, Salem, NH). All analyses were performed simultaneously and were measured in duplicate. Laboratory personnel were blinded to all clinical data. The sensitivities of the enzyme-linked immunosorbent assay kits were 31.25 pg/mL for both IGF-I and IGFBP-1, and 0.45 ng/mL for IGF-II. Intraassay and interassay variability for each assay was <10%.
Statistical analysis
Associations between the assays and pregnancy outcome were evaluated by the nonparametric Kruskal-Wallis test, since the values did not follow a normal distribution. A receiver operating characteristic (ROC) curve was constructed to determine whether any single assay or combination of assays best predicted a successful IVF outcome (live birth). A P value of < .05 was considered statistically significant. Graph Pad (Graph Pad Software, Inc, San Diego, CA) and SPSS Statistics (IBM Corp, Armonk, NY) were used for the analysis.
Results
Characteristics of the women in each group are described in Table 1 . There were no differences in regards to body mass index, number of oocytes retrieved, mature oocytes observed, number of fertilized oocytes, embryo grade, and number of embryos transferred. There was no correlation between these oocyte and embryo parameters and IGF components ( Table 2 ). However, the age distribution of women who did not become pregnant (mean age, 39.4 years; median age, 40.0 years; range, 33.0–45.0 years) and those who had a spontaneous abortion (mean age, 38.6 years; median age, 39.0 years; range, 26.0–46.0 years) were different compared to women who had a live birth (mean age, 36.0 years; median age, 35.5 years; range, 23.0–45.0 years) ( P < .001). The cause of the couples’ infertility–male factor, tubal blockage, low ovarian reserve/advanced maternal age, anovulation/polycystic ovary, endometriosis, uterine anomaly, idiopathic–did not differ between outcome groups ( P > .05). Furthermore, there was no association between cause of infertility and concentrations of IGF-I, IGF-II or IGFBP-1. The number of previous IVF attempts did not vary between outcome groups. Among the total study population, this was the first cycle in 31-42%, the second cycle in 25-29%, the third cycle in 9-13%, and greater than the third cycle in 16-31% of subjects. There were no differences in outcome between day 3 or day 5 transfers and the outcomes of the IVF cycle were comparable with the individual stimulation protocols.
| Parameter | Live birth | Biochemical | Spontaneous abortion | Not pregnant | Ectopic | P value |
|---|---|---|---|---|---|---|
| n = 54 | n = 38 | n = 45 | n = 54 | n = 35 | ||
| Age, y | 36.0 ± 0.6 | 37.4 ± 0.7 | 38.6 ± 0.6 a | 39.4 ± 0.4 a | 37.0 ± 0.7 | .0002 |
| BMI, kg/m 2 | 23.0 ± 0.6 | 24.0 ± 0.7 | 23.9 ± 0.4 | 24.3 ± 0.7 | 23.6 ± 1.0 | NS |
| No. oocytes retrieved | 13.4 ± 0.8 | 11.5 ± 1.1 | 9.9 ± 0.8 | 9.7 ± 0.8 | 11.3 ± 1.0 | NS |
| Mature oocytes | 10.1 ± 0.6 | 9.7 ± 0.9 | 8.0 ± 0.6 | 7.9 ± 0.7 | 9.1 ± 0.8 | NS |
| Embryo grade | 2.6 ± 0.3 | 3.0 ± 0.3 | 2.6 ± 0.3 | 2.5 ± 0.2 | 2.7 ± 0.3 | NS |
| No. fertilized | 7.7 ± 0.5 | 7.6 ± 0.8 | 6.5 ± 0.5 | 6.0 ± 0.6 | 6.8 ± 0.7 | NS |
| No. transferred | 3.0 ± 0.2 | 2.9 ± 0.2 | 3.1 ± 0.2 | 2.8 ± 0.2 | 3.0 ± 0.2 | NS |
| IGF component | Parameter | Spearman r | P value |
|---|---|---|---|
| IGF-I | No. oocytes retrieved | .091 | .174 |
| IGF-II | .026 | .702 | |
| IGFBP-1 | –.031 | .643 | |
| IGF-I | Mature oocytes | .058 | .386 |
| IGF-II | .075 | .259 | |
| IGFBP-1 | –.004 | .949 | |
| IGF-I | No. oocytes fertilized | .008 | .903 |
| IGF-II | .121 | .069 | |
| IGFBP-1 | .036 | .588 | |
| IGF-I | No. embryos transferred | –.073 | .277 |
| IGF-II | .008 | .900 | |
| IGFBP-1 | .080 | .230 | |
| IGF-I | Embryo grade | –.025 | .711 |
| IGF-II | .068 | .309 | |
| IGFBP-1 | –.078 | .245 |
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