Oxidative markers in cryopreservation medium from frozen-thawed embryos: a possible tool for improved embryo selection in in vitro fertilization?

PURPOSE: The present study evaluated the association between oxidative parameters in embryo cryopreservation medium and laboratory and clinical outcomes. METHODS: This prospective laboratory study was conducted in an IVF unit in a university-affiliated hospital with 91 IVF patients undergoing a frozen-thawed embryo transfer cycle. Following thawing, 50 µL of embryo cryopreservation medium was retrieved from each cryotube and tested by the thermochemiluminescence (TCL) assay. TCL amplitudes after 50 (H1), 150 (H2), and 280 s (H3) were recorded in counts per second (CPS) and the TCL ratio determined for comparison with implantation and pregnancy rates. RESULTS: A total of 194 embryos were transferred in 85 frozen-thaw cycles. Twenty-one pregnancies (24.7 %) occurred. Implantation and overall and clinical pregnancy rates were higher when the median TCL H1 amplitude was <32 CPS compared to ≥32 CPS (14.6 vs. 5.3 %, 37.5 vs. 17 %, 28.1 vs. 9.4 %, respectively). No pregnancies occurred when the H1 amplitude was ≥40 CPS. Logistic regression multivariate analysis found that only the median TCL H1 amplitude was associated with the occurrence of pregnancy (OR = 2.93, 95 % CI 1.065-8.08). The TCL ratio inversely correlated with the duration of embryo cryopreservation (r = -0.37). CONCLUSIONS: The results indicate that thawed embryos may express oxidative processes in the cryopreservation medium, and higher oxidative levels are associated with lower implantation rates. These findings may aid in the improved selection of frozen-thawed embryos for IVF.

Early embryo-endometrial signaling modulates the regulation of matrix metalloproteinase-3.

OBJECTIVE: To examine matrix metalloproteinase-3 (MMP-3) expression in human stromal cell culture after P stimulation and the effect of conditioned medium from human embryo-epithelial cells coculture on its expression and activity. DESIGN: Metabolic and endocrine studies on human tissue. SETTING: In vitro fertilization (i.v.f.) unit and endocrine research unit. PATIENT(S): Infertile patients undergoing endometrial tissue sampling for dating at the luteal phase before i.v.f. INTERVENTION(S): Endometrial sampling and collection of human embryos culture media. MAIN OUTCOME MEASURE(S): Expression and activity of secreted MMP-3 by P-induced stromal cells, and in stromal cells exposed to conditioned medium from embryo-epithelial cell coculture. RESULT(S): Expression and activity of MMP-3 in human stromal cells decrease after P induction. Following incubation of these stromal-derived decidual cells with conditioned medium from embryo-epithelial cell coculture, MMP-3 expression and activity increased in a statistically significant manner. CONCLUSION(S): Progesterone inhibition of MMP-3 expression and its support of endometrial integrity were prevented by local expression of MMP-3 in response to embryonic signaling.

Oxidative stress indices in follicular fluid as measured by the thermochemiluminescence assay correlate with outcome parameters in in vitro fertilization.

OBJECTIVE: To evaluate oxidative stress indices in follicular fluid (FF) by a novel thermochemiluminescence (TCL) assay and investigate the correlation between TCL and i.v.f. cycle parameters. DESIGN: Prospective, cross-sectional study. SETTING: I.v.f. Unit of an Obstetrics and Gynecology Department in a university-affiliated hospital. PATIENT(S): One hundred eighty-nine women undergoing consecutive i.v.f. treatment cycles during 2001. INTERVENTION(S): After oocyte retrieval, pooled FF was centrifuged and the supernatant was tested in the TCL assay. MAIN OUTCOME MEASURE(S): Maximal serum E2 levels, number of gonadotropin ampoules, retrieved oocytes, mature oocytes, fertilization and cleavage rates, number of available embryos and cryopreserved embryos, and clinical pregnancy rates and correlation with TCL indices. RESULT(S): The TCL curve slope of FF positively correlated with maximal serum E2 levels, number of mature oocytes, and number of cleaved embryos and was inversely correlated with the women's ages and the number of gonadotropin ampoules. Follicular fluid TCL amplitude at 50 seconds ranged from 294 to 711 cps, but all pregnancies (n = 50; 28.1%) occurred within the range of 347-569 cps. With 385 and 569 cps as cutoff levels for the occurrence of pregnancy, the negative predictive value beyond this range was 96% and the positive predictive value within this range was 32%. CONCLUSION(S): The TCL results may reflect the age-related increase in free radical activity and is associated with parameters of ovarian responsiveness and IVF outcome. A certain threshold of oxidative stress may be required for the occurrence of conception in i.v.f. TCL is a potential tool to evaluate, treat, and monitor antioxidant therapy in i.v.f. treatments.

Oxidative stress indices in seminal plasma, as measured by the thermochemiluminescence assay, correlate with sperm parameters.

OBJECTIVE: To examine the effect of oxidation of proteins and lipids, as measured by a novel thermochemiluminescence (TCL) analyzer, and to evaluate the correlation between TCL indices in seminal plasma and sperm parameters. DESIGN: Experimental and prospective clinical studies. SETTING: An infertility unit. PATIENT(S): One hundred forty-eight men undergoing semen analysis. INTERVENTION(S): Bovine serum albumin (BSA) and linolenic acid were oxidized and tested by TCL, protein carbonyls, and conjugated dienes assays. All participants underwent semen analysis. Seminal plasma was tested by TCL and conjugated dienes. MAIN OUTCOME MEASURE(S): Thermochemiluminescence indices before and after oxidation of BSA and linolenic acid, compared with protein carbonyl and conjugated dienes indices. Correlation between semen parameters and TCL and conjugated dienes indices in seminal plasma. RESULT(S): Oxidation of BSA and linolenic acid was marked by characteristic changes in their TCL curve pattern and an increase in the levels of protein carbonyls and conjugated dienes. Among 125 sperm-containing semen samples, the TCL curve exhibited two patterns: a positive relative ratio curve (group A, 87 patients) and a negative relative ratio curve (group B, 38 patients). Sperm concentration was lower and total motile sperm and rapid motile sperm were fewer in group B. A significant correlation was found between TCL indices, conjugated dienes, and sperm quality in group B. CONCLUSION(S): Oxidation affects TCL curve pattern of proteins and lipids in a characteristic manner. Thermochemiluminescence indices in seminal plasma closely correlate with sperm characteristics among patients with sperm disturbances, and it might serve as a tool in the evaluation, treatment, and monitoring of subfertile men.

Decreased expression of tissue inhibitor of matrix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization.

OBJECTIVE: To compare activity of matrix metalloproteinases (MMP) and expression of their tissue-specific inhibitor (TIMP) in the follicular fluid of normally ovulating women and women with the polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: IVF unit and endocrine research unit. PATIENT(S): Fourteen patients undergoing IVF treatment (seven with normal ovulation and seven with PCOS). MAIN OUTCOME MEASURE(S): Activity of MMP-2 and MMP-9 and expression of MMP-1, TIMP-1, and TIMP-2 was measured in follicular fluid of the leading follicles by using gel zymography and immunoblot analysis. RESULT(S): The activity of MMP-2 and MMP-9 and expression of MMP-1 was similar in follicular fluid of normally ovulating patients and patients with PCOS. Significantly lower expression of TIMP-1 was found in follicular fluid of patients with PCOS women compared with normally ovulating patients. CONCLUSION(S): Because MMPs and TIMPs play a role in the physical and chemical structure of the follicular compartment, the decreased expression of TIMP in patients with PCOS may be part of a compensatory process to overcome the physical properties of the thick ovarian capsule.