The Effect of Clomiphene Citrate and Letrozole in Apoptotic Pathways and Cell Cycle in Human Primary Cumulus Cells and the Protective Effect of Estradiol.

Clomiphene citrate (CC) and letrozole are ovulatory stimulants that, despite high ovulation rates, achieve low pregnancy rates. This study aimed to investigate the in vitro effects of CC and letrozole, alone or in combination with estradiol, on apoptosis in human cumulus cells. We performed a controlled prospective study using primary cumulus cell cultures from patients undergoing in vitro fertilization (n=22). Alpha-inhibin immunocytochemistry was used to assess cell culture purity and morphology. Cell viability was evaluated by MTT assay, cell cycle status by flow cytometry, and Caspase-3, Bax and SOD-2, and S26 gene expression by qPCR. Cells were treated for 24 hours in 5 conditioned media: CC, CC + estradiol, letrozole, letrozole + estradiol and control. None of the treatments affected cell viability, but letrozole reduced the mean percentage of cells in the S phase compared to control (24.79 versus 21.70, p=0.0014). Clomiphene treatment increased mRNA expression of Bax (4 fold) and SOD-2 (2 fold), which was reversed by co-treatment with estradiol. SOD-2 expression increased in cells treated with letrozole compared to control (4 fold), which was also reversed by estradiol. These findings suggest that clomiphene citrate and letrozole do not significantly affect the viability of human cumulus cells. Still, the expression of genes involved in apoptosis was modulated by these drugs alone and in association with estradiol, suggesting that CC and letrozole may have direct effects on cumulus cells beyond their known mechanisms of action.

Pro-caspase-3 is constitutively expressed in luteinized granulosa cells from women undergoing controlled ovarian stimulation for in vitro fertilization.

Apoptosis regulation in luteinized granulosa cells (LGC) during assisted reproduction procedures is still controversial. Caspase-3 is a major apoptosis mediator encoded by CASP3 and formed through cleavage of its precursor pro-caspase-3. The aim of this study was to investigate the expression patterns of pro-caspase-3 (mRNA and protein) and cleaved caspase-3 in human LGC. Thirty-five women undergoing controlled ovarian stimulation for in vitro fertilization were prospectively enrolled in the study. LGC were isolated from follicular fluid during oocyte pickup and evaluated by immunocytochemistry for pro-caspase-3 and cleaved caspase-3, and by real-time PCR for CASP3 mRNA expression. We found a positive staining of pro-caspase-3 in 77 % of the LGC (95 % confidence interval [CI] 60%-84%), whereas cleaved caspase-3 was found in only 4% of the cells (95 % CI 3%-6%). The abundance of cells expressing pro-caspase-3 was independent from CASP3 mRNA levels (r = 0.24, p = 0.255) and did not correlate with the amount of cleaved caspase-3 (r = -0.24, p = 0.186). Multivariable logistic regression showed that pro-caspase-3 positivity was not influenced by clinical characteristics such as age, cause or length of infertility, antral follicle count or hormonal drugs used to induce ovulation. These findings suggest that pro-caspase-3 is constitutively expressed in LGC, allowing quick cleavage into active caspase-3 and apoptosis triggering whenever needed in the course of gonadotropin-induced follicular development.

In vitro evaluation of cryopreserved bovine sperm and its relation to field fertility in fixed-time artificial insemination.

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed-time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H2 O2 production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H2 O2 production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H2 O2 production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation-like status of the acrosome may benefit probability of pregnancy in cows.

Clinical correlation of apoptosis in human granulosa cells-A review.

The quality of oocytes depends on interactions with surrounding granulosa cells. Granulosa cells are essential in normal follicular maturation process since they produce steroidal hormones and growth factors, and they play a crucial role in follicular atresia. The success in reproductive biology and medicine depends on reliable assessment of oocyte and embryo viability which presently mainly bases on oocyte and embryo morphology. Recent investigations have tried to establish an evaluation system for oocyte quality and to predict outcome of in vitro fertilization (IVF) based on the incidence of granulosa cells and cumulus cells apoptosis. Apoptosis of granulosa cells seems to have a negative effect on conception and pregnancy rates in IFV programs. Thus, in this review we present a brief outline of clinical correlation of apoptosis in human granulosa cells and cumulus cells, and its influence upon oocyte quality and IFV outcome. Taken together, understanding the influence of granulosa cell apoptosis on oocyte quality and maturity as well as on embryo health may ultimately allow scientists and clinicians to harness better protocols of ovarian stimulation for infertility treatments.

Implantation Failure Is Associated With Increased α-Inhibin and ß-Glycan Gene Expression in Secretory Phase Endometrium: Nested Case-Control Study of Infertile Women Undergoing IVF/Fresh Embryo Transfer.

Embryo implantation involves a complex sequence of events, and a large amount of molecules have been postulated to be involved in the interaction of embryo and endometrium. This study evaluated the endometrial expression of α-inhibin and ß-glycan in the mid-secretory phase of women scheduled to in vitro fertilization (IVF) and tested whether these markers are associated with implantation failure. We performed a nested case-control study including 52 women submitted to IVF and embryo transfer, divided into 2 groups: cases with implantation failure (n = 33) and controls with confirmed clinical pregnancy (n = 19). Endometrial α-inhibin and ß-glycan gene expression was evaluated in the mid-secretory phase of the natural menstrual cycle immediately before IVF, using real-time polymerase chain reaction. We found a higher gene expression of α-inhibin (fold increase = 2.14 ± 0.32, P < .05) and ß-glycan (fold increase = 1.44 ± 0.16, P < .05) in implantation failure patients compared to confirmed clinical pregnancy patients. The areas under the receiver operating characteristics curves for prediction of implantation failure in this context were 0.692 and 0.678 for α-inhibin and ß-glycan, respectively. The present results suggest that high expression levels of α-inhibin and ß-glycan transcripts in secretory phase endometrium are associated with a lower chance of achieving pregnancy with IVF.